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The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.

A semi-nested polymerase chain reaction (snPCR) was developed to improve the sensitivity of detection of Acanthamoeba sp. genome from corneal scrapings of Acanthamoeba keratitis patients. The snPCR was developed using a laboratory designed inner forward primer targeting the 450-bp product of the 18s rRNA-gene-based PCR. The snPCR was optimized using 11 Acanthamoeba sp. culture isolates and further applied onto 35 corneal scrapings from keratitis patients. The sensitivity and specificity of the snPCR was compared against conventional methods (smear and/or culture-gold standard) and the uniplex PCR described by Schroeder et al. Eleven out of the 35 corneal scrapings were positive by the gold standard and snPCR, whereas only 3 of these 11 were positive by the uniplex PCR. The clinical sensitivity and specificity of the snPCR was 100% when compared with the gold standard. DNA sequencing was performed on first round amplicons of four culture isolates and one specimen, and all of them were identified as genus Acanthamoeba when compared with the GenBank database sequences. Application of snPCR on the 11 culture isolates yielded amplicons ranging 120-160 bp in size, indicating sequence variation among the different culture isolates. The clinical sensitivity of snPCR was higher than the conventional methods and the uniplex PCR reported earlier.

The wound healing activity of topically app lied extract of bark of Ziziphus jujuba was evaluated in albino rat by excision wound model for a period of 24 days. In this study, the rats were divided into four groups, each groups continued healing process with 5 and 10% w/w methanolic extract of Zizip hus jujuba mg/cm 2 . Test duration was 24 days, wound diameter measurement using digital image analysis on days 0, 8 th , 16 th , 21 st and 24 th with biopsy of two rats from each group were taken . The results revealed that treatment with low - dose group of met hanolic extract of Ziziphus jujuba (5%) less shrinkage of wound area and also from the perspective of Histopathological change. In the second and third week, tissue had better organization than most other groups (p

Oestradiol acts both as a mitogen and as an inducer of differentiation of target cells. The cellular responses to oestradiol are generally mediated through the regulation of gene expression, although nongenomic modes of action are also documented. The present observations show that the regulation of keratin gene expression in rat vaginal epithelial cells is under the influence of oestradiol. It is observed that oestradiol regulates both the qualitative and quantitative expressions of keratin polypeptides in a time dependent and sequence specific manner. These regulatory effects are the result of modulations in transcriptional, post-transcriptional and translational processes of these genes, brought about by the hormone in vaginal epithelial cells.

We synthesized a novel water-soluble porphyrin THPP and its metalated derivative Zn-THPP having excellent triplet excited state quantum yields and singlet oxygen generation efficiency. When compared to U.S. Food and Drug Administration approved and clinically used sensitizer Photofrin, THPP showed ca. 2-3-fold higher in vitro photodynamic activity in different cell lines under identical conditions. The mechanism of the biological activity of these porphyrin systems has been evaluated through a variety of techniques: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, comet assay, poly(ADP-ribose)polymerase (PARP) cleavage, CM-H(2)DCFDA assay, DNA fragmentation, flow cytometric analysis, fluorescence, and confocal microscopy, which confirm the apoptotic cell death through predominantly reactive oxygen species (ROS). Moreover, THPP showed rapid cellular uptake and are localized in the nucleus of the cells as compared to Hoechst dye and Photofrin, thereby demonstrating its use as an efficient sensitizer in photodynamic therapy and live cell NIR nucleus imaging applications.

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB.We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKβ and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.

Alpha-crystallin, a major eye lens protein, bears homology with small heat shock proteins (sHsps) and exhibits molecular chaperone-like activity. Structural perturbation by temperature or low concentrations of denaturants leads to enhancement of its chaperone-like activity. We have earlier demonstrated similar enhancement of chaperone-like activity using biologically compatible solutes such as arginine hydrochloride and aminoguanidine. The purpose of the present study is to get an insight into the mechanism of the arginine induced enhancement of chaperone-like activity of alpha-crystallin.The effect of arginine hydrochloride on the chaperone-like activity of alpha-crystallin at 25 degrees C was studied using DTT induced aggregation of insulin as a model system. Changes in the accessibility of the thiol group near the end of the alpha-crystallin domain in the absence and the presence of arginine hydrochloride were studied using dithiobisnitrobenzoic acid. Fluorescence resonance energy transfer studies were performed to investigate changes in the dynamics of the subunit assembly. Urea induced denaturation studies of alpha-crystallin were carried out to investigate structural destabilization of alpha-crystallin, if any, in the presence of arginine hydrochloride.Arginine hydrochloride increases the chaperone-like activity of alpha-crystallin several fold towards DTT induced aggregation of insulin at room temperature. Our study shows that both the extent and the rate of accessibility of the thiol group are increased in the presence of arginine. Fluorescence resonance energy transfer experiments show that arginine hydrochloride significantly increases the subunit exchange between the oligomers of alpha-crystallin. Arginine induced structural perturbation and loosening of subunit assembly of alpha-crystallin leads to overall destabilization of the protein as reflected by the urea denaturation study.Arginine perturbs the tertiary and quaternary structure of alpha-crystallin and enhances the dynamics of the subunit assembly leading to enhanced chaperone-like activity. Thus, in addition to size, surface hydrophobicity, and charge distribution, the dynamics of the subunit assembly appears to be one of the critical factors that can modulate the chaperone activity.

We examined the effect of heat stress on localization of two sHsps, alphaB-crystallin and Hsp25, and of Hsc70, a member of a different class of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, alphaB-crystallin and Hsp25 are found in the cytoplasm; only alphaB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that alphaB-crystallin and Hsp25, but not Hsc70, colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C. Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This differential entry of alphaB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin A/C network or stabilizing this specific network.

A method is given for the optimum design of linearly elastic trusses which can be partitioned into a series of statically determinate substructures. In the case of such structures the design problem is formulated as a problem in optimal control theory. The member areas of substructures are treated as control variables and the interacting forces between substructures are taken as the state variables. Transformation equations for state variables are derived using equilibrium and displacement compatibility conditions. The design problem is envisaged as a problem of determining optimal controls to minimize the weight of the truss. This necessitates the solution of a number of smaller problems in sequence, instead of a single problem of larger dimension. Solution of the resulting optimal control problem can be obtained using the method of local variations.

This note presents the solution to the problem of the absolute minimum volume of reinforcement for strength criterion. It is for the case of clamped square reinforced concrete slabs, using Hillerborg’s strip method and the necessary and sufficient conditions for an absolute minimum to exist as proposed by Rozvany and Charret based on the Prager and Shield hypothesis, for uncurtailed and curtailed reinforcement layouts.

A finite-difference method is developed for obtaining the natural frequencies and mode shapes for rectangular plates, of abruptly varying stiffnesses causing re-entrant corners, by assuming average curvatures at junctions. This eliminates the use of fictitious points. It is shown that the discrete model analogue proposed by Witteveen 1 in 1966 for flexural analysis of such plates is a degenerate case of this method. The method is applied to the case of plates with simply supported and clamped boundary conditions. Numerical examples are presented to show the convergence and the versatility of the method. Checks are made for the limiting cases of plates of constant stiffnesses.

By replacing the continuous system of the plate with a discrete system of beams and torsional panels with finite degrees-of-freedom, a grid framework model is formulated for obtaining the critical buckling load of plates whose stiffness changes abruptly in two directions. This approach is then generalized to consider the effect of lateral deformations by a mathematically consistent first order finite-difference method. However, it is found that the effect of Poisson's ratio on the critical buckling load is negligible. The advantage in the discrete formulation is that no fictitious points need be considered, thereby making it possible to easily deal with cases of nonhomogeneity, complex boundary conditions, etc. An advantage over the finite element method is that the number of equations are far less, since there is only one unknown at a grid point. Numerical examples for different boundary conditions are presented to show the convergence and versatility of the method.

A general problem in the optimal design of structures for loading and other environmental conditions wherein structures material-interplay is to be given due recognition falls into the category of multifactor optimization problems. This can be visualized by noticing that a single objective function cannot be identified in such aforementioned design situation, since the design is to meet optimally several structural and environmental objectives. Also different candidate materials and configurations, have different degrees of suitabilities to each of the design requirements. The present algorithm is an attempt to generate a suitable design procedure to choose the best material out of an available set of materials to meet several design criteria optimally by seeking a trade off between the conflicting design objectives. In the algorithm that is generated, the candidate material properties, the field equations relating the material properties are the input information. The weightage of design requirements with the associated utility measures for each material for each criterion (either on a deterministic or probability basis) according to the subjectivity of the designer, serve as a basis to rate different materials with reference to their suitability to the design situation. A material is rated by its total expected utility value. This method is compared to existing methods available so far and it is shown that it obliviates some of their limitations. An example worked out and the general flow chart is discussed m detail in the paper.

A grid frame work model is extended to obtain the natural frequencies of square plates with stiffened square openings. This technique eliminates the use of fictitious points at the re-entrant corner point, junction point, etc. on the stiffened opening. Numerical results are presented for the first time for the cases of plates with simply supported and clamped boundary conditions in order to show the convergence and versatility of the method. Also, this investigation provides a general feeling about the changes in fundamental frequencies that occur when stiffeners are introduced.

A method is given for the analysis of nonlinear trusses which can be partitioned into a series of statically determinate substructures such that each substructure is coupled to two adjacent substructures. In the case of such structures, the number of independent variables to be determined in order to evaluate the forces in all the members is considerably reduced. The method is the same as the state space analysis herein used for controlled systems. The formulation given results in a set of operator equations with the interacting forces between the substructures as dependent variables and the properties of truss elements as independent variables. The method is applied to trusses having conservative, nonlinear elements undergoing small deformations. The resulting set of equations are solved using the Newton-Raphson technique.

alphaB-crystallin, a small heat shock protein, plays an important role in muscle homeostasis. It gets up-regulated during muscle differentiation and mice lacking alphaB-crystallin die prematurely with extensive muscle wastage. We have examined the role of alphaB-crystallin in muscle development using C2C12 myoblasts as a model system. Over-expression of alphaB-crystallin delays the muscle differentiation program significantly. C2C12 myoblasts over-expressing alphaB-crystallin (CRYAB-C2C12) display defect in cell-cycle exit upon induction of differentiation. During differentiation, CRYAB-C2C12 cells exhibit sustained level of cyclin D1 and delay in p21 and myogenin expression as compared to C2C12 cells. We find less accumulation of MyoD in CRYAB-C2C12 cells than in C2C12 cells. In vivo protein stability studies reveal faster ubiquitin-proteasome-mediated MyoD degradation in CRYAB-C2C12 cells (t(1/2)=1.42 h) than in C2C12 cells (t(1/2)=2.37 h). Immuno-precipitation experiments showed that MyoD gets ubiquitinated at earlier time points in CRYAB-C2C12 cells than in C2C12 cells. Our data reveal alterations in the synthesis and degradation of MyoD in CRYAB-C2C12 cells. The level of alphaB-crystallin as well as its Ser-59 phosphorylated form increases with increasing time of differentiation. Our studies show, inter alia, that alphaB-crystallin modulates myogenesis by altering MyoD level and provide an interesting insight in its role in myogenesis.