Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

Analysis of genetic diversity among selected grasspea (Lathyrus sativus L.) genotypes using RAPD markers

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.     

Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide

Background  

The de novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (ΔPhe) comes in use for spawning well-defined structural motifs. Introduction of ΔPhe induces β-bends in small and 3₁₀-helices in longer peptide sequences.     

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats

Molecular Biology Reports - On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose...     

Influence of biotic and abiotic factors on the persistence of a Beauveria bassiana biopesticide in laboratory and high-rise poultry house settings

Recent studies suggest the potential for use of oil formulations of the entomopathogenic fungus, Beauveria bassiana, as residual sprays for control of house flies in poultry production facilities. The current study investigated the influence of biotic and abiotic factors on biopesticide persistence. We found that flies physically removed and deactivated conidia, with higher fly densities and greater cumulative exposure hastening the decline. Nonetheless, very low densities of viable conidia were still able to cause rapid mortality, suggesting the potential for relatively long re-treatment intervals as fly populations are controlled. Considering abiotic factors, we found that fungal spray treatments remained viable for up to 13 weeks under laboratory conditions. Periodic exposure of flies to the spray residue showed high levels of mortality, with very little decline in mortality rate over time. Equivalent treatments placed in a commercial poultry house showed much more rapid decline. One trial at the end of summer showed conidia to remain viable up to seven weeks. However, repeats during the winter months revealed decay in 1–2 weeks, with fly mortality rates influenced accordingly. The exact reasons for the more rapid decay remain unclear but could be linked to high concentrations of ammonia in the basement areas, especially during winter when ventilation is minimal. The combined data suggest the potential for adaptive treatment regimes with weekly spray intervals in conditions with very high fly populations and/or high ammonia levels, and potentially monthly spray intervals when fly populations and ammonia levels are reduced.     

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.     

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP(6)- and inositol pyrophosphate (InsP(7))-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP(6) enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.