A bacterial invasin induces macrophage apoptosis by binding directly to ICE.

Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. Deletion mutants in the invasion invasion plasmid antigen B (ipaB) of Shigella flexneri are not cytotoxic. Here, we localized IpaB to the cytoplasm of macrophages infected with S. flexneri. Purified IpaB induced apoptosis when microinjected into macrophages, indicating that IpaB is sufficient to induce apoptosis. Using a GST-IpaB fusion protein as a ligand in affinity purification, we isolated four IpaB binding proteins from macrophages which were identified as the precursor and the mature polypeptides of interleukin-1beta converting enzyme (ICE) or a highly homologous protease. We found that IpaB binds directly to ICE and this enzyme is activated during S. flexneri infection. Furthermore, specific inhibitors of ICE prevented Shigella-induced apoptosis.     

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg¹⁸⁰–Gly¹⁸¹–Asp¹⁸² and Arg²⁶⁹–Gly²⁷⁰–Asp²⁷¹. The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.     

Statistical optimization of critical medium components for lipase production from Yarrowia lipolytica (MTCC 35)

Statistical optimization is an effective technique for the investigation of complex processes with minimal number of experimental runs. In this study, statistical approach was used to study the optimization of media components for lipase production from Yarrowia lipolytica MTCC 35. Mahua cake, glucose, MnCl₂ and KH₂PO₄ were screened to be the most significant variables among the nine medium variables that were tested to determine influence on lipase production by Plackett–Burman design. Central Composite Design was used for further optimization of these screened variables for enhanced lipase production. The determination coefficient (R²) value of 0.922 showed that the regression models adequately explain the data variation and represent the actual relationships between the variables and response. The optimum values of investigated variables for the maximum lipase production were 6.0% Mahua cake, 2.0% glucose, 0.2% MnCl₂ and 0.2% KH₂PO₄. The maximum lipase production (9.40 U mL^?1) was obtained under optimal condition.     

Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection

Bovine viral diarrhea virus (BVDV) infects cattle and may lead to persistent infection (PI). The PI animals harbor BVDV throughout their life and become immune tolerant against BVDV. Thus, diagnosis of this virus in herd is highly important. Recombinant E2 protein expression (using pET-32a in Escherichia coli) was confirmed by SDS-PAGE and Western blotting; then purified by Ni⁺ affinity chromatography. Chickens were immunized with BVDV-E2 protein, and IgY antibodies were extracted from egg yolk by PEG-6000. The peak titer of anti-BVDV-E2-IgY was 1:128,000 after the fifth immunization. IgY-based enzyme-linked immuno sorbent assay (ELISA) and immunochromatographic assay (ICA) were further developed. Coincidence of ELISA and ICA test with RT-PCR was 95.45 and 90.91%, respectively. The anti-BVDV-E2 IgY could be used in routine screening of BVDV infection. Besides, it can also be applicable while licensing and/or using live vaccines; screening of imported products containing bovine serum and strong surveillance of BVDV outbreaks.     

Role of counterion condensation in folding of the Tetrahymena ribozyme. I. Equilibrium stabilization by cations

Folding of RNA into an ordered, compact structure requires substantial neutralization of the negatively charged backbone by positively charged counterions. Using a native gel electrophoresis assay, we have examined the effects of counterion condensation upon the equilibrium folding of the Tetrahymena ribozyme. Incubation of the ribozyme in the presence of mono-, di- and trivalent ions induces a conformational state that is capable of rapidly forming the native structure upon brief exposure to Mg2+. The cation concentration dependence of this transition is directly correlated with the charge of the counterion used to induce folding. Substrate cleavage assays confirm the rapid onset of catalytic activity under these conditions. These results are discussed in terms of classical counterion condensation theory. A model for folding is proposed which predicts effects of charge, ionic radius and temperature on counterion-induced RNA folding transitions.     

Pair potentials for protein folding: choice of reference states and sensitivity of predicted native states to variations in the interaction schemes

We examine the similarities and differences between two widely used knowledge-based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa-Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676-688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner.     

Impaired clearance of primary but not secondary Brugia infections in IL-5 deficient mice

Eosinophilia in blood and tissues has been strongly associated with helminth infections for over a century. In vivo depletion of IL-5, a cytokine crucially involved in eosinophilopoiesis with an antibody or through genetic manipulation, reproducibly abrogates helminth-induced eosinophilia, but renders mice permissive only in some models of parasite infection. In the current study, we compared the ability of IL-5(-/-) and B6(+/+) mice to clear intraperitoneal infections with Brugia pahangi L3. IL-5(-/-) mice had statistically significantly higher worm burdens than B6(+/+). This was true for primary infections, in young as well as old mice, suggesting that IL-5 deficient mice are more permissive to Brugian infections. This increase in permissiveness seemed to correlate well with the drastically reduced eosinophil numbers in the peritoneal cavity, the site of infection. In secondary infections, primed IL-5(-/-) mice cleared infections in an accelerated manner, comparable with B6(+/+) mice. These observations suggest that IL-5 induced eosinophilia is more important in the control of a primary infection in na?ve mice than a secondary infection in primed mice.     

Charge states rather than propensity for beta-structure determine enhanced fibrillogenesis in wild-type Alzheimer's beta-amyloid peptide compared to E22Q Dutch mutant

The activity of the Alzheimer's amyloid beta-peptide is a sensitive function of the peptide's sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimer's amyloid beta-peptide relative to that of the wild-type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of beta structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Abeta(10-35)-peptide in aqueous solution. The simulation results indicate that the propensity for formation of beta-structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of "flickering" of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of beta-structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.