Evidence for <Emphasis Type="Italic">Wolbachia</Emphasis> symbiosis in microfilariae of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> from West Bengal,India

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.     

Synthesis and biological evaluation of non-peptide alpha(v)beta(3)/alpha(5)beta(1) integrin dual antagonists containing 5,6-dihydropyridin-2-one scaffolds

Small constrained non-peptidic molecules consisting of a polyfunctionalized rigid core, carrying appendages corresponding to arginine and aspartic acid side chains, have been recently reported to be promising for drug development. In this work, the 5,6-dihydropyridin-2-one was envisaged as a scaffold to turn into potential integrin ligands, introducing a carboxylic acid and a basic appendage. The synthesis and the antiadhesion activity of a small library of peptidomimetics capable to recognize alpha(v)beta(3) and alpha(5)beta(1) integrins has been herein reported.     

BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood

By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.     

Virulence of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> using soybean oil formulation for control of the cotton stainer bug, <Emphasis Type="Italic">Dysdercus peruvianus</Emphasis>

The cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) is an insect pest that causes heavy losses in cotton plantations. The need to reduce the use of insecticides for control of this pest has increased steadily, and Metarhizium anisopliae (Ascomycota: Clavicipitaceae) could be an important biopesticide candidate to control this pest. The effect of M. anisopliae on D. peruvianus nymphs and adults using formulations with soybean oil and Agral^® was evaluated. Formulation using 10% soybean oil added to 10⁸ conidia mL^?1 (grown on used and reused rice) was the most effective for nymph and adult, causing 100% mortality 6 and 7 days after exposure, respectively. The SEM analysis of infected insects showed that M. anisopliae conidia were able to adhere anywhere on the exoskeleton, but were more abundant between the joints. Using the same rice for two batches of growth may be an option for improving commercial conidial production of M. anisopliae and may reduce overall costs. Its effect on D. peruvianus adults opens a new possibility for using this fungus as an alternative to chemical pesticides and the use of M. anisopliae in association with integrate pest management.     

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

GeMS: an advanced software package for designing synthetic genes

A user-friendly, advanced software package for gene design is described. The software comprises an integrated suite of programs—also provided as stand-alone tools—that automatically performs the following tasks in gene design: restriction site prediction, codon optimization for any expression host, restriction site inclusion and exclusion, separation of long sequences into synthesizable fragments, T_m and stem–loop determinations, optimal oligonucleotide component design and design verification/error-checking. The output is a complete design report and a list of optimized oligonucleotides to be prepared for subsequent gene synthesis. The user interface accommodates both inexperienced and experienced users. For inexperienced users, explanatory notes are provided such that detailed instructions are not necessary; for experienced users, a streamlined interface is provided without such notes. The software has been extensively tested in the design and successful synthesis of over 400 kb of genes, many of which exceeded 5 kb in length.     

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.