Our efforts in protein simulation originated from our early theoretical considerations of the effect of hydrogen bonds on protein reactivity, and of the physical origin of hydrophobic interactions and their effect on protein reactivity.  Applications of these ideas to experimental studies of bovine pancreatic ribonuclease A led to the identification of three tyrosyl...aspartate interactions which serve as distance constraints on the folding of the backbone chain, and thus, with the aid of an empirical potential function, help to simulate the three-dimensional structure.  This motivated our initial development of an all-atom force field and methods for its global optimization to compute protein structure.  Initial results from applications of this approach led to structures of the cyclic decapeptide gramicidin S, models of the fibrous protein collagen, and the three-helix bundle of the 46-residue protein A, all of which were verified by experiment.  These results and the extension of the all-atom model to a united-residue (UNRES) one, to be able to apply simulation methods to compute structures and folding pathways of proteins larger than protein A, will be discussed, together with results with UNRES in CASP blind tests.

Twenty-five years ago, how proteins folded into organized structures on the basis of their sequence was a great mystery. By characterizing the energy landscapes of proteins with tools from the statistical mechanics of disordered systems like spin glasses, a "new view" of the folding process became possible. Energy landscape theory provided an incentive to pursue heroic new experiments and to carry out difficult computer simulations addressing protein folding mechanisms.

Many aspects of folding kinetics revealed by these studies can be quantitatively understood using the simple idea that the topography of the energy landscape is that of a "rugged funnel".

Energy landscape theory provided a quantitative means of characterizing which amino acid sequences can rapidly fold. Algorithms based on energy landscape theory have been used to successfully design novel sequences that fold to a given structure in the laboratory.

Energy landscape ideas have begun to transform the prediction of protein structure from sequence data from being an art to being a science. The success of energy landscape- based algorithms in predicting protein structure from sequence will be highlighted. While there is still much to learn about folding mechanisms and much work to do achieving universally reliable structure prediction, many parts of what used to be called "the protein folding problem" can now be considered solved.

The detailed characterization of the overall free energy landscape associated with the folding process of a protein is the ultimate goal in protein folding studies. Modern experimental techniques provide accurate thermodynamic and kinetic measurements on restricted regions of a protein landscape. Although simplified protein models can access larger regions of the landscape, they are oftentimes built on assumptions and approximations that affect the accuracy of the results. We present new methodologies that allows to combine the complementary strengths of theory and experiment for a more complete characterization of a protein folding landscape at multiple resolutions. Recent results and possible applications will be discussed.

The conversion of the cellular prion protein (PrP), into its altered conformation – the PrP^Sc – is believed to be the major cause of prion diseases. Although PrP is until now the only identified agent of these diseases, there are several evidences that other molecules can modulate this conversion. We have identified that interaction of PrP with double-stranded DNA lead to a higher beta-sheet content, with similar characteristics of PrP^Sc and have structurally characterized the PrP:DNA complex. RNA molecules can also interact with PrP and potentially modulate PrP^C to PrP^Sc conversion or even bind differentially to both PrP isoforms. In this work we have investigated the interaction of recombinant full-length PrP and two PrP amino terminal deletion mutants with distinct RNA sequences or total RNA extracted from cultured cells. We have shown that PrP interacts with RNA with nanomolar affinity and aggregates upon this interaction. RNA does not induce aggregation of the PrP N-terminal deletion mutants, indicating that the N-terminal region is important for this process. Cell viability assays showed that PrP:RNA aggregates are toxic to cultured cells. These results indicate that RNA can modulate recombinant prion protein conversion into toxic aggregates.  Support: CNPq, FAPERJ, CAPES, IMBEBB, L’ÓREAL.

Heme proteins are highly versatile molecules that perform a multitude of tasks in biological systems, including oxygen storage and transport, electron transport and ligand sensing. The seemingly simple ligand binding reaction to heme proteins is actually quite complicated, as seen from the presence of several intermediate states in kinetic experiments. Time-resolved and cryo-crystallography on carbonmonoxy myoglobin (MbCO) under photolyzing conditions have related kinetic intermediates to different photoproduct structures, in which CO resides in different protein internal cavities (1, 2). With a variety of infrared and visible spectroscopy techniques, we have investigated the functional role of these cavities in the ligand binding reaction (3). Recently, we have applied the same experimental approach to investigate NO binding to both ferrous and ferric Mb to evaluate the effect of the ligand itself and of the iron oxidation state on the migration within the protein. Moreover, we have extended the investigation towards other heme proteins, including the monomeric mini-hemoglobin of Cerebratulus lacteus (CerHb) and the dimeric hemoglobin of Scapharca inaequivalvis (Scapharca HbI). Similarities and differences in the behavior between different ligands and heme proteins will be discussed.   References 1.         Ostermann, A., Waschipky, R., Parak, F. G., and Nienhaus, G. U. (2000) Ligand binding and conformational motions in myoglobin, Nature 404, 205-208.2.         Schmidt, M., Nienhaus, K., Pahl, R., Krasselt, A., Anderson, S., Parak, F., Nienhaus, G. U., and Srajer, V. (2005) Ligand migration pathway and protein dynamics in myoglobin: a time-resolved crystallographic study on L29W MbCO, Proc Natl Acad Sci U S A 102, 11704-11709.3.         Nienhaus, K., Deng, P., Kriegl, J. M., and Nienhaus, G. U. (2003) Structural Dynamics of Myoglobin: The Effect of Internal Cavities on Ligand Migration and Binding, Biochemistry 42, 9647-9658. 

Protein misfolding has been implicated in a large number of diseases, which are now grouped under the name of protein folding disorders (PFDs). In these diseases, large quantities of wrongly folded proteins undergo aggregation, destroying brain cells and other tissues. Such disorders include Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington’s disease, and type II diabetes, among other well-known diseases. To approach the changes in hydration, packing and volume both when proteins fold correctly or when folding goes wrong leading to the protein folding disorders, we have used several biophysical and structural tools, including hydrostatic pressure. Partially folded conformations, which are stabilized by pressure, are usually at the intersection between productive and off-pathway folding. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP^C) can be altered into a misfolded, b-sheet-rich isoform, the PrP^Sc (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. Prions seem to have other accomplices that chaperone their activity in converting the normal, cellular form into the disease-causing isoform – among chaperone candidates, a nucleic acid is the most likely. We find that binding of PrP to nucleic acid is followed by a decrease in solvent accessible surface area and in hydration. Our recent studies on the application of high pressure on amyloidogenic proteins will be discussed as regards to the changes in hydration and volume. The pressure studies reveal that amyloid fibrils and aggregates of PrP, a-synuclein and transthyretin excludes molecules of water. The use of high pressure has contributed to the identification of the underlying mechanisms of these misfolding diseases and to develop new therapeutic approaches  Acknowledgments: Supported by FAPERJ, CNPq, and FINEP.

We will present an analytical model of studying the statistical fluctuations of conformational dynamics of single molecules. We show both time and spatial dependent diffusion play important roles in determining the kinetics. At very high temperatures, the systems approaches Poisson statistics. At lower temperature, the system approches log normal statistics. At even lower temperature, the system approaches a power statistics. At lowest temperature, the system approaches again to Poisson statististics. We explain in details the origin of the resulting statistics from the energy landscape perspectives. We also performed microscopic detailed simulations to show the universality of the anaylytical results.

The cell plasma membrane is the site of numerous vital interactions between the cell and its environment. Remodeling of its composition and shape is driven by a plethora of interactions between its lipid and protein components with external or cytoplasmic ligands. Much progress has been made towards understanding the complexity of this interface. Yet, most interactions are highly dynamic and of nanometer scale. Because current optical techniques are restricted by the diffraction limit, novel approaches to confine the excitation volumes have been devised. Zero mode waveguides (ZMWs) are optical nanostructures of sub-wavelength dimensions that have been used in combination with fluorescence correlation spectroscopy (FCS) for focal volume confinement and study of single-molecule interactions at physiologically relevant concentrations. The use of these structures on plasma membranes of RBL mast cells is reported. A requirement for the effective application of ZMWs to plasma membranes is that the membrane penetrates the nanostructures and enters the evanescent field. We demonstrate that cell membrane invaginates into the nanostructures in an actin filament-dependent but microtubule-independent fashion. It is also observed that there is a strong correlation between actin-EFGP filament formation in the ZMWs and membrane invaginations, further supporting the notion of a directed exploration of the nano-structured substrate. Finally, we show that FCS autocorrelation curves from cell membrane labels can be obtained with single molecule resolution and compare them with curves obtained from model membranes and freely diffusing molecules. The demonstration of cell membrane invagination into ZMWs is an important first step towards the application of the optical nano-structures to cell membrane studies and the delivery of highly localized stimuli for signal transduction elucidation.

The remarkable mechanical properties of living cells and tissues are
attributed to a dense meshwork of semiflexible biopolymers known as
the cytoskeleton. The cytoskeleton is responsible for a multitude of
essential biological processes, among them intracellular transport,
cell locomotion, contractility and mechanotransduction. Physical
understanding of the cytoskeleton requires a combination of in vitro and in
vivo approaches. We show striking similarities between the nonlinear
mechanics of living cells and those of reconstituted networks and solutions
of biopolymers. The agreement brings hope of an understanding
of biomechanics in physical terms. We digress on the possible microscopic
mechanisms and introduce new theoretical approaches.

Micro-organisms play a central role in every ecosystem and in the global biomass cycle. They live in almost every habitat, generally forming biological communities with strong interactions. Nevertheless, nearly 99% of them have not yet been successfully cultured in vitro. Microbiology is a subject with practical applications in many fields: medicine, environmentalism, biotechnology and food industry, among others. Therefore, new approaches and new tools have to be developed in order to understand the behaviour of micro-organisms in complex systems (ecosystems, cultures, bioreactors). Theoretical and practical methodologies to study microbial communities at a mesoscopic scale (10⁰-10⁶ cells) are required to deal with such systems. Individual-based Modelling (IbM) has become a widely used tool for describing complex systems made up of autonomous entities such as ecosystems and social networks. IbMs have proven to be adequate because: a) they are bottom up approaches that describe the system’s behaviour as a whole by establishing procedural rules for the individuals and for their interactions, and thus allow more realistic assumptions for the model of the individuals than continuous models do; b) they permit the introduction of randomness and individual variability, so they can reproduce the diversity found in real systems; c) they supply a holistic description of the system and its emerging properties. IbMs are adequate to deal with microbial communities in non-steady states, such as lag phase and decay phase. And spatially explicit IbMs are appropriate to study laboratory and natural microbiological systems with spatial heterogeneity, such as the soil or composting systems. Experimental techniques to study the real system in the mesoscopic scale (such as laser-scattering, flow citometry and microcalorimetry) need to be used in order to validate and complement these models.  In this paper, we review the IbM methodology applied to microbiology. Specifically, its application to the study of bacteria, yeast cultures and Plasmodium falciparum infected erythrocytes in vitro cultures. Last, it is important to highlight that physical and geometric constraints must be taken into account whenever applying IbM to microbiology.    

We present an investigation of the molecular basis of the modulation of oxygen affinity in heme proteins using computer simulation. QM-MM calculations are applied to explore distal and proximal effects on O₂ binding to the heme, while classical molecular dynamics simulations are employed to investigate ligand migration across the polypeptide to the active site. Trends in binding energies and in the kinetic constants are illustrated through a number of selected examples highlighting the virtues and the limitations of the applied methodologies. These examples cover a wide range of O₂-affinities, and include: the truncated-N and truncated-O hemoglobins from Mycobacterium Tuberculosis, the mammalian muscular O₂ storage protein: myoglobin, the hemoglobin from the parasitic nematode Ascaris lumbricoides, the oxygen transporter in the root of leguminous plants: Leghemoglobin, the Cerebratulus lacteus nerve tissue hemoglobin, and the Alcaligenes xyloxidans cytochrome c’.

Novel therapies are required for the treatment of acute liver failure. Bioartifical liver (BAL) systems are under development to offer better clinical performance than ordinary therapies, because they should be able to replicate in part the critical functions of the natural organ.
 Our design consists in a cartridge composed of a parallel bundle of hollow fibers housed within an external casing which incorporates hepatic spheroids (cells assembled together with great physiological activity). The hollow fibers are those usually used for microfiltration, but in our case we synthesized pore-filled ones with polyAcrilamide hydrogel. Blood or plasma normally flows through the fiber lumens, and spheroids are in the extracapillary space.
 
One of the main considerations in the design of a BAL must be the appropriate supply of oxygen to a huge amount of cells. In comparison to many other cell types, hepatocytes have elevated levels of carbohydrate, protein, and xenobiotic metabolism and are underpinned by a very high, specific oxygen consumption rate (OCR).
 
In the core of living tissues where there are no blood vessels, oxygen transport occurs solely by diffusion. In cellular tissue artificially cultivated, this situation imposes severe restrictions on the system; necrosis due to oxygen deficiency will occur in regions where the partial oxygen pressure is lower than a certain critical value.
 
The volumetric liquid mass transfer coefficient (k_La) is a useful parameter to characterize bioreactors capacity for aeration. This is a critical factor in this reactor design. From different available methods to determine k_La,  we choose the sulphite oxidation method because it has a kinetic in the range of our system, thus is able to be adapted to dynamic conditions, simulating a continuous culture system.
In this work we show the description of a system to measure transport of oxygen through the cartridge with hydrogel filled membrane. Equations and a model are proposed to explain the oxygen transport and simulate the physiological conditions and make some concrete proposals for the optimal design of bio-hybrid systems consisting of cellular tissue on a hollow fiber scaffold.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ligand gated calcium channel that mediates calcium release from intracellular stores such as the endoplasmic reticulum (ER). Although there is a large body of experimental work on IP3R´s, a comprehensive understanding of its kinetics is still lacking. A hierarchy of intracellular calcium signals due to calcium release through IP3R´s can be observed in Xenopus laevis oocytes using fluorescence microscopy and calcium-sensitive dyes. This hierarchy goes from spatially localized signals, such as “blips” and “puffs”, to global ones, such as calcium waves that propagate across the whole cell.  Although calcium puffs constitute the building blocks of global calcium signals in oocytes, they already involve the simultaneous release of calcium through several IP3R´s in a cluster.  In this work we analyze some properties of calcium puffs using a series of experiments done in oocytes with wide-field fluorescence microscopy. We then combine this analysis with simple mathematical models in order to infer single IP3R´s properties from the collective behavior of IP3R´s during puffs.  In particular, we determine that the duration of calcium release during a puff is independent of the puff´s amplitude. Although it has been observed that the mean open time of single IP3R´s is relatively insensitive to the concentration of cytosolic calcium [Mak et al, 2001], we show that the independence between puff amplitude and duration is not a direct consequence of this property of single channels.

Backgroud and aims: Hepatic outcome after paracetamol (acetaminophen) intoxication is compromised by tissue injury and release of hepatic enzymes and by production of pathological mediators. The Platelet-Activating Factor (PAF) is an endogenous lipid-mediator, which plays a key role in catalysing various pro-inflammatory processes associated with acute liver failure. The aim of this study was to investigate the effects of PAF inactivator, recombinant PAF-acetylhydrolase (rPAF –AH) on post paracetamol-treatment functional outcome of the liver in the rat.
Methods: Fifty male Wistar rats were divided into two groups: the control group received by gastric tube a toxic dose of paracetamol (3.5g/Kg BW) suspended in saline solution and the rPAF-AH-treated group received the same dose of paracetamol followed by a dose of rPAF-AH (5mg/Kg BW) intraperitoneally. The animals were sacrificed at time points of 20, 24, 28, 32 and 40h after paracetamol treatment. The hepatic injury was evaluated at the different times interval after intoxication by determination of serum enzyme activities of AST, ALT, ALP and LDH. Hepatic levels of malondialdehyde (MDA), serum cholesterol/HDL cholesterol fraction and serum superoxide dismutase (SOD) activity were also measured as indicators of tissue damage and as parameters of oxidant-antioxidant balance.
Results: In control group of rats paracetamol was found to cause oxidative stress followed by acute hepatic injury. Compared with the control group, the addition of PAF inactivator had a significant effect in tissue recovery. The positive effects of rPAF-AH were expressed by: (1) reduction of oxidative stress evident by low levels of MDA and cholesterol/HDL cholesterol fraction and increase of SOD activity and (2) high decrease of hepatic injury as showed by all serum enzyme activities (AST, ALT, ALP and LDH) measured.
Conclusions: The use of PAF inactivator enhances liver’s recovery from paracetamol intoxication. rPAF-AH attenuates the severity of experimental  liver injury and can provide important means of improving liver function following paracetamol intoxication. These results suggest that PAF plays an important role in the pathogenesis of paracetamol-induced liver injury and may act as a signaling mediator in the initiation and amplification of the hepatic inflammatory process.

Two theoretical single-molecule
related nanosensor problems are investigated:

(I) Utilizing fluorophore-quencher dressed DNA constructs for
nanosensing applications: An important feature of double-stranded DNA
is the ease with which its component strands can come apart and
rejoin. It has been demonstrated recently, by fluorescence
correlation methods, that the fluctuations of DNA-bubbles (the DNA
breathing) can be explored on the single molecule level. We have
developed a new discrete dynamical description of the DNA breathing
dynamics in terms of a master equation (T. Ambjornsson et al, Phys. Rev.
Lett. 97, 128105 (2006)). Employing recent experimental DNA stability
data we calculate experimentally measurable quantities such as
the autocorrelation function of a fluorophore-quencher labeled basepair; these quantities
depend on temperature, NaCl concentration,and DNA sequence. Good agreement with the experimental results is found.

(II) Tagged particle motion in 1d systems (single-file diffusion):
Excluded volume effects (i.e. particles cannot pass each other) are
pronounced in a system of diffusing particles in one dimension. Of
particular interest is the diffusive motion of a (flourescently) tagged particle.
it has been found previously (theoretically and
experimentally) that the mean square displacement for a tagged
particle scales as t^(1/2) (single-file diffusion), rather than t as
for unconstrained diffusion. The prefactor in front of the t^(1/2)
depends in a specific way on the concentration of particles, thereby
allowing for the the tagged particle to serve as a nanosensor. The
result above is valid for an infinite system; we have recently
solved exactly (T.Ambjornsson and L.Lizana, in preparation) the case
of N diffusing excluding particles in a "box" of FINITE size, and find that
the t^(1/2) behaviour then represents a quasiasymptotic regime,
whereas for very long times a(non-trivial) equilibrium distribution
or the tagged particle position is reached.

Reaction-diffusion theory has played a very important role in the study of pattern formations in biology. However, a group of individuals is described by a single state variable representing population density in reaction-diffusion models and interaction between individuals can be included only phenomenologically. In this paper, we propose a new scheme that seamlessly combines individual-based models with elements of reaction-diffusion theory and apply it to predator-prey systems as a test of our scheme.  In the model, starvation periods and the time to reproductive maturity are modeled for individual predators. Similarly, the life cycle and time to reproductive maturity of an individual prey are modeled. Furthermore, both predators and prey migrate through a two-dimensional space. To include animal migration in the model, we use a relationship between the diffusion and the random numbers generated according to a two-dimensional bivariate normal distribution. Despite the simplicity of this model, our scheme successfully produces logistic patterns and oscillations in the population size of both predator and prey. The peak for the predator population oscillation lags slightly behind the prey peak. The simplicity of this scheme will aid additional study of spatially-distributed negative-feedback systems.

Substrate-dependent Homophilic Cadherin Interactions.  Cadherins are transmembrane, cell surface proteins.  There is ample consensus that Cadherin molecules are the primary mediators of cell adhesion (Shapiro, Curr Opin Struct Biol 2003).  This has been demonstrated in a variety of relevant situations: cell sorting during embryogenesis, maintaining the architecture of tissue in adult organisms, cell-cell recognition, and in several pathologies.  The microscopic mechanisms that lead to initial recognition and final adhesion are less well understood.  To clarify this problem, experiments measuring Cadherin-Cadherin interactions have been made both between Cadhering-coated surfaces using the Surface Force Apparatus (Israelichvili, Surf Sci Rep 1992) (Leckband, Langmuir 2003) and, at the microscopic level using atomic force microscopy (Zypman, Encyc Nanosci Nanotech 2004), between individual Cadherin molecules (Silberzan, Langmuir 2006).  An additional problem appears when measuring forces across these length scales.  Namely, how to understand if the measurements are consistent and ultimately, how to predict from the experimental forces, the actual forces that will take place between real cells in real physiological conditions.  To clarify this issue, we propose an algorithm that evaluates microscopic Cadherin-Cadherin forces in terms of the corresponding plane-plane interactions.  This algorithm is based on the generalizations of the Derjaguin approximation (Zypman, J Phys: Cond Matt 2006).  We use the algorithm to compare real experimental data in the two limits.  In addition, we use our results to predict the forces between membranes and Cadherin coated membranes.  In this case the membranes correspond to an intermediate scale, with radius of curvature of the order of 1μm.  This is of particular interest since there is evidence (Riveline, PNAS 2004) that membrane and acto-myosin tension promote clustering of adhesion proteins.  Finally, we propose a theoretical model for the microscopic Cadherin domain-domain forces from which we build plane-plane, molecule-molecule and curved surface-substrate forces.  These forces are compared with the simpler generalization of Derjaguin approximation.

The biological consequences of ionizing radiation exposure (IR) such as cell death and mutagenesis are related to the broad variety of damages induced in cellular DNA. IR generates isolated DNA lesions including single strand breaks, abasic sites and oxidized bases as well as clustered damages. Bistranded clustered DNA damages are defined as two or more strand breaks, abasic sites and oxidized bases on opposite strands within a few helical turns. These types of damages are difficult to repair and probably constitute key factors in genome instability. Previous studies have shown that the relative levels of each type of clusters are different in isolated kilobasepair DNA and human cells. Since the repair processing of clustered damages is complex and still unknown and it is probably affected by the relative frequencies of each type of cluster, we have investigated the induction of abasic clusters, oxypurine clusters and double strand breaks by γ-irradiation in Saccharomyces cerevisiae as the first step for further repair studies. The wild type haploid strain BY4742 of Saccharomyces cerevisiae was used. Stationary phase cells were washed with double distilled water several times, resuspended in water at a concentration of 1 x 10⁸cells/ml and kept on ice before and after irradiation. Cell samples were irradiated with a ⁶⁰Co source (dose rate: 0.8 Gy/s) in a dose range from 0 to 400 Gy. After irradiation, aliquots of cells were plated on nutrient solid medium to assay survival. For DNA isolation, cells were embedded in agarose, lysed and digested with Proteinase K. Clustered damages were determined by digestion with damage-specific enzymes, neutral gel electrophoresis, electronic imaging and length average length analysis. Our results show that clustered damages increase linearly with the dose of radiation. The percentage of complex damage in yeast was different from that previously observed in T7 DNA and in human cells.

Mammalian cells obtain energy from glucose, continuously supplied by the extracellular fluid. Glucose transport through the plasma membrane is facilitated by a protein family of transporters (GLUTs). Glucose transport is affected in pathological states, and modulated by physiological, chemical and pharmacological agents. Regulation of glucose transport occurs at three levels: (1) genetic expression of the transporter, (2) membrane recruitment of transport units from intracellular stores and (3) changes in the transporter structure. Michaelis-Menten kinetics for enzyme reactions has been successfully applied to experimental data: the initial transport velocity (V₀) expressed as function of the concentration gradient across the membrane allowed calculations of maximal velocity (Vm) and glucose affinity for the transporter (Km). A usual model of facilitated diffusion includes four species linked in a cycle by four partial steps: (1) the ligand  (L) binds to the transporter (T) at the outer side of the membrane, (2) the T-L complex transconforms so that L now faces the inner side, (3) L dissociates from T inside, and (4) T undergoes another transconformation reorienting their binding sites, to reinitiate the cycle. The dynamic behavior of the model was simulated with a computer program. We studied the effect of changes of the rate constants of the model, the dissociation constants of the binding sites, and the concentration of extracellular glucose, on the initial and maximal transport velocities, the apparent affinities of glucose and the turnover number of the transporter. Our results agree with V₀ Vm, Km and turnover numbers calculated from experimental data. They adjust to Michaelis-Menten kinetics. V₀ is a hyperbolic function of external glucose concentration and of the rate constants of binding and transconformations. V₀ increases with the increase of the transconformation rate constant of the forward reaction. V₀ increases when the ratio of the forward and backward rate constants of the transconformation reactions approaches 1. V₀ increases when the ratio of the outer and inner dissociation constants of glucose approaches 1.

We developed exact solutions for a particular class of cooperative kinetic model, the K-allosteric family of Monod-Wyman-Changeaux (MWC). The model defines a macromolecule (M) who fluctuates between two limit states under allosteric control (Relaxed and Tense),  with n identical sites for a ligand and (positive homotropic) cooperativity effect. The K-allosteric family has different ligand affinity for the R (greater affinity) and T states but equal specific activity for all occupied sites.  All solutions are parameterized on the number of sites, the ligand relative affinity, the global states equilibrium constant and the normalized ligand concentration. Furthermore we use the bit-measured informational entropy function of Shannon (H) to reflect the molecular asymmetry at each stationary state. We explored the solutions of the saturation fraction as function of ligand concentration, the Hill plot and corresponding informational entropy. Since activity remains proportional to the saturation fraction, there will be no problem to assume any kind of related specific biological activity. 
 We were interested in comparing under different conditions the value of the n-Hill parameter with the H pattern.  For the simulated conditions, the solutions exhibit a high sensitivity of the H function, who reflects accurately minor fluctuations of the model parameters, even those which does not induce significant changes on classical representations as Lineweaver-Burk or Hill plots.
 Some general conclusions are obtained as a result of our parameter space exploration: the H plot behaves more sensitive at higher ligand concentration, the allosteric control deeply affects the H function and overall bit changes stands out as the number of sites (n) increases. Furthermore the bit scale of H or the number of molecular distinguishable species of M, allows a comprehensive interpretation of the order-disorder transitions induced by parameter changes. As a final conclusion, we suggest that the H plot qualifies as a valuable modeling tool.

At present when the environmental pollution raises the public concern in all over the world, the disclosure of a cell marker for detection of environmental pollution becomes one of the global problems in modern Life Sciences. Therefore, the clarification of the nature of metabolic cascades, through which the biological effects of environmental factors are realized, is extremely important, as the lack of our knowledge in this field is the main barrier for estimating their hazardous effects from the point of public health. In spite of the fact that water is the main medium where the major part of biochemical processes takes place and an extremely low energy is necessary for changing its dissociation, an adequate attention has not been paid by investigators to its role as a messenger through which the biological effects of external and internal signals on the cell are realized.  During the last two decades our laboratory tries to clarify, whether the cell hydration serves as a universal and extra sensitive target for biological effect of various environmental factors. It was shown that the biological effect of extremely low concentration of biological active substances and weak physical signals (magnetic and electrical fields, infrasound, background radiation and etc.) unable to activate the ionic channels and receptors in the membrane, on cells and organism were realized by modulation of the level of cell hydration. There were close correlation between cell hydration and its anabolic (proliferative) and catabolic (anti-proliferative) types of metabolisms. Membrane proteins, having enzymatic, chemoreceptive and ionoforetic properties, could be either in functionally active or inactive (reserved) states; the number of functionally active molecules in the membrane was determined by membrane active surface. Based on these data was suggested that neuronal abnormal hydration was the main reason for the generation of nociceptive signals and cell pathology. In present work the original data on multidisciplinary aspects of the metabolic regulation of cell hydration in norm and pathology and its physiological significance in regulation of cell membrane functional activity; intracellular signaling and cell death; dehydration therapy of various cell disorders would be discussed.

Bichromophoric cyanine dyes (BCD) possess attractive spectral characteristics for application in photodynamic therapy (PDT) and high affinity to model and natural biological systems. Recently we have demonstrated their high photocytotoxicity toward colon adenocarcinoma cells (HT29). Here we report on comparative photocitotoxicity of a bichromophoric cyanine dye (BCD) and Photogem, the commercial compound already applied in PDT toward melanoma cells (B16F10) - one of the most resistant and aggressive cancers.
The melanoma cells were cultured in RPMI media supplemented with 7,5% heat inactivated horse serum and 1% penicillin at 37 ºC in a water-jacketed CO₂ incubator. The day before the experiment, the cells were seeded at 5x10⁴ cells/ml, the cell concentration calculated using a Neubauer chamber.
The photocytotoxicity test was based on the MTT assay. The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide (MTT) was reduced in live cells producing a colored compound used to determine cell viability. For MTT assays, cells were seeded in RPMI containing 7,5% FBS into wells of 96-well ELISA-type plates and exposed to a range of drug concentrations from 0.01 to 20 mM for time intervals 0,5 - 24 h in the dark or 0,5 - 6,5 h under illumination. The plates with MTT were incubated in the dark for 4 h, and then water-insoluble MTT-formazan crystals were dissolved in SDS, and absorbance at 570 nm was measured in an ELISA plate reader. To illuminate the samples a 250 W halogen light projector was used.
Both compounds manifested low cytotoxicity in the dark. Under visible light the same photocytotoxicity level for the same irradiation dose was reached for lower BCD concentrations as compared with those of Photogem. Moreover, BCD possessed shorter incubation time. We think these results encouraging to consider BCD promising for PDT application.Support: FAPESP, CNPq, CAPES Brazilian agencies

            Cyanine dyes with two chromophores (BCD) are promising photosensitizers for Photodynamic Cancer Therapy (PDT) because of their specific characteristics, such as high optical absorption in the visible spectral region, high triplet-state quantum yield and high affinity to biological and model structures (DNA, micelles). Recently we have demonstrated their high photocytotoxicity toward cancer cell cultures, which prompted us to investigate the interaction of BCD with lipid Langmuir monolayers as a simplified membrane model. Stable Langmuir monolayers were formed by co-spreading dipalmitoyl phosphatidyl choline (DPPC) and BCD with relative dye concentrations from 0.2 to 50%. BCD was present in the monolayers in all stages of their formation, as confirmed by monitoring the surface pressure and potential isotherms. Monolayer formation was visualized with Brewster angle microscopy and monitored by measuring the BCD optical absorption spectra in situ. For all BCD concentrations, the surface pressure and surface potential isotherms became more expanded, with higher surface pressure at the liquid-expanded to the liquid-condensed phase transition. With Brewster angle microscopy we observed earlier formation of phospholipid clusters in the presence of BCD than for a pure DPPC monolayer. Thus BCD on the one hand stimulated the cluster formation, and on the other hand increased electrostatic repulsion between them (higher pressure at the plateau). The cluster profiles were essentially the same for the pure DPPC monolayer and for the one containing BCD, which indicated that BCD molecules did not penetrate the cluster interior but were bound on the frontier. Therefore it is reasonable to expect that BCD molecules will not penetrate the hydrophobic region of the membrane and thus will not affect cell structure, decreasing probability of negative side effects. The changes in the BCD absorption spectra revealed BCD aggregation during monolayer formation. All observed effects were more pronounced in the presence of 0.2M NaCl in aqueous subphase.
            Support: FAPESP, CNPq, CAPES Brazilian agencies.

Bioactivity of nitroheterocyclic compounds (NHCC) is well documented. Some of NHCC are widely applied in clinics as antiseptics and antibiotics. During two last decades their anticarcinogenic and radiosensibilization activity was demonstrated. These compounds are cheap and available. Some NHCC possess intense absorption in the near UV and visible spectral region. Recently we have demonstrated cytotoxicity increase under visible light for some NHCC. Here we present the results on three NHCC photocytotoxicity: 2-(5´-nitro-2´-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1´-methylbutyl] carbamoyl] quinoline (Quinifuryl), 1-nitro-9(3-3-dimethylaminopropylamino) acridine (Nitracrine) and 1-[[[5-nitro-2-furanyl]methylene]amino]-2,4-imidazolidinedione (Nitrofurantoin) toward malignant and non-transformed cells in vitro.
Their cytotoxicity was measured in the dark and under 350-450 nm light. All drugs demonstrated elevated cytotoxicity when illuminated. Cell mortality expressed in LC(50) values after 1 h illumination was 7-35 times higher than that after 1 h dark incubation of cells with drugs. Total toxic effect calculated as a sum of direct cell death and cell proliferation arrest exceeded 80% after 1 h cell illumination in the presence of drugs in concentrations as low as 0.2 mM for Quinifuryl, 0.02 mM for Nitracrine and 1.0 mM for Nitrofurantoin, respectively.
The detailed study on Quinifuryl phototransformation has demonstrated that under visible light it formed three types of bioactive species: anion superoxide radical, NO and singlet oxygen. Recently we have observed NO liberation from Nitrofurantoin under visible light. We associate high photocytotoxicity of these compounds with the formation of these active species.
Support: CAPES, FAPESP, CNPq Brazilian agencies.

We have developed theoretical and computational aspects, implemented in public-domain computer programs, that allow the prediction of hydrodynamic and other solution properties macromolecules and bioparticles in dilute solution. For rigid particles, rigid-body hydrodynamic theory and bead models are the basis of the programs of the HYDRO suite, which allow the calculation of solution properties from structures ranging from coarse-grained models to atomic-detail structures. Calculated properties include hydrodynamic coefficients (sedimentation, diffusion, viscosity), along with more complex properties, like fluorescence anisotropy decay and NMR relaxation. For flexible structures, the complex interplay between hydrodynamics and conformational statistics is adequately treated by means of  Brownian dynamics simulation of bead-and-connector models. Again, we have implemented these aspects in the SIMUFLEX suite of programs, which can be used to treat a variety of problems, ranging from the calculation of the above mentioned overall properties, to the simulation of single-molecule phenomena. We have also developed a methodology, implemented in program HYDFIT, for structural search or parameter estimation by fitting experimental data – including various solution properties, and eventually samples of varying molecular weights – to the computational predictions. This communication will describe the general features of the three software packages, along with examples of their applications. 
 
References
 
·        http://leonardo.fcu.um.es/macromol/
·        J. García de la Torre et al, "HYDRO: A Computer Software for the Prediction of Hydrodynamic Properties of Macromolecules". Biophys.  J. 67, 530-531 (1994).
·        J. García de la Torre et al "Calculation of hydrodynamic properties of globular proteins from their atomic-level structure". Biophys. J. 78, 719-730 (2000).
·        J. García de la Torre et al, “Calculation of solution properties of flexible macromolecules. Methods and applications”. Eur. Biophys. J. 32, 477-486 (2003).  
·        A. Ortega and J. García de la Torre, “Equivalent radii and ratios of radii from solution properties as indicators of macromolecular shape, conformation and flexibility”, Biomacromolecules, in press (2007). 
 

Xenopus laevis melanophores are one of the cellular systems commonly used to study the function of molecular motors in vivo. An unexpected but common observation in trajectories of single organelles moving along microtubules is that the organelle frequently revert its direction. This observation was explained by the switching of the motor responsible for the transport but the mechanism by which motors with a given polarity turns on and motors with opposed polarity turns off is not know. In this work, we explore the mechanism of in vivo coordination of microtubule motors by using a fast and precise tracking method. We analyze the interval in the trajectories where reversals of directions are observed and postulate a model to explain the reversals in living cells.

Formal dynamical models that capture the complexity that characterizes biological systems are needed to study cell type determination and other morphogenetic processes. We put forward a gene network model that integrates the available experimental data for a widely documented gene regulatory module in the plant Arabidopsis thaliana. This model enables a dynamical explanation for cell fate attainment in leaf and root epidermis and allows testing hypothesis, generating predictions, and constricting future experiments. The network model was extended to a discrete spatio-temporal one that also considers cell communication. This model recovers cellular spatial patterns similar to those observed in the epidermis of both wild type and mutant Arabidopsis. It is known that positional cues affect cellular patter formation in this system, yet the effect and nature of these remains unclear. Our coupled networks model can be used to test the role of particular chemical or structural (e.g. domain geometry or border effect) aspects that could constitute positional information or contribute to the robust emergence of the observed cell arrangements. Finally, even though this model focuses on a particular plant system, it advances the discussion of some general evolutionary aspects. For instance, our results suggest that the alteration of anatomical aspects during evolution can give rise to novel phenotypes even when certain genetic modules remain unchanged.

Over the past decade, fluorescence fluctuation spectroscopy (FFS) has been established as one of the most versatile fluorescence-based techniques for acquiring information on the dynamics of molecular processes both in vitro and in vivo conditions. In severals applications of FFS the system under study is close to a surface, i.e. in processes involving a cellular membrane. The proximity to a surface has a strong influence on FCS curves and can lead to erroneous interpretation of data if this is not carefully analyzed. The main purpose of this study was to quantitatively investigate this effect and how this could be reflected in FFS experiments, as well as to indicate how this effects can be used to extract additional information about the mobility properties of the investigated molecules.

Neuroglobin (Ngb) and Cytoglobin (Cyb) are two recently discovered human heme proteins whose function is still unclear. In contrast with hemoglobin or myoglobin, their heme is hexacoordinated. Proposed functions for these proteins include oxygen transport, reactive oxygen species detoxification, hypoxia protection, and redox state sensing. For any of the proposed functions, distal histidine dissociation from the hemic iron is required. In addition, a disulfide bridge between two cysteine residues is found in both proteins and might have an important effect on the dissociation of the distal histidine. >With the aim of understanding this hexa-penta coordination transition, we have performed 50ns molecular dynamics simulations in explicit water for ferrous Ngb in the hexacoordinated and pentacoordinated states. We evaluated the effect of the redox state by means of performing simulations of the protein with a disulfide bridge. We found that protein oxidation promotes a decrease in the barrier for pentacoordination and stabilizes the pentacoordinated species. The key step to obtain the pentacoordinated protein appears to be a distal histidine rotation which is concomitant with an upward displacement of the E helix. In order to compare the results for Ngb with another protein that displays an hexacoordinated heme, molecular dynamics simulations of Cyb in the reduced and oxydized forms were performed. Recent spectroscopic and kinetic results have shown that the process of hexacoordination is enhaced in several globins when exposed to high pressures. In order to study the effect of high pressure on hexacoordination, we performed molecular dynamics simulations of Ngb at high pressure for each of the states mentioned below. High pressure simulations show a decrease of protein flexibility, in special an increase in the rigidity of the CD loop. The results presented in this work allow a better understanding of the molecular mechanisms involved in the hexacoordination process.

There is recent evidence suggesting that nitrite anion (NO₂^-) represents the major intravascular NO storage molecule whose transduction to NO is made possible by reduction to NO catalyzed by deoxy hemoglobin (Hb). In this work, we provide a detailed microscopic picture for the possible mechanisms of this nitrite reductase (NIR) activity of deoxy-Hb by performing a combination of classical MD and hybrid quantum-classical (QM-MM) simulations. Our results suggest that two alternative mechanisms could be operative: in the first one, which is similar to the proposed mechanism for bacterial NIRs, nitrite or nitrous acid coordinate to the heme through the N atom. This pathway involves one or two proton transfer processes involving His E7, depending if the active species is nitrite or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and  methemoglobin. In the second mechanism, nitrite coordinates to the heme through the O atom. This pathway requires only proton transfer step from His E7, and leads directly to the formation of a hydroxo Fe(III) complex and NO.

The parasite Trypanosoma cruzi, the etiological agent of Chagas´ disease is transmitted into the blood flow of the victim via bites from a different number of insects. One of them, the Rhodnius prolixus has developed a series of insidious mechanisms that make the invasion by T. cuzi a very efficient process. R. prolixus´ saliva contains a heme protein named Nitrophorin 4 (NP4) which is injected into the victim´s blood stream after the bite. This protein binds nitric oxide in a pH dependent fashion, stabilizing NO for long periods in the insect salivary glands (pH=5), that is then released when the saliva is injected into a victim due to the increase in pH (pH=7.4). NO causes vasodilatation, which leads to improved feeding by the insect. In this work we present in the first place theoretical results using a QM/MM scheme in which the active site is treated at the DFT level, while the rest of the protein is modeled using the Amber force field, to compare the NO affinity in the NP4 for the conformations at different pHs. We have also performed classical multiple steering molecular dynamics simulations to obtain the free energy profiles associated with the ligand escape from the active site in the two conformations. Our results show that: (i) ligand affinity is similar in both conformations. (ii) the release of NO for this protein is mainly determined by the differences in ligand migration patterns in the pH=5 with respect to the pH=7.4 conformations. These differences are mainly due to structural differences of the AB and GH loops.

Serine protease inhibitors (serpins) are proteins that inactivate serine proteases, enzymes that catalyze the hydrolysis of peptide bonds. The reactive center loop (RCL) of the serpin binds to the serine in the active site of the protease where the enzyme makes a cut. Then like a spring-loaded mousetrap, the metastable serpin traps the protease through a conformational change in the complex by incorporating the RCL into a nearby â sheet. We use fluorescence resonance energy transfer (FRET) to study the reaction between the serpin á₁-antitrypsin (A1AT) and the protease rat trypsin (Rtryp). We also employ a recently developed in vitro assay to confine the reacted complex in optically trappable aqueous nanodroplets emulsified in an immiscible perfluorinated matrix with lower index of refraction in a confocal microscope. A droplet containing a complex is captured and positioned at the confocal spot using optical tweezers, and the molecules are excited with a visible laser and the fluorescence detected. Furthermore, the nanodroplets can be easily mixed to permit observation of the reaction in real time in a two-channel flow cell. A droplet with A1AT is flowed into one channel while a droplet with Rtryp is flowed into the other. At a Y junction, each droplet is grabbed with separately controlled optical traps, then fused and immediately interrogated. Fluorophore positioning at appropriate amino acid sites are designed to provide dramatic changes in FRET intensity over the course of the reaction, yielding information about distances and dynamics. An additional mixing strategy using a pair of piezoelectric controlled injectors to deliver one aqueous nanodroplet at a time into the solvent matrix will be presented as well. A drop from one injector contains Rtryp and a drop from the other contains A1AT. The two then can be mixed using optical trapping and interrogated via laser. Additionally, polarization anisotropy of Rtryp and A1AT molecules and complexes will be compared to enhanced green fluorescent protein (EGFP) and free dye to determine any rotational constraints introduced by the water-solvent interface.

In order to understand the basic mechanisms of living cells, it is essential to elucidate the complexity of fundamental interactions and dynamical processes between their underlying molecular building blocks, such as DNA, RNA, and proteins. In particular, high resolution, non invasive, optical methods can provide us with insights into such molecular machineries.
In this work I will present a new optical experimental technique which is highly sensitive and minimally invasive that allows, in particular, study of real time dynamics of molecular motors in the single molecule limit. The extremely high temporal (~30 microsecond) and spatial (subnanometer) resolutions provided by the new method may be key to studying various interactions and dynamical mechanisms of molecular motors and Brownian dynamics. I will describe the basic physical principles of the technique, and present our approach to the study of microtubules traveling on kinesin-coated surfaces or nanochannels with the aim of understanding cooperative effects of the motors on the motility of microtubules.

We study E.coli RecA protein, from preparation and purification of protein monomers to structural and functional studies of protein polymers formed on DNA – nucleoprotein filaments. RecA protein is a multirole one, where DNA strand exchange by forming nucleoprotein filament during homologous recombination and cleavage of SOS response repressors are the most prominent two. The first role, where we are interested into structural details (and structure makes function, very much so in this case) also came recently into focus with the work of Zahradka et al. (Nature 2006), where reassembly of shattered chromosomes in D. radiodurans is completed by RecA-dependent crossovers. RecA is a relatively small protein, MW = 37,842, with 352 amino acid residues. Only by polymerizing within nucleoprotein filaments it achieves its function. The RecA polymers have been crystallized and structure determined to atomic resolution by XRD, however the structure of RecA-DNA complex is not solved, and the exact path of DNA within the nucleoprotein filament is not known, although it has been extensively studied by SANS, electron microscopy or NMR. We form RecA nucleoprotein filaments using very short, monodisperse, 146 bp long DNA. Such 50~75 nm long filaments are shorter than their respective persistence length – i.e. they should behave as straight rods. A monodisperse colloidal system of helical rodlike particles is capable of forming liquid crystal. This, indeed, might be the most ordered possible preparation of nucleoprotein filaments. Studying it by optical and electronic microscopies and XRD will allow further insight into the function of RecA. There are several routes of investigation: the nucleation and growth process observed for short filaments, their structural parameters and the properties of the liquid crystalline phase they could form. * permanent position: Institut za fiziku, Zagreb, Croatia

Ruthenium (II) complexes presenting the dppz (bypirido[3,2-a:2’,3’-c]phenazine) ligand, are able to intercalate between DNA base pairs with high binding affinity (K_b>10⁶ M^-1).^[1,2] They also present the ability to emit when intercalated into DNA but are largely quenched in water solution, this effect is called light switch.^[3] These features focus the attention on the usage of these complexes in diagnostic applications which target nucleic acids.
Once intercalated the Ru(II) complex may present many intercalative geometries, which have been proposed and demonstrated experimentally by different approaches including NMR studies.^[4] The existence of such intercalative geometries  brings the idea of differential exposition for the dppz ligand to the solvent. This gave a possible explanation for changes in emition rate over time.
Because of the relevance of such dynamical behavior, molecular dynamics calculations were performed to get insight into the specific interactions between a Ru(II) complex and the base pairs in the intercalation site from a structural and energetic point of view. The modeled system included the Ä-[Ru(bpy)₂dppz]²⁺ complex intercalated into the 6^th and the 7^th base pairs of a DNA dodecamer. 2ns simulations were carried on in physiological conditions. Effects in changing the DNA sequence were explored comparing the behavior of two sequences: 5’-CGCTTGGTTGCG-3’ and 5’-CGCTTCGTTGCG-3’.
Important sequence dependence behavior was observed in structural properties both on the intercalative geometries and in the DNA conformation. Van der Waals and electrostatic interactions between the Ru(II) complex and base pairs seems to play a mayor role in the dynamical behavior observed.
References.
[1] P. U. Maheswari, V. Rajendiran, M. Palaniandavar, R. Parthasarathi, V. Subramanian, J.Inorg.Biochem. 2006, 100: 3.
[2]  Jin-Gang Liu, Quian-Ling Zhang, Xian-Fa Shi, Liang-Nian Ji, Inorg. Chem. 2001, 40: 5045.
[3]  Y. Jenkins, A. E. Friedman , N.J. Turro, J.K. Barton.  Biochemistry 1992, 31: 10809.
[4] C. M . Dupureur, J. K. Barton. Inorg. Chem. 1997, 36: 33.

Local anesthetics are able to induce pain relief by binding to the sodium channel of excitable membranes, blocking the influx of sodium ions and the propagation of the nervous impulse. Ropivacaine (RVC) is an amino-amide, enantiomerically pure, local anesthetic largely used in surgical procedures, which present physico-chemical and therapeutic properties similar to those of bupivacaine, but decreased toxicity and motor blockade. The present work focuses on the preparation and characterization of nanospheres containing RVC. 0.2% RVC was incorporated in poly(d,l-lactide-co-glycolide – PLGA, 50:50) nanospheres, prepared by the nanoprecipitation method. Characterization of the nanospheres were conducted through the measure of pH, particle size, zeta potential. The pH of the nanoparticle system with RVC was 6.58. The average diameters of the RVC containing nanospheres was 162.7±1.5 nm and their zeta potential were negative, with values of about -10.81±1.16 mV, which promoted good stabilization of the particles in solution. Since this polymer drug-delivery system can effectively generate an even less toxic RVC formulation, this study is fundamental to characterize a potentially novel pharmaceutical form for the treatment of pain with RVC, a newer and safer long-acting local anesthetic than bupivacaine.
Acknowledgment: Fapesp
 

We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels (IP₃Rs) that underlie calcium puffs observed in Xenopus Laevis oocytes using confocal microscopy and fluorescent indicators. To this end, we use a simplified version of the algorithm presented in Ventura et al., 2005 [1]. An analysis of 130 puffs in 11 cells let us conclude that calcium release comes from a region of size non larger than 76±6nm. The duration of the observed releases is peaked around 10ms and the underlying Ca²⁺ currents range between 0.13 and 1.1pA. All these parameters are independent of IP₃ concentration. We relate the obtained current amplitude distribution with the number of open channels in a cluster during a puff, N_p, which we assume to be given by a Poisson distribution. We find that low currents scale linearly with N_p, while large currents scale with N_p^1/2 as predicted by Thul and Falcke, 2004 [2]. The different scaling behavior of I with N_p depending on how large I is is interpreted in terms of the mean distance that separates the open channels during each release event and the consequent endoplasmic reticulum local depletion. The border between both behaviors occurs at 4-8 open channels, separated by a distance of about 24nm-14nm. Using this model we can estimate the number of channels that opened during   the puffs that we analyzed. This number depends on the value of the single IP₃R current that we consider and can be as large as 32. The mean inter-IP₃R distance in a cluster is less sensitive to the single IP₃R current estimate and is less than 20nm.

References

[1] Ventura, A.C., L. Bruno, A. Demuro, I. Parker, S. Ponce Dawson, A Model-Independent Algorithm to Derive Ca²⁺ Fluxes Underlying Local Cytosolic Ca²⁺ Transients, Biophys. J. 88 (2005)  2403-2421.[2] Thul, R. and M. Falcke. Release currents of IP3 receptor channel clusters and concentration profiles. Biophys. J. 86 (2004) 2660-2673

In this work the role of glutamine synthetase (GS) regulation and glutamate dehydrogenase (GDH) activity on metabolic control of glutamine and glutamate synthesis from ammonia and oxoglutarate in Escherichia coli was studied. Both enzymes form a linear pathway, which can have also a cyclic topology if glutamate-oxoglutarate amino transferase (GOGAT) activity is included. We modelled the metabolic pathways in the linear or cyclic topologies using a coupled non-linear differential equations system. To simulate GS regulation by covalent modification, we introduced a relatioship that took into account the levels of oxoglutarate and glutamine as signal inputs, as well as the ultrasensitive response of enzyme adenylylation. Thus, by includiing this relationship or not we were able to model the system with or without GS regulation. On the other hand, GDH activity was changed manually. The response of the model in different stationary states, or under the influence of input exhaustion or oscillation, was analyzed in both topologies. Our results indicate a metabolic control coefficient for GDH ranging from 0.40 in the linear topology without GS regulation, to less than 0.08 in all other situiations, with a default free GDH concentration of  4 μM. Thus, in these conditions GDH seemed to have limited influence on metabolic control of this pathway, being cyclic topology the more robust. When GS was regulated, system robustness was higher in both topologies and against all perturbations. Furthermore, we found that effects of regulation against perturbations depended on the relative values of the glutamine and glutamate output first-order kinetic constants, which we named k₆ and k₇, respectively. Effects of regulation were markedly observed only when k₆ ≤k₇. Hence, GS regulation seemed important for metabolic stability in a changing environment, depending on the cell metabolic regime.

Genetic oscillations play a major role in different cellular processes, as for example in circadian clocks, during the cell cycle, and patterning in developing embryos. Due to the stochastic nature of gene expression, the period of such oscillations is subject to fluctuations. The precision of the oscillator can be characterized by the quality factor. We study the precision of genetic clocks in a simple but general stochastic description of feedback oscillators, in which functional molecules are produced by an assembly line consisting of many identical steps. The initiation rate of this assembly is regulated by its products via a negative feedback. We show that high quality is possible for certain parameter ranges even when the number of molecules is low and amplitude fluctuations are large. We relate our results to circadian clocks in bacteria, where high quality oscillations have been observed in single cell experiments. Furthermore, we discuss effects of stochastic amplification steps on precision to account for translational bursting.

     The myelin membrane can be fully solubilized in solvent and spread at the air-water interface forming a monolayer that conserves all the compositional complexity of the natural membrane. This model membrane shows a complex pressure-dependent surface pattern that changes from two liquid phases, that coexist at low pressure, to a viscous fractal phase embedded in a liquid phase, at tighter packing. This system allowed us to study the individual or combined roles of  the major myelin protein components, Myelin Basic Protein (MBP) and Folch-Lees Protelipid protein (PLP), in the structural dynamics of the interface. The effects of each single protein on the mixture of the whole myelin lipids were determined, and two different surface pressure-dependent behaviors were found.  MBP induces the  segregation of two liquid phases at low pressures and, on compression, it becomes excluded from the film and finally localizes in aggregated structures adjacents to the interface. PLP, on the contrary, organizes as a fractal or mixes homogeneously depending on the surrounding monolayer; when included in the protein-free myelin lipid film it  forms a fractal at all surface pressures but it remains mixed in the MBP-induced liquid phase. The resultant surface topography and dynamics is regulated by combined near-equilibrium and out-of-equilibrium effects. PLP appears to act as a surface skeleton for the whole components while MBP couples the structuring to surface pressure-extrusion and adsorption processes.

In this study, a comparison among membranes capable of emulating biological systems was made. These systems are solid supported lipid bilayers onto gold substrates. The gold surfaces were initially covered with a chemisorbed monolayer of negatively charged mercaptopropionic acid molecules, on top of which it was attached a planar lipid bilayer via electrostatic interactions, by phospholipid vesicle fusion, employing a mixture containing 80 % Egg Yolk Phosphatidylcholine and 20 % positively charged Dimethyldioctadecylammonium chloride (DODAC). Resulting supported bilayer lipid membranes (s-BLMs) were doped with the ionophores: a) Gramicidin and b) Valinomycin. These systems were utilized to study the transport kinetics of different ions by electrochemical impedance spectroscopy in a wide frequency range and for varying ion concentrations in solution. Experimental data were modelled according to a set of fundamental microscopic equations describing the kinetic pathways. Rate constants for the ion transport were determined by means of a fitting procedure, involving a complex non-linear least squares algorithm. Experimental and simulated data are in reasonable agreement despite some simplifying assumptions used in the simulations. The experimentally observed dependences of the membrane conductance on concentrations of the transported ion and the ionophore confirm the theoretical propositions. Derived values for the rate constants of association and dissociation of the permeating ionophore – ion complex agree with literature data. The fit of experimental data to the model allows, in Gramicidin case, checking the accepted sequence of conductivity for alkaline cations. Further work based on the proposed theoretical model is expected to allow other predictions to be made. We believe that this approach may be useful to characterize reconstituted protein/lipid systems, in studies of membrane interfacial phenomena and in sensor applications.

The scattering and hydrodynamic properties of rigid or flexible biological macromolecules of different nature, size and shape can be calculated by a bead modeling methodology (e.g. by Debye modeling or Garcia de la Torre´s approach) [1]. The particle under analysis has to be represented by an assembly of spherical elements yielding an overall size and shape resembling the target molecule as much as possible. This may be achieved either by a filling-model strategy (using all beads, equal- or unequal-sized) or a shell-modeling approach. The latter approach is hydrodynamically more rigorous and avoids problems like overlapping of beads. However, filling models are required for the concurrent prediction of scattering and hydrodynamic properties, since the usage of all constitutive elements is a necessary prerequisite for handling scattering problems. In this context, two major problems arise, in particular with hydrodynamic modeling: (i) the interaction of overlapping beads of unequal size, (ii) the multitude of beads when applying huge numbers of coordinates or groups. While the first problem has been solved by us by applying a special interaction tensor, the second problem requires usage of effective reduction steps. For example, far-reaching reduction procedures are required for constructing bead models of huge proteins for hydrodynamic modeling from their atomic-level data. As an initial step, the data stored in data banks are transformed to initial models based on atoms or atomic groups or amino acid residues. The ´Running Mean´ approach can be used for the generation of models of reduced bead number (by increasing the bead size or compression index). ´Cubic Grid´ or ´Hexagonal Grid´ approaches are equally successful, provided the beads of unequal size are located on gravity centers; locating the beads on lattice points yields less satisfactory results, irrespective of the choice of beads of unequal or equal size. The described procedures were tested with proteins of different size and symmetry, including the giant earthworm haemoglobins and bacteriophage capsids, and by comparing predicted and observed scattering and hydrodynamic properties (R_G, D, s, [η]). [1] Zipper et al., in: Analytical Ultracentrifugation: Techniques and Methods (Scott et al., eds.) RSC, Cambridge, 2005, pp. 320-371. 

Metallo-â-lactamases (MâLs) are responsible for one of the main mechanisms of bacterial resistance to â-lactam antibiotics. There is currently no therapeutically useful inhibitor against this class of enzymes. This fact reveals the importance of studying in detail their catalytic mechanism and the substrate binding mode.
The catalytic mechanism used by MâLs to hydrolyse â-lactams is not fully understood
but previous studies, including mutagenesis and crystallography with â-lactam derivatives have shown that Lys 224 is implicated in substrate binding and catalysis. This residue lies in the active site but is not directly involved in metal coordination.
To examine the proposed role for this residue, well conserved among different MâLs, we obtained several mutants of MâL BcII in position 224. BcII is the Mâls isolated from Bacillus cereus, an opportunistic pathogen. This MâL binds two Zn(II) ions in its active site and posseses a wide substrate spectrum that includes penicillins, cephalosporins and carbapenems.
Using a PCR-based mutagenesis approach, we replaced Lys 224 with Ala, Asp and Arg. We obtained the steady-state kinetic parameters for the mutants against different â-lactams, we studied their metal content and explored the active site structure by Co(II)-substitution. Our results show that the metal content of the mutants is not affected, nor is the catalytic efficiency markedly compromised, with the greatest effect being a 2-order decrease relative to wild type. This is in contrast with similar experiments performed on other MâLs and indicates that in BcII, this position is not essential for substrate binding or catalysis.

Molecular dynamics (MD) allows determining the detailed dynamics of proteins, providing data which are not accessible through experiments. One of the basic assumptions of MD is that the nuclei move on the potential energy surface determined by the electrons, and that this surface can be calculated as the sum of bonded and non-bonded interactions between the atoms of the system. In particular, covalent bonds are usually considered as harmonic oscillators. Because of this treatment, standard MD is not appropriate to describe bond breaking and forming processes, as those occurring during enzymatic reactions.
The enzyme methylamine dehydrogenase catalyzes the oxidation of methylamine to formaldehyde and ammonia. The rate determinant step of the catalyzed reaction consists of a proton transfer. The experimental evidence suggests that this transfer occurs by tunneling, assisted or promoted by the fluctuations of the protein.
We have determined the barrier for this proton transfer performing quantum calculations on active site models [1]. Even useful, these calculations do not allow seeing how the fluctuations of the protein environment affect the probability for the transfer of the proton. In order to appreciate this effect we have developed an AVB parameterization [2] for the system. The parameterization works along with the AMBER94 force field as implemented in TINKER.
In our presentation we will show that the AVB parameterization accurately reproduces the results of quantum calculations for active site models. Then, we will present the results obtained with the whole enzyme. In particular we will discuss how the protein environment changes the height and width of the barrier for the reaction. Finally we will show how these changes can affect the probability for proton transfer using a model in which the proton is considered as a wave packet in 3D.
 References:
[1] G. Pierdonimici-Sottile, J. Echave and J. Palma, Int. Journal of Quantum Chemistry, 105 (6), 937-945, (2005).
[2] Grochowski P, Lesyng B.; Bala P.; McCammon J. A. ,International J. of Quantum Chemistry 1996, 60, 1143-1164.

Organization of the cytoplasm of eukaryotic cells depends on the function of molecular motors that move organelles and other cellular cargoes along microtubules and microfilaments to their correct destination in the cytoplasm. Despite detailed knowledge of the structure and the working cycle of many individual motors in vitro, are not well known about the molecular mechanisms involved in the precise spatial and temporal control of the motors during organelle transport. Moreover, several works showed that the cytoplasm has viscoelastic properties but little is known about how transport is developed in this environment. In this context, we are exploring the properties of the cytoplasm of Xenopus laevis melanophores which are considered model cells to study the mechanism and regulation of intracellular transport. To characterize the micromechanical properties of Xenopus laevis melanophores we studied the motion of fluorescent tracers in the cytoplasm of these cells. With this aim, we developed a technique for fluorescent particle tracking that is based on an inverted microscope Zeiss IM35, adapted with an LED of wavelength of 470nm as excitation source. The tracking method is based on pattern recognition algorithm developed by our group. We performed control experiments with fluorescent particles of different sizes and brightness to study the performance of the system.

â-lactamases constitute the major bacterial defense mechanism against â-lactam antibiotics. These enzymes hydrolyse the â-lactam ring rendering the antibiotics ineffective. Metallo- â-lactamases (MâLs) comprise a group of â-lactamases that require Zn(II) for function, in contrast to the well-known Serine-â-lactamases (SâLs) whose activity relies on a serine residue in the active site. MâLs are considered a great threat for human health since they have a wide substrate spectrum and cannot be inhibited by therapeutically useful drugs, effective against SâLs. Rational design of such drugs relies on the thorough knowledge of the chemical nature of the hydolysis intermediates. Imipenem belongs to the family of carbapenems, the last generation of â-lactam antibiotics. In contrast to SâLs, MâLs are efficient carbapenemases. Generally, imipenem hydrolysis gives rise to a mixture of species with a cleaved â-lactam ring and a rearranged double bond. We have studied the reaction of BcII, the Bacillus cereus MâL, with imipenem. We found that BcII is stereospecific rendering only one major hydrolysis product. We used stopped-flow and rapid freeze quench EPR (RFQ-EPR) experiments to study in detail the catalytic behaviour of BcII. We used Co(II) as a spectroscopic probe for Zn(II) in order to characterise reaction intermediates and delineate a minimal kinetic pathway for imipenem hydrolysis by BcII. With both techniques, we could detect transient species with distinctive spectral features. Rapid kinetics sudies revealed that BcII uses a branched mechanism for imipenem hydrolysis and that a stable enzyme-product adduct is formed. Direct evidences of the formation of such an adduct were obtained with UVVis, EPR and NMR spectroscopy.

Whilst the hydrophobic effect is, for many systems, one of the most relevant interactions, it may be said that in the case of biological systems this effect becomes of determinant importance.
            The dependence of this interaction with pressure and temperature presents a abnormal conduct, behaviour which we have been able to reproduce not only qualitatively, but also quantitatively.
            From our results, we may state that the hydrophobic interactions are responsible for the clustering of non-polar particles in solution.
            We have worked analyzing firstly the clustering of Lennard-Jones particles in water, and secondly the non-clustering of these particles when the solvent is replaced by Lennard-Jones particles.
            This simple model is analyzed by means of Molecular Dynamics Simulation, which allows us to change easily the conditions of the system.
            The results obtained show notable agreement with the available experimental data, which establishes a clear validation for both model and method.

The spatio-temporal properties of intracellular Ca2+ signals have been extensively characterized in Xenopus laevis oocytes using optical techniques. In these cells, the signals arise upon  Ca²⁺ entry through IP3 receptors (IP3R\\´s) which are  Ca²⁺ channels. These studies have revealed a hierarchical organization of release events which is consistent with the IP3R\\´s being organized in clusters of tens of channels with a typical inter-cluster distance of a few μm. In this way, the smallest release events can be associated to Ca²⁺ liberation through single IP3R\\´s,  Ca²⁺ ``puffs\\´\\´ correspond to the concerted opening of several IP3R\\´s within a cluster and global waves involve cluster-cluster interactions via  Ca<sup>2+</sup>-induced  Ca<sup>2+</sup> release. Thus, ``puffs\\´\\´ constitute ubiquitous ``elementary events\\´\\´ of intracellular  Ca²⁺ signaling, which can both have local signaling functions in their own right, and serve as building blocks from which global signals are constructed.It is thus of interest to develop an effective IP3R-cluster model that could reproduce the observed properties of puffs and that could be used as the basic  Ca²⁺ release unit for  Ca²⁺ waves and other signals that spread over regions which are much larger than the cluster size. In this work we address the question to what extent an effective model that is able to reproduce the observations obtained under certain experimental conditions can still describe the cluster dynamics in other settings. More specifically, we analyze how the presence of different amounts and types of buffers modulate the response of the cluster. Using the model introduced in [1], which takes into account the behavior of the individual IPR`s in a cluster in a simplified way, we show how to take buffers into account and analyze how the puff amplitude and inter-puff time distributions change depending on the buffer characteristics. This analysis allow us to understand some of the non-intuitive observations reported in [2], in particular, why the addition of slow (Ca²⁺ trapping) buffers can lead to an amplification of the amplitude of puffs.
[1]  D. Fraiman, B. Pando, S. Dargan, I. Parker, S. Ponce Dawson (2006). Biophys. J., 90: 3897.      [2] S. Dargan, I. Parker (2004). J. Physiol. 553: 775.

One of the commonly observed outer surface components of cell envelops of prokaryotic organisms, archaea and bacteria, are crystalline arrays of proteinaceous subunits, known as surface layers. Isolated S-layer subunits of numerous organisms are able to assemble "in vitro", either in suspension or liquid surface interfaces, such as lipid films, liposomes and solid supports. In a previous study we demonstrated that S-layer proteins from Lactobacillus kefir and Lactobacillus brevis are able to reassemble on positively charged liposomes, enhancing significantly its stability towards different stress factors, as low pHs, bile salt and pancreatic extracts. In the present study, we investigate with fluorescence anisotropy < r > with diphenylhexatriene (DPH) and generalized polarization (GP) with 6-dodecanoyl-2-dimethyl-aminonapthalene (Laurdan), the changes in the lipid order induced by the attachement of S-layer proteins from lactobacilli to liposomes composed by cholesterol: stearylamine: and soy lecithin. The results show that the GP value of S-layer coated liposomes gradually decreases with increasing temperature, as expected, for the increase in mobility at the membrane interface (where Laurdan is localized) which leads to an increase in the dipolar relaxation in this region of the membrane. The GP values for S-layer coated liposomes from L. brevis and L. kefir exhibit higher values than control liposomes indicating that s-layer proteins are able to modulate the lipid order. Similar results were obtained using anisotropy measurement, as expected the value of < r > decreased with temperature increase. The difference in anisotropy values between high and low temperatures was progressively reduced with S-layer proteins, suggesting that the proteins increase the liposomes rigidity, specially at high temperatures. In summary S-layer proteins from both lactobacilli testes are able to increase order and rigidity of liposomes studied. These result could be explain why liposomes coated with S-layer proteins exhibit an important increase in their stability showing that S-layer-liposomes could be used at drug targeting and delivery systems for oral vaccines.

As is known, growing cotton hairs are a single cellular system, in which one the growth process of cotton hairs is accompanied by motion of a cell liquid - cytoplasm. It is showed, that the motion cytoplasm in a cotton fiber has laminar nature. In future laminar motion of cytoplasm in growing cotton hairs is simulated by flow of a viscous cell liquid along a capillary with diameter d and length l.  For a stationary fluid flow the pressure loss on overcoming of friction is determined by Darcy -Weisbax formula . Taking into account, that mean length of cotton hair is 30 mm and also that the average velocity of cytoplasm flow ~ 30mkm/s it is possible to estimate, that pressure losses on generation and growth one hair on the ovary seed up to length l=30 mm with hair diameter d=25mkm is 1-10cm . Change of the flow direction on separate sites of a capillary tube always results to additional pressure losses. In our case of cytoplasm motion with a small Reynolds’s number ~0.0001 pressure drop in the field of a bending of cotton hair can result to originating a new outgrowth length l in a point of a bending.  If to take into consideration, that on the mean part one seed average quantity of cotton hairs is 500-800 /mm² and take in the mind that all surface of a seed, for example, for sorts 108-F is about 135 mm², quantity of cotton hairs on all seed surface can be about 105 pieces. Thus, common pressure loss , responsible for formation and the growth of cotton hairs from one mature ovary seed is ~105 -106 cm . of a water pile. This value can use as an energy potential of one cotton seed.

 Department of Analytical Chemistry and Physical chemistry, University of Buenos Aires, Cátedra de Química General e Inorgánica, Junín 956 2º, 113, Buenos Aires, Argentina.
 
e-mail: caroperdo@yahoo.com.ar.
 
Previous results of our laboratory have shown that the interaction of an aqueous soluble enzyme, Rennet from Mucor miehei with different lipid environments is dependent on the changes in the superficially active water confined between the phospholipids (1). The changes on this kind of water are related with the nature of the acyl chains, the hydration layer and the dynamical exposure of the C=O groups.
A different exposure of the hydrocarbon chains to water may affect the surface free energy of the organized water. For this reason, in the present work we analyze the surface properties of the lipid bilayer in the liquid crystalline state by means of the interaction with the  fluorophore 1,8 anilinonaphtalene sulfonic acid (ANS) which interacts with the hydrocarbon region. The highest fluorescent intensity correlates with the highest hydrophobic environment. These changes were studied in liposomes of phosphatidylcholines (MLV´s) as a model of a lipid membrane.
The emission spectra were measured in a spectrofluometer (Perkin Elmer S5). The titration with ANS was done for different lipid concentrations, at constant temperature: 25±0.5 ºC.
The results show a lineal relation between ANS and the phospholipid composition at the highest value of wavelength of absorption (l).This value shifts to higher wavelengths with increasing lipid concentrations.
To reveal the changes in the water molecules penetration in the hydrocarbon region intercalating fluorophores at different depths of the bilayer and taking into account the previous results of surface pressure, we studied the fluorescent changes cited before in the presence of the enzyme Rennet.
 
 
References.
 
(1)   Martini M.F.; Disalvo E. A, BBA (in press)
 

Prodan (2-dimethylamino-6-propionylnaphthalene) is a hydrophobic fluorescent probe widely used in lipidic systems. This probe has been shown to be highly sensitive to lipid  phases, and this sensitivity is related to the probe´s microenvironment polarity and viscosity. With the objective of understanding what this dye can sense in the lipid bilayer, a study in several solvents was conducted. In the present study, Prodan fundamental state was theoretical and experimentally analyzed and its structural and electronic properties were studied. This molecule´s geometry in the fundamental state was determined using DFT with B3LYP functional, and the electric dipole and the energy absorption for the first excited state were determined using two different methods. In the first one, the solvent is considered to be continuous (PCM) and, in the other method, it is considered to be discreet, in which case the sequential Monte Carlo/Quantum Mechanics (S-MC/QM) methodology was used. These calculations were made using different solvents which covered a large range of polarity (2 to 80). Comparing both methods it was possible to conclude that the first one does not describe the interaction solute-solvent so well. For example, it gives a dipole of  ~ 6,10D for Prodan in all solvents. But in the discreet method it was possible to observe that the dipole in water was really around 10D. The results from theoretical methods have also  been compared with the experimental results.

Changes in physiological properties (i.e: tissue water content, thickness) of human skin, subcutaneous fat, or deeper tissues can provide significant information from a clinical point of view. In some cases, these variations produce changes in the dielectric properties of the media under measurement. Open ended coaxial lines operating at microwave frequencies have been successfully used for measuring dielectric properties of layered materials. Their wide frequency response allows the study of different dielectric relaxation processes, providing relevant information not achievable with other kind of probes. As a non-invasive technique, it is well suited for application in biological measurements in vivo, without damaging the media under test.
In search of suitable models, several authors have developed mathematical, empirical and numerical approaches. In particular, in this work a simple exponential approximation is applied and compared with a theoretical one. The resulting approach is useful to represent measurements in vivo of body areas that can be dielectrically modeled by layers. A fast procedure for estimating changes in the relaxation processes of layered biological tissues in a wide frequency spectrum is developed, using time domain measurements and Fourier analysis. Time Domain Reflectometry (TDR) and System Identification tools are used to obtain the frequency response. For both techniques the media under measurement is modeled as a causal linear time invariant (CLTI) system. The technique developed here is applied to measurements of bovine cortical and cancellous bones between 10 and 1300MHz. The results obtained are validated with frequency measurements and literature data.
Application of the proposed methodology depends on a previous knowledge of the kind of tissues under study and hence, on the expected values their dielectric properties can take. Deviations from these values can suggest abnormal conditions.

The immobilization of biologically active molecules is important for diagnostic screening as well as for micro-technology. Protein microarray enables us high-throughput analysis of protein-protein, substrate-enzyme, DNA-protein, RNA-protein, and ligand-protein interactions. A wide variety of protein immobilization methods are available and are sensitive for various factors such as its stability, structure and surface condition. We have developed a method of immobilization of protein based on an insect virus strategy.
Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called polyhedron in which progeny virion is occluded. The polyhedra produced by some insect viruses could be useful platform for protein immobilization without lost of biological activity. Previous works showed that Bombyx mori cypovirus (BmCPV) outer capsid protein, VP3, was the immobilization signal for incorporation of protein molecules into polyhedra [1, 2]. Fusion of the enhanced green fluorescent protein (EGFP) to the C-terminal of VP3 resulted in its incorporation into polyhedra when the chimeric protein co-expressed with polyhedrin during BmCPV infection [1].
In this study, we focus on spectroscopic analysis for protein-protein interactions and biological activities. Interaction between the protein and the tagged with EGFP protein was observed by fluorescence spectroscopy. Our observations indicate that the immobilized protein molecules remain the biological activities on the surface of polyhedra. We also discuss the possible application of our method for protein microarrays.

[1] Ikeda, K. et al., J.Viol. 2001, 75, 988-995.
[2] Ikeda, K. et al., Proteomics 2006, 6, 54-66

 Thermal, mechanical and dynamic properties of concentrated sugar-water mixtures, and their temperature dependence, have become subjects of interest, because they govern the storage stability and processing characteristics of a wide variety of food and pharmaceutical products. In view of the high water sugar mole ratio of its stable pentahydrate, the  thrisaccharide raffinose appears most relevant. The literature describing such systems is limited. The present work was undertaken with the aim of contributing to an improved characterisation of the binary water-raffinose system. The  dielectric permittivity of raffinose pentahydrate has been studied in the supercooled liquid and glassy regions, in the frequency range 300Hz to 7MHz. At least two relaxation processes were identified, and was assumed to be secondary ones. Both were Cole-Cole type. One of them resulted in a non-Arrhenius process, with (apparent) activation energies between 0.6 and 3.8kJ/mol. The other, was assumed to follow an Arrhenius equation and an (apparent) activation energy of 77.5kJ/mol was estimated. 

Biological information originated from physicochemical interactions of biopolymers and the results of co-evolution. We tried to prove this statement experimentally. When an RNA polymerase is given, the strongest promoter sequence is determined physicochemically. Using an isothermal amplification of hairpin DNA/RNA, we drove a natural selection-type evolution reactor taking the specific growth rate as the fitness. We used HIV-1 RT and ThermoT7 RNAP at 50°C in 1.4M trehalose. Starting from the randomized promoter pool, we observed selection or evolution process to the strongest promoter at 50°C. Although the selection process showed one-step convergence for a simple random pool, we observed a neutral evolution between two context-dependent selection processes for not so simple pools. These processes showed convergence-divergence-convergence of the promoter sequence. However, an RNAP itself has been evolved on the Earth. Therefore both RNAP and promoter are results of co-evolution, which is not necessarily deterministic. We have developed various in vitro virus(IVV) species, which are protein-mRNA/cDNA fusions and amplify, mutate and evolve in a test tube. Recently we developed a new IVV linked at N-terminus and 5’-terminus. We are planning to make an IVV displaying RNAP that transcripts its own genotype and co-evolves with promoter. A natural selection-type evolution reactor using this transcriptase IVV will show an autonomous evolution, which can create new algorithms,  rather than a directed evolution. 

Sporothrix schenckii expresses several factors that appear to be necessary for its invasiveness. Amonts these factors are the conversion or morphogenesis of S. schenckii yeast cells to a filamentous growth pseudohyphae and /or hyphae, the production of phospholipases and secreted proteinases, and host-cell recognition by cell surface adhesions.(Sandoval-Bernal  et al). A number of different factors can either trigger or inhibit morphogenesis for example pH and temperature or the presence/absence of specific nutrients (Rodriguez del Valle et al). However unknown if the  fungal interaction with  the host cells trigger the morphogenesis. The focus of this work its  present new information on the morphogenesis of S. schenckii,  particularly how it relates to signal  patways involved in the regulation of morphogenesis with the interaction epithelial. Our result shown yeast cells, grown a 37°C in a epithelial culture medium such as C-DMEM, germinate within a few hours and grow as hyphae provided that the pH of medium is near neutrality. We discuss the role of adherence and colonization of epithelial cells by the organism to regulate  its conversion from a yeast form to filamentous growth.
 This research was supported by grants N0.18  from DINPO-UG  to MSL.

The metallo-â-lactamase (MâL) BcII from Bacillus cereus was first crystallized with only one Zn(II) ion tetrahedrally coordinated to three His residues and a bridging H₂O/OH^- molecule (3H-site). Spectroscopic experiments and a second crystal structure later indicated that BcII was also capable of binding a second metal ion, which is trigonal bipyramidally coordinated to Asp120, Cys221, His263, the bridging H₂O/OH^- and an additional water molecule (DCH-site). We recently carried out a study on Co(II) uptake by BcII which indicated that three equivalents of Co(II) are bound sequentially, with macroscopic binding constants K_D1 = 0.12 ± 0.06 ìM, K_D2 = 0.16 ± 0.04 ìM and K_D3 = 94 ± 12 ìM. Besides, an exchange of the metal ion between the 3H and the DCH-sites occurs on mono-Co(II) BcII. The identity of the catalytically relevant species of BcII and the mechanism of action of this enzyme are still matter of debate. In this work we studied penicillin G hydrolysis catalyzed by Co(II)-BcII through stopped-flow rapid-scanning experiments and Rapid Freeze Quench-EPR experiments, at different Co(II)/BcII ratios. Our results indicate that both the mono-Co(II) and di-Co(II) forms of BcII are active, and that penicillin G hydrolysis proceeds by way of a branched kinetic pathway that involves Co(II) dissociation from the dinuclear enzyme-substrate complex to give rise to a mononuclear enzyme-substrate adduct. This model implies that mono-Co(II) BcII is active, in contrast to the previously reported conclusions drawn from kinetic experiments carried out in the presence of excess Co(II), which suggested that di-Co(II) BcII was the only active species. Our data also indicated that binding of penicillin G to mono-Co(II) BcII gives rise to an enzyme-substrate complex with the metal ion localized in the DCH site. This proposal contrasts to the generally accepted hypothesis that the mononuclear active species would bear a single metal ion bound to the 3H site. Acknowledgments: This work was supported by grants from ANPCyT and HHMI to AJV. LIL was recipient of a doctoral fellowship from CONICET.

Dengue virus is a mosquito-borne virus responsible of serious illness in humans. It belongs to the Flaviviridae family together with other important human pathogens. Dengue is an enveloped virus with a single-stranded RNA genome which is directly translated in the cytoplasm into a large viral polyprotein. This polyprotein is processed by the viral protease NS3 which also possesses helicase and NTPase activities that are involved in viral replication. The ATPase activity is intrinsic to viral and cellular RNA helicases and is required for unwinding the double-stranded RNA substrate. Observing individual mechanistic cycles of these motor proteins is central for the understanding of their cellular functions and a source of valuable information for drug design. Thus, we have initiated a program to characterize the interaction of NS3 with nucleotide triphosphates (NTPs) and nucleic acids (RNA).
In previous studies, we had successfully tested the protease and helicase activity of a recombinant full length NS3 protein in vitro. In this study we use this protein to characterize the kinetics of NTPase activity in vitro.
We show that NS3 catalyzes the hydrolysis of γ-phosphate present in the four nucleotides tested (ATP, CTP, GTP and UTP). The substrate curve for each of the nucleotides followed a single hyperbolic function. The specificity for each nucleotide is analyzed according to the Vmax/Km value obtained.
We found that Mg²⁺ is an essential activator of NTPase activity, reaching saturation at 2mM. No inhibition of enzyme activity was observed at Mg²⁺ concentrations higher than 10mM. In the absence of Mg²⁺, Mn²⁺ could also activate NTPase activity.

The monomeric D-Glucose/D-Galactose-Binding Protein(GGBP) is synthesized by Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with the sulfhydryl-reactive probe 2-(4’-iodoacetamidoanilino)naphthalene-6-sulfonic acid (2,4-ANS), at room temperature, glucose (Glu), galactose (Gal) and lactose (Lac) increased the fluorescence intensity (FI) of the ANS bound to GGBP in a concentration-dependent manner, with dissociation constants 1 mM, 0.4mM and 0.3mM, respectively. The binding affinity of GGBP-ANS for Gal was similar to the value published previously, however the effect on ANS FI was the opposed and might have been an inespecific effect induced on an already unfolded protein. In order to investigate this hypothesis we evaluated ANS and intrinsic (Trp) fluorescence of GGBP-ANS as a function of urea concentration (between 0 and 8M) and temperature (15-65ºC), in the presence and in the absence of 10 mM Glu. The effect of Glu on the FI of Trp and ANS of GGBP-ANS complex at 50ºC. The following main conclusions were reached:
1- At room temperature, the binding affinity for Glu was twice that for Gal and Lac.
2- a) The protein seemed able to follow an unfolding process, suggesting that the original sample was not deteriorated. b) With urea, Trp spectra were red shifted (as expected up on unfolding) while ANS spectra were blue shifted. This suggested that the conformational change that exposed Trp to water internalized ANS in a hydrophobic environment. c) Temperature induced a decrease in the FI of Trp (as expected) and an increase in the FI of ANS, both in the presence and in the absence of Glu. d) Glucose affected Trp fluorescence. This might have been either an effect on the protein conformation (inducing an unfolding perhaps by afecting the water activity/structure) or a dielectric effect. 3- At 55ºC we found that Glu decreased the FI of ANS as reported in the literature.
_{Acknowledgements: SeCyT-UNC, Conicet and Foncyt for the finantial support. PDC is a fellowship holder from Foncyt and MAP is a career investigador from Conicet.}

Papain is a thiol protease widely used in wound treatments and as absorption enhancer. However due to a natural unfolding mechanism the enzyme has a limited stability. In this way, its applications are restricted once it cannot be applied in medicines or cosmetics. The main purpose of this work is to analyze complexes formed by papain and hydroxypropyl-b-cyclodextrin (HP-b-CD) and papain b-cyclodextrin (b-CD) by thermal analysis and citotoxicity studies in order to verify a possible increase in the enzyme stability. Papain was diluted with phosphate buffer (pH 6.0) in presence of cystein to reach a final concentration of 1x10^{-5 M}. Cyclodextrin was added in order to obtain a 1:100 and 1:200 molar ratio solution (papain/cyclodextrin). The samples were incubated at 50°C under mechanical agitation of 90 rpm. Aliquots were collected as a function of time (0, 24 and 48 hours) and submitted to lyophilization. A control solution was also prepared and lyophilized following the same procedures containing only papain. The lyophilized samples (1:100) were submitted to DSC analysis, with a nitrogen flux of 50mL/minute, from 10ºC until 300ºC. The samples (1:100 and 1:200 molar ratio) were tested after 48 hours of complexation in order to assess their citotoxicity by using in vitro MTS method with culture cells at 24 and 48 hours contact. The test bases itself in the cell viability as a parameter for citotoxicity. A shift in the endothermic peaks was observed for both complexes, corroborating with the citotoxicity presented where the complexed enzyme performed less citotoxicity than papain itself. At higher molar rations the complex presented citotoxic effects as similar as papain isolated. The HP^-b-CD showed more significant thermal changes if compared to the b-CD as occurred with the citotoxicity tests performed.

Nitric oxide (NO) is a free radical involved in the regulation of several physiological functions, including vasorelaxation, neurotransmission and immune response. Its small size and slightly hydrophobic character let it diffuse through lipid membranes and lipoproteins with ease. In membranes and lipoproteins, the reaction between NO and O₂ is accelerated because of the higher solubility of both gases in the hydrophobic phase than in water, leading to the formation of oxidizing and nitrosating species. Herein, we extended these studies to proteins, and found that human serum albumin (HSA) accelerated the reaction 9 times per g/mL of added protein. To discern wether the protein surface or its core was responsible for this acceleration, we assessed the ability of different proteins with more polar surfaces to accelerate this reaction. Ovalbumin, lysozyme and to a lesser degree trypsin, also accelerated this reaction. In addition, we studied the pH dependence of the acceleration by HSA, and found a sharp decrease below pH 5, following the acid transition of HSA to an open conformation, with higher surface area and less volume. All these results suggest that the acceleration is due to interactions with the protein core rather than with its surface, though specific surface effects could not be excluded. Besides the accelerated formation of oxidizing and nitrosating species, the acceleration of NO reaction with O₂ could be reflecting the higher solubility of both gases within the hydrophobic cavities of proteins. We calculated that both gases will be ~2 times more soluble within HSA than in water, which corresponds to an association constant, K_a = 110 M^-1, that indicates that 1 every 15 NO molecules in plasma will be associated to HSA. One of the consequences of this weak association will be a slower macroscopic diffusion of NO and O₂, explaining some reports in the literature.

To study how the conservation of protein structure constrains sequence divergence, we have developed the Structurally Constrained Protein Evolution (SCPE) model. The SCPE simulates sequence divergence with special attention to the conservation of protein structure. These simulations allow the derivation of site-specific substitution matrices that we found outperform protein evolution models that do not consider protein structure explicitly. Recently, we improved the performance of SCPE taking into account the quaternary structure of a protein and consequently the intersubunit interactions present in these oligomeric complexes.

Here we show that the quaternary SCPE can discriminate native-like oligomeric structures from a set of decoys. To this end, we obtained a set of oligomeric decoys using SYMMDOCK and CLUSPRO docking programs for a set of oligomeric proteins. For each oligomeric decoy, using quaternary SCPE simulations, we obtained a whole set of site-specific substitution matrices. As SCPE is a model that takes into account protein structure, each oligomeric decoy defines a given model of protein evolution. Using each set of the substitution matrices, we estimated the maximum likelihood (using HYPHY program) for a corresponding set of homologous sequences and a given evolutionary tree. We then selected the best oligomeric decoy as the one producing the best maximum likelihood estimation. Our results indicate that the quaternary SCPE could be used to improve the selection of native-like complexes obtained using docking processes. In addition, preliminary results indicate that the same protocol could be used to estimate the oligomeric degree of a given protein.

Microcin J25 is a 21 aminoacid antibiotic peptide with a distinctive lasso-structure produced by Escherichia coli and active against Escherichia coli, Salmonella enteritidis serovars and Shigella strains. It has been shown that the target in E. coli is the b subunit of the RNA polymerase. However, it’s also known that in Salmonella newport cells MccJ25 inhibits the plasmatic membrane’s respiratory enzymes, suggesting a dual mechanism of action.
To study with more detail the antibiotic activity of the peptide, a fluorescent labeled microcin was sinthetizated. To achieve this, a mutant peptide containing lysine instead of isoleucine(13) was purificated, the MccJ25 I13K. For the labelling reaction, the mutant peptide was incubated with the amine-reactive probe fluorescein isothiocyanate, which form a thiourea bond with free amine groups. A mixture of peptide and fluorescent probe 1:3 ratio (w/w) was incubated in alcaline medium at room temperature during two hours in the dark.
The purification of the labeled peptide was carried out in two steps. First, the non reacted fluorescent probe was eliminated by a C-8 hydrophobic chromatography. Then, the methanolic fraction was purified by HPLC on a C18 column in order to separate the labeled peptide from the non labeled one. The purified fluorescein-labeled microcin showed antibiotic activity against MccJ25 sensible-strains similar to the native peptide.
Uptake assays of the microcin derivative with several strains were performed following the maximum emission fluorescence at 520nm. A bacterial suspension was incubated with the peptide at sublethal concentrations at 37°C in a TRIS buffer medium supplemented with glucose. Aliquots were taken at different times and the residual peptide in the supernadant was determined. The sensible strains Salmonella newport and E. coli AB1133 (pGC01) and the resistant strains E. coli SBG 231 y E. coli AB 259 (pTUC 200) were used. The independent effect of 100 mM 2,4 dinitrophenol, 200 mM vanadate or heat plus azide on the derivative peptide uptake were studied.
The results indicates that MccJ25 incorporation is an active mechanism ATP driven in the different strains tested, and the incorporation is seen both in sensitive and resistant strains.

In immunology it is important to understand mechanisms of the immune response and for this purpose it is useful to handle experimental data in terms of a mathematical model. Thus, having at our disposal present-day immunochemistry techniques we can both arrange data in the context of mathematical models with common differential equations and predict the behavior of the system of immune response beyond the reach of experiments. A combination of immunological approach and mathematical modeling was undertaken in order to study immune responses to tropical infections, including malaria. Using immunoblotting and immunoelectrophoretic methods it was possible to obtain characteristic times and constants of such periodic disease as malaria. The following assumptions were taken during mathematical modeling. B lymphocyte precursors are delivered to lymph nodes with a constant rate, they circulate there for some time, then, if they do not encounter “their” antigen, die off and get eliminated from the organism. The interaction with an antigen causes the transition of these cells into the second state, that is quickly proliferating immature plasma cells. It is supposed in the model that proliferating cells quickly propagate in the presence of the antigen. After the elimination of the antigen from the organism those cells lose their capacity of rapid proliferation but their amount keeps high long enough afterwards (memory cells). Intermediate cells already can produce some quantity of specific proteins-antibodies but the main producers are mature plasma cells which are generated from intermediate cells as a result of the secondary interaction with the antigen. Modelling of dynamic behavior revealed several regimes and critical points where the treatment may be taken. Intertwined experimental and theoretical approach represents here a nice way of studying interactions between the host and the pathogen.

The objective of this work is to explore the causes of the formal convergence between the matrix models of biological memories and the vector space models used in the design of search engines that act on the World Wide Web. I describe how, formally, the term-by-document matrix (a usual mathematical representation of a set of codified documents) can be interpreted as an associative memory. The dimensionality reduction performed by the Latent Semantic Analysis (LSA) acting on the term-by-document matrices on the one hand, and the basic theory of the matrix biological memories by the other, implies in both cases the generation of a statistical ‘conceptualization’ of data using little dispersed weighted averages. As we recently showed (Valle-Lisboa and Mizraji, Information Sciences, in press), the success of the matrix procedures of information extraction requires the detection of thematic clusters. I describe here a matrix memory model that creates thematic blocks using multiplicative contexts. These thematic memories are supported by modular networks and are acceded using passwords represented by vectorial semantic contexts. These memories assume contextualized outputs. Usually, a cognitive query produces an output that is distributed between many memory modules due to the anatomic connectivity. Yet, I show that the context modulation of inputs and outputs provokes that the neural information emitted by a memory module is only accepted by memories having the appropriate semantic context in their databases. Consequently, a given query can trigger different associative trajectories according to the sequence of contexts involved. This view implies that the biophysical neuronal dynamics happening during a cognitive process activated by a neural query, coexist with a dynamics of semantic neural vectors governed by the structure of the neural databases and by the semantic contexts involved in the neural queries.
 

We have investigated the formation of DMPC (dimyristoyl phosphatidylcholine) layers on top of hydrophilic and hydrophobic silicon substrates. The layers were prepared using the solution spreading method from DMPC dilutions in the range of 0.1 to 2.0 mg/mL in isopropanol and chloroform. The surface morphology and stability of supported phospholipid were investigated by contact mode atomic force microscopy. The experiments were performed ex-situ, just after solvent evaporation, after a hydration step, and in-situ with immersion in a buffer solution. For ex-situ measurements, in general, very thick deposits with 60 nm or higher were observed. On the other hand, in-situ measurements displayed much thinner layers with thicknesses, in most cases, corresponding to a DMPC bilayer. Isopropanol in hydrophilic Si produced uniform layers with large terraces. On the other hand, the use of chloroform resulted in layers with relatively high density of holes and unfilled or filled cylinders, depending on the amount of phospholipids. For hydrophobic Si and chloroform, the topography of the surface was strongly dependent on the region where the images were obtained, indicating a low lateral uniformity with thicknesses varying from 1 to 20 bilayers. The DMPC dilution also influenced significantly the surface morphology of the deposits. The observed structures were stable for several hours during the ex-situ and in-situ measurements, indicating that the solution spreading method is adequate for the preparation of self-assembled DMPC thick layers on Si, a very important step towards fundamental studies as membrane protein insertion and development of biosensor on a technological substrate. Acknowledgments: Financial support from FAPESC, PRONEX, PROSUL, FINEP, CNPq and CAPES are greatly acknowledged.

The interaction between antigens or antibodies and surfaces is the crucial step in the development of reliable solid phase immunoassays. Site-oriented specific adsorption, obtained by modifying the protein, the surface or both, provides a stable and strong interaction with the appropriate orientation to favor the surface immunoreaction. Recombinant antigens are commonly engineered to terminate with a stretch of six histidines (His₆), incorporated in the primary sequence to facilitate the purification. The high affinity between the His₆ end of the recombinant antigen and Ni²⁺ ions on the sorbent surface, may be exploited to promote site-oriented specific adsorption of antigens. The aim of this study is to optimize the adsorption of the recombinant protein His₆-H49 on a Ni²⁺ silica modified surface. This particular protein is able to recognize antibodies in sera of chagasic patients (1); thus, the designed biofunctional surface will be applied to diagnose Chagas’ disease.
The study was performed in different steps to achieve the optimal conditions to favor the coordination between the His₆ end of the recombinant antigen and the Ni²⁺ ions on the surface. In the first step, the coordination properties of His₆ with Ni²⁺ ions in solution were determined by spectroscopic techniques and potentiometric titrations. (2). On these bases, H49 was engineered with the His₆ end on the N-terminal of the recombinant protein. Sencondly, the adsorption-desorption process of His₆ on silica and silica modified was studied by reflectometry as a function of the pH and the concentration. Based on these results, the adsorption-desorption process of His₆-H49 was performed at pH 8 and low degree of surface coverage to induce the coordination between His₆ end and the metal ions on the surface and to minimize non-specific adsorption. With this step by step strategy, a strong, stable and site-oriented antigen-surface interaction is achieved avoiding conformational and biological activity perturbations.
 Referencias:
[1] Paranhos-Bacalla G.S., Santos M.M.R., Cotrim P.C., Rassi A., Jolivet M., Camargo M.E., Franco Da Silveira J., Paras. Immunol. 1994, 16, 165-169.
[2] Valenti L. E., De Pauli C. P., Giacomelli C. E., J. Inorg. Biochem. 2006, 1000, 192-200.
 * Corresponding author: Email: valenti@fcq.unc.edu.ar, Phone: 0054 351 4334169

As a preliminary stage, maps of distribution of relative concentrations of free radicals were obtained for the beginning of cleavage, blastula, gastrula and neurula. The distinct regionalization was found in the beginning of cleavage: the concentration of free radicals in cortical layer and dorsal half of embryo is lower than in central area and ventral half, respectively. At early blastula stage this regionalization is preserved in its general features. The region of embryo characterized by active free radical processes corresponds to the presumptive endo- and mesoderm in the period of inductive interaction.
The possible participation of regional changes of oxidative metabolism in the fertilized egg in determination of cytoplasmic localization of morphogenetic potencies is discussed.
The localization of free radical processes and changes in their level during common frog and fish development have been studied by means of grafted copolymerisation, autoradiography and dihydrorhodamine fluorescence.
Exposing fish embryos to ultraweak cell radiation during 24 hours can decrease or increase ROS level in tissues. At later stages there were variations observed in free radicals concentration, which are discussed as being related to the determination and morphogenesis of some rudiments of embryo. A local peak of free radicals concentration was also found out in the eye rudiment just before the onset of differentiation of its components.

Supported lipid membranes on the surface of gold gives relevant information related to phase transition, stability and morphology of the layers and are promising structures for the development of biosensors. The modification of gold with organic molecules that act as spacers to link macromolecules such as phospholipids provides a biomimetic system where hydrophilic domains can accommodate proteins and makes available studies of charge transport through this complex layers ^(1-3).
In this work we have studied the structure and electrochemical properties of a well order Au(111) surface modified with dithiothreitol (DTT) at room temperature and at 60° C in order to elucidate the possible configurations of this layer to prepare lipidic bilayers by vesicle fusion.
XPS and electrochemical measurements made at room temperature allow us to infer that DTT forms a monolayer in an up-right position with one of the two sulfur atoms bound to the Au through one S-Au bound and the terminal S-H group facing the interface. The STM images show a flat surface with a disorder layer on it. At 60° C the DTT layer has been partially oxidized to sulfonate groups. In this experimental condition the vesicle fusion produce the formation of phospholipidic bilayer membranes as has been extensively characterized by in-situ AFM measurements.
[1]   Lingler S., Rubinstein, I., Knoll, W., Offenhausser, A., Langmuir, 1997, 13, 7085-7091.
[2]  Tyrrell, M., Kokkoli, E., Biesalski, M., Surf. Science, 2002, 61-83.
[3]   Biosensors, B.Eggins, John Wiley & Sons, England, 1999
Acknowledgments: Financial support from CONICET, ANPCyT (PICT 02-11111), CIC BsAs, and FAPESC, PROSUL-CNPq and CAPES are greatly acknowledged.

Molecular machines described below are meant to be such molecular systems that make use of conformational mobility. Components of molecular machines move mainly by means of restricted diffusion. Whereas it is true to consider biological molecules-motors (various ATPases, bacterial flagellar complexes, membrane transporters) as machines, one may facilitate their studying by applying simpler models from macromolecular chemistry. As an example of molecular machines of nonbiological nature catenanes (compounds with two interlocked molecular rings) can be proposed. Thus, for example, model catenane ((2)-(cyclo-bis(paraquat-p-phenylene))-(1(2,6)-tetrathiafulvalena-16(1,5)naphtalena-3, 6, 9, 12, 15, 17, 20, 23, 26, 29-decaoxatnacontaphane)-catenane) changes its redox status when an electric field is applied, and rotation of the rings takes place. It occurs with fixation at certain moments of the influence. To find out characteristic properties of rings movements under various external conditions, molecular dynamics (MD) simulations were carried out. Three cationic forms of the catenane were first subjected to geometrical optimization and quantum chemical calculation. Then at different temperature conditions and under varying magnitudes of external electrical field these catenane states were run in MD calculations.
The movements of different time-scales were characterized either by statistical functions of rotation angles and angular velocities and by spectral decomposition of the same quantities. Large-scale rotation came to light the best in Fourier spectra, bending movements occuring on times scales of nanosecond order – with help of correlation analysis. The dielectric relaxation concerned with "trembling" of the rings occurs on smaller time scales.
The total rotational movement of the rings in catenane co-conformers is stochastic but for one of the rings it includes selected degrees of freedom with the prevalence of large-scale rotational movements in frequency contribution to the total spectrum. For the most charged forms rotation through angles more than 180º was absent or occured once at the transition from one charged form to another. Probably, it is concerned with the strong influence of redox-status. Summarizing results allowed developing an idea about possible mechanism of the catenane’s functioning.
The work was supported by RF Federal Agency Rosnauka (02.513.11.3086), RF Federal Agency on Education, Russian Foundation for Basic Research (06-04-08136, 07-04-01169), US CRDF (2803).

In first super weak luminescence of separate living cotton hair-cell was investigated for cotton plant variety  Tashkent - 1, C- 4727, Gossipium hirsutum, Turfan guza G.herbaceum, C-6063 G. barbadense L. and wild form G. raimondii   This phenomena under optical microscope Neophot-2 was observed with using so named replica–reprint technique for preparation living cotton cell. For this purpose the polymer solution (gelatin) was used for visualization of track luminescence in bulk of deposited polymer film on cotton cell-hair.  It is showed that luminescence for lifeless and mature plant cells were not generated but maximal luminescence is detected from apical parts of cotton hairs in early growth stage (4-6 days after flowering). By us it is showed that luminescence have cone like form difference variety of cotton plant. This fact indicates to a focusing of radiation connected with morphological and structural features of apical part of cotton cell-hair at early stages of their evolution. The electromagnetic nature of cotton cell luminescence has been showed by experiments with using of the photoelectric multiplexer (PEM) sensing to an ultraviolet radiation. At insertion in darkness of cotton seed-bud on a window PEM the dark current increased on 6-8 %. Actually radiation flux from developing cotton hair is more high-power, as through a window of PEM the small part of radiation was fixed only. Therefore on the basis of a large experimental material obtained by many investigators on miscellaneous biological objects it is possible to talk that the luminescence of cotton hairs are high energy ultra-violet irradiation.

Hyppocampal atrophy (HA) is a constant observation in long lasting depression. However, the clinical correlates of this change and its connection with the duration of illness or treatment resistance remain under discussion. Furthermore, it is well known that antidepressant drugs are neuroprotective and may lead to neuronal sprouting and neurogenesis in the hippocampus. Depressive disorder is accompanied by impairment of cognitive function. A known empirical finding is an increase in reaction times on a lexical decision task (to identify words which may be positive, negative, or neutral). Also, a bias in response times towards negative words was found in disphoric young students but not in patients with advanced major depression. An additional elevation in the response time is usually found in patients with major depression with respect to young disphoric individuals. We investigated the effect of HA on depression with a neural network model known to reproduce the phenomenology of temporal delays. We assume that time delays can be considered as a measure of illness correlated with the rest of symptomatology. We simulate HA by destroying synapses in memories attributed to hyppocampus. After atrophy in the model, an increase in the time response for both positive and negative words is observed. We also found that atrophy diminishes the bias, as was observed in patients with major depression.  These findings strongly suggest an active role of HA in the severity of depression measured through the increment of response times. Simulating the effect of cognitive therapy we found that, after an early improvement, the reversion is undercut by the reaction time levels reached by atrophy. This suggests that cognitive therapy alone cannot reverse severe or evolved depression where HA is known to be present. We simulated the effect of antidepressants provoking a reversion of the atrophy. In our model, cognitive therapy applied post reversion of HA reverts depressive manifestations. Assuming that clinical manifestation becomes evident after a threshold level in reaction times, the model also gives a plausible explanation of the frequent relapses of illness and treatment resistance observed in evolved major depressive condition.

Graph-theoretical methods have recently been used to analyze certain properties of social networks. In a previous work we have investigated the growth of an Uruguayan academic network, the Biology Section of the Program for the Develpment of Basic Science (PEDECIBA), by means of the construction of graph representations of the network at different times. In that approach, nodes represented scientists, and edges linked scientists that participated together in a master´s thesis evaluating committee. Graphs constructed in this way achieve connectedness early in their evolution, exhibit the small-world property (i.e., high clustering with short path lengths), and show power-law distributions of connectivities, clustering coefficients and centrality. Evidence of preferential attachment of new nodes and of new links between old nodes was also found in these ´committees´ graphs. To what extent these findings depend on the way the graphs are built?  In the present work we address this question by constructing a different graph representation of the same data set and comparing the properties of the new construction with those found for the previous one. The graphs representation used in this work focus on the central role of the thesis advisor to relate other committees´ members. We create ´advisor graphs´ consisting of subgraphs of the previously built ´committees´ graphs that retain the same set of nodes but only a subset of the original edges. These graphs show qualitatively the same properties as the previous ones; they are small-worlds with power-law relationships that develop early into fully connected graphs, showing preferential attachment in their growth dynamics. Nevertheless there are some quantitative differences such as an important decrease in clustering. Therefore it can be concluded that there are topological properties observed throughout the growth of the network that do not depend on the representation chosen, but reflect intrinsic properties of the academic collective under study.

Our third millennium medicine is faced with the strong challenge of abating medical error. The role of medical errors as important factors in the health outcomes of patients is well known. Diagnostic errors also constitute the second leading cause for malpractice suits against hospitals. Among individual causes of error such as forgetfulness, inattention, poor motivation, carelessness, negligence and recklessness, a singular place occupy cognitive errors, those arising from defective mental processes. A more accurate assessment of the mechanisms underlying cognitive errors is necessary to develop successful interventions to improve patient safety. Human memories are associative, and their general behavior is well captured by distributed associative memory models. In a recent article we described how to instruct expert systems in medical diagnosis with associative memories. The system can incorporate the clinical experience, building in that way a representative database of historical data that captures the frequency of presentation of the different nosological entities within a population. A minimal model trained with real data succeeded in diagnosing late-onset sepsis in neonates within an intensive care unit. In this communication we show that the associative nature of human memory implies an inherited bias towards certain kinds of cognitive errors in diagnosis. We instructed a memory with autoassociations of orthonormal vectors representing diseases. Each input vector was multiplied with a sum of the vectors representing the signs and symptoms characteristic of that disease using the Kronecker product. This procedure yields an autoassociative memory with overlapping contexts. The instruction based on individual cases inserts different frequency coefficients weighting the same symptom in different diseases. It also provides a coefficient that multiplies the autoassociated diseases by its own accumulated prevalence. The model predicts an evolution of the kind of bias depending on the experience gained by the practitioner, with morals for medical education assigning a role to the early instruction of rare diseases and to the value of casuistic literature. As outstanding causes of error enlightened by the model emerge epidemiological masking and the effect of an early erroneous labeling, this last seeming to act as a kind of cognitive illusion.

Several mRNAs are targeted to specific cellular locations where translation must occur because the protein must function there. This concept has grown in cells like fibroblasts and extended to neuron dendrites and in the last years to axons, in spite to the traditional dogma of their lack of protein synthesis. Dendrites and axons are different because they have different locally synthesized proteins. The mRNAs coding for these proteins are targeted to dendritic spines or Periaxoplasmic Ribosomal Plaques (PARPs) in axons. Targeting is codified in specific sequences in the 3’ or 5’ UTRs of their mRNAs. Myosin-Va or Kinesin are some of the motor proteins that transport the mRNAs to their target. Other proteins (linkers) make possible the mRNAs binding to motors proteins, forming the so-called Ribonucleoproteins (RNP), that sometimes also share ribosomes. mRNAs contained in RNPs are not translatable. Translation inhibition has been related to linker proteins. In our Lab, we demonstrated that PARPs share not only ribosomes (Electron microscopy immunocytochemistry, EM-ICh), but actin mRNA (in situ hybridization, ISH), Myosin Va coding mRNA (ISH), as well as some linker proteins (ZBP, actin mRNA binding protein and HuD, a Tau mRNA binding protein). We also showed that RNPs are present in the axonal core (EM-ICh), probably associated to the microtubule network. By immunoprecipitation we showed that some RNPs are composed by Myosin Va associated to its own coding mRNA, plus others. EM-ICh showed that ribosomes not only could be conveyed to the axon from neuronal soma, but from surrounding associated Schwann cells. The latter raised the possibility that macromolecular trans-cellular transfer could be other kind of motor driven traffic, contributing to convey the protein synthesis machinery to the axon. Intracellular macromolecules traffic -as big cities traffic-, is a complex system that has specific routes, energy consuming molecular motors, and signals that determine the transport, destiny, functions and so on. The determination of its regulation and components is a growing subject now. Alterations of normal traffic determine already described pathologies, but would be also pathogenic factors in neurodegenerative diseases like Charcot-Marie-Tooth (CMT) syndrome, Amyotrophic Lateral Sclerosis (ALS), as well as in others.

During embryonic development, the positional information provided by concentration gradients of maternal factors directs pattern formation by providing spatially-dependent gene expression. In Drosophila, the hunchback (hb) gene translates the positional information provided by the smooth maternal Bicoid (Bcd) gradient into a sharp expression pattern. There are at least three different aspects to how the gap gene hunchback (hb) reads the maternal gradient of Bicoid (Bcd) protein. First, positioning locates the anterior Hb expression domain at ~50% egg length in wild-type embryos, and can be shifted by mutations which vary bcd dosage. Second, sharpening turns the initially shallow border of the early Hb domain into a sharp step-function. Third, precision maintains precise location of the domain border, despite natural environmental variability (e.g . Bcd variability, temperature, etc). Fitting expression patterns (for wild-type and mutant embryos, as well as for artificial hb promoter constructs), we have developed a predictive bistable regulatory model for hb activation. Experiments and computations indicate that the three aspects of hb activation are relatively independent. Positioning depends mainly on Bcd concentration, while sharpening requires both Bcd and Hb. Bcd serves to raise Hb to a threshold activation level, allowing the hb self-regulation mechanism to generate bistability, with a sharp on-off expression pattern. Our results indicate that bistability can play a central role for threshold reading mechanisms of positional information during embryonic development. Other work in our group indicates that Hb precision may require other (gap) factors in the segmentation network.

While DNA is an essentially stiff molecule, it acquires a semiflexible nature as increasing
its size in the course of evolution.
It is obvious that a living matter is driven by a highly integrated complex system.
Although many integrant parts have been identified with their structures and mutuality, phys
ical mechanism dominating a biological system still remains in a black box.
In fact, even a fundamental property of DNA as a semiflexible polyelectrolyte chain is far f
rom being comprehended.

In this paper, we concentrate our attention to the folding problem of DNA, especially,
a discontinuous condensation (a coil-globule transition) of a monomolecular DNA chain, which
 is involved in a life cycle of a phage virus and also living cells.
This condensation originates from a segmental attractive interaction mediated by various con
densing agents.
Through the studies over decades, it has become clear that a monomolecular condensate of DNA
 often takes a toroidal structure, and it sometimes shows a partially folded (rings-on-a-str
ing) structure as an equilibrium structure. We show that these phenomena are described in a
common theoretical framework, namely a double equilibrium of the formation of loops and thei
r aggregation along a one-dimensional chain.
We develop a formulation of the free energy of a ring structure based on the wormlike chain
model, and discuss the transitional behavior of the chain conformation with respect to sever
al physical parameters.
An experimental result and the applicability of our formulation to it are discussed especial
ly with respect to the dependence on the ionic strength.
In the experiment, a partially folded structure of DNA only appears in a high-salt condition
.
The present formulation reproduces the experimental trend in a satisfactory manner.

The additive, synergistic or antagonistic  effects of  the combination of physical and chemical genotoxic agents on cellular populations depend on the agent’s nature and doses, and metabolic moment of the biological target. We studied the lethal and mutagenic interactions between the following agents: γ-rays, cisplatin (Pt), etoposide (E) and their combinations (γ+Pt, γ+E, Pt+E and γ+Pt+E) on haploid strains of Saccharomyces cerevisiae. It is known that γ-rays induce  DNA strand breaks, base loses and their oxidative modification; Pt produces adducts, and cross-links; etoposide inhibits topoisomerase II.
The SC7K lys2-1 wild type strain and the mutant top2-1, deficient in topoisomerase II, were treated in exponential phase, into nutrient medium. The absorbed doses and concentrations were: γ-rays (Co60: D=120Gy, Ï=107cGy/min); Pt (0.6-3mM, 40 min) and E (0.1-2mM, 40 min). Lethality- and mutation frequencies, as well as interaction magnitudes, were determined as a function of doses. At the molecular level, DNA double strand-breaks were quantified by pulse field electrophoresis and laser densitometry.
For the SC7K strain, the observed lethality induced by Pt and E, acting as single agents, was similar. The highest induced mutagenic effect was observed for Pt exposure.  Regarding the used agents combinations, the highest mutation frequency corresponded to γ+Pt. In SC7K, the lethal and mutagenic interaction for  γ+Pt or γ+E was synergistic and absorbed dose dependent. The Pt+E combination resulted in low lethal and mutagenic synergistic interactions, independently of used concentrations. The described interactions at the cellular level corresponded to the molecular quantifications of DNA  fractionation.
The top2-1 mutant strain was insensitive to etoposide, as was expected.  Lethal interactions events in case of: γ+E, Pt+E, and γ+Pt+E  suggest independence of etoposide. This  fact indicates an important role of topoisomerase II in the mentioned interactions in the wild type strain. In top2-1 the combination γ+Pt determined an antagonistic lethal interaction. 
It is known that in Clinical Oncology, the concurrent treatment of radiotherapy, cisplatin and etoposide  is performed in certain solid tumors. Present results provide a critical base regarding  these concurrent treatments.
We are indebted to Assistant Prof. G. Paolini (INCA-UdelaR) and to Fundación Manuel Pérez for support.

The mechanical properties of soft tissues (isotropy, non-linearity) are highly dependant on their living or dead state. Thus in vivo  instrumentation of visco-elasticity are of importance from a biological or medical point of view. In the field of medical imaging, measurement techniques of these mechanical properties have been intensively developed during the last 15 years. They are called static elastography, transient elastography or magnetic resonance elastography. In this work, we present the time reversal elastography.
This method  is an application of the one channel time reversal (TR) mirror in reverberant cavities. In these experiments, a TR wave is shown to refocus back to its source, colapsing into a spot limited by the Rayleigh criterion. Thus, measuring the focus spot dimension provides a wavelength estimation which is in turn linked to the mechanical properties of the medium. Transposed in soft solids, the TR experiment is shown to give an accurate estimation of the shear elasticity. Indeed, the elastic waves are mechanicaly generated by a shaker working in the low frequency range (50-300 Hz). At such low frequencies, the vectorial field measured using a non invasive ultrasonic method developed in the field of transient elastography is dominated by shear waves.
Theory of one channel TR has been developped for scalar wave field in perfectly reverberant cavity. Some studies in a gelatin based phantoms were conducted in order to define the limits of these assumptions and to validate the technique. Its robustness as regard to the source position, the emitted frequencies, the field component and the solid reverberation are carrefully studied.
Experimental validation in gelatin based phantom is demonstrated. Results in human muscles are also presented.

The mechanical properties of tissues present interesting features for their basic study at high temperatures. From the point of view of medical applications, local temperature changes in tissue while irradiating with ultrasound therapy is an important parameter of treatment’s control. Diagnostic ultrasound is a promising way to non-invasively estimate temperature changes in tissues. However, this technique has some limitations. First, it assumes a linear relation between temperature changes and tissue strain, which is valid in a small range of temperature change (< 10 °C), but ultrasonic therapy reaches higher values. Secondly, when tissue temperature reaches high values (>50 °C), tissue undergoes structural changes and the backscattering signals are modified such as temperature estimate algorithms fails. In this work, we address the first of these issues. Investigations performed in agar based phantoms and bovine skeletal muscle show that the temperature range of validity of the linear model depends on the starting point (baseline). Preliminary investigations at room temperature have produced optimistic results. A range between 10 ºC and 15 ºC of temperature increase was estimated with good accuracy. However, if the baseline is the body temperature, it is more limited and adaptive estimation is needed to get quantitative values.

Serum albumin is the most abundant protein in the circulatory system. At neutral pH, the crystal structure of this 66 kD protein reveals a heart-shaped molecule organized into three similar structural domains (1). These are alfa helical and include loops and a large number of disulfide bonds. It was described that aqueous solutions of BSA present a monomer-dimer equilibrium with a dissociation constant of 10 mM at 25 degrees and pH 6 (2). Despite the BSA acid-base transition had been extensively characterized, no previous work had accurately described how these changes are reflected in hydrodynamic properties of the different oligomeric structures of this molecule. In this work, we analyze the effects of pH on the oligomeric states of BSA using static and dynamic light scattering combined with size exclusion chromatography. In these studies, BSA monomer (Sigma (St. Luois, MO)) at 10mg/ml was prepared using phosphate buffer 10 mM with NaCl 100 mM. The same buffer was used to equilibrate the Sepharosa S-200 column at different pHs. All solutions were prepared using bidistilled water filtered through a 20 nm filter and degassed before use. Elution chromatogram of monomeric BSA showed four different oligomeric forms. The molecular masses of these molecules were obtained from static light scattering data using the three angle detector demonstrating that these species are the monomer, dimer, trimer, and small quantities of higher molecular weight oligomer. At lower pHs only dimer and monomer were detected. Hydrodynamic radii and diffusional coefficients of monomer and dimer were calculated from the dynamic light scattering data. At lower pHs hydrodynamic radii becomes higher and diffusional coefficient becomes smaller. Dimer and trimer were collected and reinjected. In both cases, we observed the monomer, dimer, and trimer, demonstrating the reversible character of the forces stabilizing these oligomeric forms.
With grants from UBA, CONICET, and ANPCyT.
Referencias:
 
[1] Carter D., Ho JX. Advances in Protein Chemistry, 1994, (45) 153-203
[2] Levi V., Gonzalez Flecha FL. Biochimica et Biophysica Acta, 2002, (1599) 141-148

Statistical prediction about typical parameters of biological tissues can be made through ultrasonic signals. Characterization of tissue from backscattered ultrasound signal using the spectral autocorrelation (SAC) provide information about the scatterer distribution in a biological tissue. Periodicity manifests itself as spectral peaks in SAC. The spacing between spectral peaks of autocorrelation in the bi-frequency plane is appropriate to estimate the mean scatterer spacing (MSS), in a statistical procedure. SAC capabilities in characterizing periodicities in A-scans due to regularity in the scatterer distribution, was shown in a theoretical frame by Donohue et al (1993). The relationship between the scatterer spacing and the spacing of spectral peaks was established using a stochastic model of the echo-formation process from biological tissue. In modeling, the distribution of scatterers is governed by a Gamma distribution (Gamma-pdf). The degree of regularity increases when the Gamma-order parameter value increases. For low Gamma-orders SAC components reveal information that is not seen in the PSD.
            The attenuation of acoustic wave propagating in a wide variety of lossy media obeys a power law dependence on frequency. In medical ultrasound, for tissue absortion, the exponent was encountered to be in the range between 1 to 1.7  (Bambert 1986). Frequency domain form for wave equations with losses gives place to a dispersion equation for which the wave number solution is complex.
            We introduce attenuation and dispersion in a stochastic echographic model. In the signal formation process, the amplitude of each echo is degraded due to their trip in the absorbent medium. The model developed in the frequency domain, as a summation of reflectivity amplitudes with appropriate retarded phases, also filtered by an attenuation transfer function.
            Theoretical formulation of SAC predictions are reviewed for the proposed model described above. Gamma-pdf  is used to describe the distribution of scatterers.
            We perform statistical experiments with simulated signals in which the attenuation coefficient obey a linear with frequency law. The emitted pulse is Gaussian with 3.5 MHz of central frequency and 1.5 MHz of bandwidth. The performance of SAC in estimate periodicity is tested varying signal parameter values.

In natural environments, microorganisms encounter complex mixtures of carbon sources. These are consumed in different proportions, depending on their available amounts. When present at low concentrations, many sources are consumed simultaneously. In contrast, at relatively high concentrations, one of the available sources is selected, as suggested by the principle of diauxic growth. The list of biochemical reactions that form an organism’s metabolism can be reconstructed from the sequence of its complete genome. These genome-scale metabolic networks can be used to determine, in silico, the stoichiometric capabilities of organisms, including their ability to grow in different environments and the effect of mutations. One strategy to perform this type of calculations is applying optimization criteria to the reconstructed network. In previous contributions, we have shown that the experimental pattern of carbon source consumption, in a mixture of two sugars at low concentrations, can be reproduced assuming that the microorganism uses all its stoichiometric capabilities to maximize growth rate. However, maximization of growth rate does not lead to the diauxic behavior found at high concentrations. Here, starting with the core metabolic network in E. coli, we determine the patterns of utilization of mixtures of two sugars, when present in different amounts and proportions. The optimization target used is the minimization of the number of active reaction steps, constrained to obtain a rate of ATP production greater than a certain lower bound. We found four patterns of nutrient consumption. When both sugars are at very low concentration, the minimum rate of ATP production could not be achieved. If the concentrations of the two sugars increase but are still low, both carbon sources are simultaneously consumed. For high concentrations of both sugars, the pattern depends on their proportions. The "preferential" sugar, after reaching a certain characteristic threshold value, is the only carbon source used, independently of the concentration of the other sugar. On the other hand, the "alternative" sugar is the only source used, only if it has reached its threshold but the preferential sugar has not. Therefore, the optimization criterion applied can account for the two main patterns of sugar utilization found experimentally.

The Hydroxyapatite (HAP) was produced by chemical precipitation controlling the pH and maturation time. Powder X-Ray difractometry (XRD), Optical Microscopy, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to characterize the samples. The crystalline structure of the samples was evaluated using standard Rietveld´s refinement method. Optical micrographs revealed grains sizing from 5 to 10 µm. SEM images indicated the material has homogeneous morphology and also showed that the micrometer-sized grains are agglomerates of nanometer-sized particles, and this latter result agrees quite well with the sizes of the crystallites obtained from the XRD pattern. The AFM images confirmed that the agglomerates were HAP particles in nanometer scale, with average size of 32,65 nm. Porous samples of HAP were also prepared with addition of different concentrations of cassava starch. Pores with diameters ranging from 1 to 5 µm were formed in the sample with 10%wt of cassava starch, mimicking the bone morphologic structure.

Liposome complexes have received significant attention for a variety of biochemical and biomedical applications including drug targeting and drug delivery and tumor imaging and diagnostics. Semiconductor nano-crystals, also known as quantum dots, are now beginning to be used in similar biochemical experiments. Like fluorescent dyes, these quantum dots have the ability to reliably fluoresce at pre-engineered wavelengths. However, these nano-crystals have lifetimes significantly longer comparable to fluorescence dye counterparts. We have successfully encapsulated approximately 10nm CdSe nano-crystals inside approximately 100nm liposomes and studied the resulting complex using fluorescence resonance energy transfer (FRET) microscopy. Further studies were performed using transmission electron microscopy (TEM) showing the details of the encapsulation, and Raman spectroscopy to examine their structural details. Our nano-manufactured quantum dot liposome complexes do not bleach over periods of hours and are general enough to allow the addition of drugs targeted for the vectored cells thus offering the ability to both image and medicate simultaneously over a long period of time.

The Atomic Force Microscopy (AFM) (1) has been used as a force sensor to measure unbinding forces of single bound complexes in the nanonewton range. The force spectroscopy measurements can be applied to study both inter- and intramolecular interactions of complex biological and synthetic molecules. In a single force-curve experiment, the tip is brought into contact with the surface. During tip-surface approach the cantilever deflection angle remains constant until the tip contacts the surface from when an increasing repulsive force develops. Subsequent tip-surface retraction leads to relaxation of the repulsive force. And then, the adhesion is measured directly from the bend of the cantilever upon retraction.
            Although the AFM has been extensively used as a force sensor, the commercially available cantilever is limited to silicon and silicon nitride. Those materials reduce the adhesion sensitive with specific surface and/or molecule. Different methods have been developed to functionalize a cantilever in order to modify AFM cantilever by covalent linking of organic monolayer. However, no method has presented a control way to form that monolayer. In previous work, we have described a method to functionalize the silicon (Si) tip (AFM tips) with carboxylic groups by radio frequency (RF) plasma treatment by applying acrylic acid (AA) vapor at 100 W plasma discharge for 5 min (2).
Here, we have used the AFM functionalized tip with carboxylic groups to immobilize directly the LexA protein. The LexA is a protein of 202 amino acids which compound the Escherichia coli SOS system. The SOS system includes phenomena such as enhanced capacity for DNA repair and mutagenesis and inhibition of cell (3).
We were able to measure the unbinding of peptide bonds between free amino groups of LexA protein and the carboxylic groups on the silicon surface.
 References:
(1) Binnig G, Quate CF and Rohrer C: Atomic force microscope. Phys. Rev. Lett. 56: 930-933, 1986.
(2) Vilani, C., Costa, L.T., Simão, R., Achete, C.A., Functionalization of AFM tip by RF plasma treatment – applied to polymer interface characterization (in preparation)
(3) Little, J.W. and Mount, D.W. (1982).The SOS regulatory system of Escherichia coli. Cell 29, 11-22.

The study was designed to provide experimental and computational information on some nervous segments involved in the phototransduction after exposure to microwaves of 9,75 GHz and 0.5 mW/cm2. The biological material was consistent with Drosophila melanogaster that due to the visual system complex organization as well as to the high genome plasticity become a biological model system in the studies dedicated to human physiology and pathology. The present study was focused on the electrophysiological response of the neural cells from the visual system following microwave impact during the early ontogenetic stages of the studied adults. Electroretinographic recordings were carried out on eight days old Drosophila adults developed from larvae and pupae irradiated for two hours. In comparison to the control insect lot, provided by the same genetic line and grown in similar conditions, significant changes in the ERG signal were evidenced. Both in high and low intensity light the ratio of the amplitudes of the two main components of the ERG signal was changed, even atypical shape of the ERG signal being revealed in some cases. The dynamic analysis carried out on the ERG data series was also able to give qualitative and semi-quantitative information on the degree of complexity characterizing the visual neurons in the investigated individuals. Fourier analysis, wavelet maps, surrogate data and other computational tests have been applied successfully aiming to make the difference between the contributions of each type of visual neurons to the ERG signal in the exposed lot versus the control one. The higher influence of microwave exposure on the ganglion cells n comparison to the photoreceptor ones was assumed, the non-thermal microwave effects being supposed to have the major role in the ERG changes revealed during the experiment.

Cadherins are transmembrane proteins, with an extracellular segment made of a tandem of five cadherin-like domains (EC1 to EC5). The outermost domain, EC1, is important for the specificity of cadherin-mediated cell adhesion. X-ray crystallography studies with C- and E-cadherin have revealed that EC1 can participate in the formation of structurally similar molecular complexes. In both cases the complexes are formed by the insertion of the side chain of the conserved residue W2 of one molecule into, an also conserved, hydrophobic pocket of the partner molecule. This is structure known as the strand dimer interface. Even though the general characteristics of the structures are similar, in the C-cadherin homophilic complex the conserved residues W2 are separated 15 Å whereas this separation is 25 Å in the E-cadherin complex. These distances characterize a closed and an open configuration of the cadherin adhesive complexes, respectively. Steered Molecular Dynamic (SMD) simulations were used to investigate the forced dissociation of homophilic complexes of both C- and E-cadherin, in the two configurations. The open configuration in C-cadherin and the closed configuration in E-cadherin were induced by applying a suitable force while keeping the length of the complex constant. The forced dissociation was induced using a disrupting force of 500 pN. For C-cadherin the rupture times of the adhesive complex in the closed and open configuration were 1.02 ± 0.07 and 1.17 ± 0.07 ns, respectively. On the other hand, for E-cadherin the rupture times of the adhesive complex were 1.61 ± 0.22 and 1.04 ± 0.17 ns for the closed and open configuration, respectively. These results show that the adhesive complexes of both C- and E-cadherin can exist in at least two configurations with a hierarchy of strengths specific to each cadherin.