UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Studies on the Mechanism of Action of Miconazole: Effect of Miconazole on Respiration and Cell Permeability of Candida albicans

The antifungal drug, miconazole nitrate, inhibits the growth of several species of Candida. Candida albicans, one of the pathogenic species, was totally inhibited at a concentration of approximately 10 μg/ml. Endogenous respiration was unaffected by the drug at a concentration as high as 100 μg/ml, whereas exogenous respiration was markedly sensitive and inhibited to an extent of 85%. The permeability of the cell membrane was changed as evidenced by the leakage of 260-nm absorbing materials, amino acids, proteins, and inorganic cations. The results we present clearly show that the drug alters the cellular permeability, and thus the exogenous respiration becomes sensitive to the drug.

Studies on the Mechanism of Action of Miconazole: Effect of Miconazole on Respiration and Cell Permeability of Candida albicans

The antifungal drug, miconazole nitrate, inhibits the growth of several species of Candida. Candida albicans, one of the pathogenic species, was totally inhibited at a concentration of approximately 10 µg/ml. Endogenous respiration was unaffected by the drug at a concentration as high as 100 µg/ml, whereas exogenous respiration was markedly sensitive and inhibited to an extent of 85%. The permeability of the cell membrane was changed as evidenced by the leakage of 260-nm absorbing materials, amino acids, proteins, and inorganic cations. The results we present clearly show that the drug alters the cellular permeability, and thus the exogenous respiration becomes sensitive to the drug.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

5-Methyl-2-Thiouridine in the tRNA of Candida tropicalis and Its Localization in Lysine tRNA

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.

Velocity ratio in the analysis of linear dynamical systems

The transfer matrix method is known to be well suited for a complete analysis of a lumped as well as distributed element, one-dimensional, linear dynamical system with a marked chain topology. However, general subroutines of the type available for classical matrix methods are not available in the current literature on transfer matrix methods. In the present article, general expressions for various aspects of analysis-viz., natural frequency equation, modal vectors, forced response and filter performance—have been evaluated in terms of a single parameter, referred to as velocity ratio. Subprograms have been developed for use with the transfer matrix method for the evaluation of velocity ratio and related parameters. It is shown that a given system, branched or straight-through, can be completely analysed in terms of these basic subprograms, on a stored program digital computer. It is observed that the transfer matrix method with the velocity ratio approach has certain advantages over the existing general matrix methods in the analysis of one-dimensional systems.

Review of continuum, finite element and hybrid techniques in the analysis of stress concentrations in structures

When a uniform flow of any nature is interrupted, the readjustment of the flow results in concentrations and rare-factions, so that the peak value of the flow parameter will be higher than that which an elementary computation would suggest. When stress flow in a structure is interrupted, there are stress concentrations. These are generally localized and often large, in relation to the values indicated by simple equilibrium calculations. With the advent of the industrial revolution, dynamic and repeated loading of materials had become commonplace in engine parts and fast moving vehicles of locomotion. This led to serious fatigue failures arising from stress concentrations. Also, many metal forming processes, fabrication techniques and weak-link type safety systems benefit substantially from the intelligent use or avoidance, as appropriate, of stress concentrations. As a result, in the last 80 years, the study and and evaluation of stress concentrations has been a primary objective in the study of solid mechanics. Exact mathematical analysis of stress concentrations in finite bodies presents considerable difficulty for all but a few problems of infinite fields, concentric annuli and the like, treated under the presumption of small deformation, linear elasticity. A whole series of techniques have been developed to deal with different classes of shapes and domains, causes and sources of concentration, material behaviour, phenomenological formulation, etc. These include real and complex functions, conformal mapping, transform techniques, integral equations, finite differences and relaxation, and, more recently, the finite element methods. With the advent of large high speed computers, development of finite element concepts and a good understanding of functional analysis, it is now, in principle, possible to obtain with economy satisfactory solutions to a whole range of concentration problems by intelligently combining theory and computer application. An example is the hybridization of continuum concepts with computer based finite element formulations. This new situation also makes possible a more direct approach to the problem of design which is the primary purpose of most engineering analyses. The trend would appear to be clear: the computer will shape the theory, analysis and design.

Application of D transforms in the analysis of nonlinear distributed parameter systems

The scope of application of Laplace transforms presently limited to the study of linear partial differential equations, is extended to the nonlinear domain by this study. This has been achieved by modifying the definition of D transforms, put forth recently for the study of classes of nonlinear lumped parameter systems. The appropriate properties of the new D transforms are presented to bring out their applicability in the analysis of nonlinear distributed parameter systems.

Influence of anvil supports and overhangs in dynamic analysis of three point bend testing

Optimum design of dynamic fracture test rigs demands a thorough appreciation of beam vibration under impact. Analyses invariably presume rigid anvils, and neglect overhang effects. The beam response predicted analytically and numerically in this paper highlights the significant role of anvil rigidity and beam overhangs on the impact dynamics of three point bend (3PB) specimens.

Quantitative In Situ Analysis of Deformation in Sliding Metals: Effect of Initial Strain State

Using in situ, high-speed imaging of a hard wedge sliding against pure aluminum, and image analysis by particle image velocimetry, the deformation field in sliding is mapped at high resolution. This model system is representative of asperity contacts on engineered surfaces and die-workpiece contacts in deformation and machining processes. It is shown that large, uniform plastic strains of 1-5 can be imposed at the Al surface, up to depths of 500 mu m, under suitable sliding conditions. The spatial strain and strain rate distributions are significantly influenced by the initial deformation state of the Al, e.g., extent of work hardening, and sliding incidence angle. Uniform straining occurs only under conditions of steady laminar flow in the metal. Large pre-strains and higher sliding angles promote breakdown in laminar flow due to surface fold formation or flow localization in the form of shear bands, thus imposing limits on uniform straining by sliding. Avoidance of unsteady sliding conditions, and selection of parameters like sliding angle, thus provides a way to control the deformation field. Key characteristics of the sliding deformation such as strain and strain rate, laminar flow, folding and prow formation are well predicted by finite element simulation. The deformation field provides a quantitative basis for interpreting wear particle formation. Implications for engineering functionally graded surfaces, sliding wear and ductile failure in metals are discussed.

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.

Proline-containing β-turns in peptides and proteins: Analysis of structural data on globular proteins

Tetrapeptide sequences of the type Z-Pro-Y-X were obtained from the crystal structure data on 34 globular proteins, and used in an analysis of the positional preferences of the individual amino acid residues in the β-turn conformation. The effect of fixing proline as the second position residue in the tetrapeptide sequence was studied by comparing the data obtained on the positional preferences with the corresponding data obtained by Chou and Fasman using the Z-R-Y-X sequence, where no particular residue was fixed in any of the four positions. While, in general, several amino acid residues having relatively very high or very low preferences for specific positions were found to be common to both the Z-Pro-Y-X and Z-R-Y-X sequences, many significant differences were found between the two sets of data, which are to be attributed to specific interactions arising from the presence of the proline residue.

An algorithm to find distant repeats in a pair of protein sequences

Distant repeats between a pair of protein sequences can be exploited to study the various aspects of proteins such as structure-function relationship, disorders due to protein malfunction, evolutionary analysis, etc. An in-depth analysis of the distant repeats would facilitate to establish a stable evolutionary relation of the repeats with respect to their three-dimensional structure. To this effect, an algorithm has been devised to identify the distant repeats in a pair of protein sequences by essentially using the scores of PAM (Percent Accepted Mutation) matrices. The proposed algorithm will be of much use to researchers involved in the comparative study of various organisms based on the amino-acid repeats in protein sequences. (C) 2010 Elsevier B.V. All rights reserved.