Efficient synthesis and biological evaluation of proximicins A, B and C

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.     

Both the 5' and 3' LTRs of FIV contain minor RNA encapsidation determinants compared to the two core packaging determinants within the 5' untranslated region and gag

This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.     

Microstructural mechanics of collagen gels in confined compression: poroelasticity, viscoelasticity, and collapse

BACKGROUND: Collagen gels are important as platforms for in vitro study of cell behavior and as prototypical bioartificial tissues, but their mechanical behavior, particularly on the microscopic scale, is still poorly understood. METHOD OF APPROACH: Collagen gels were studied in step (10% strain in 0.05 s) and ramp (0.1%/s strain rate for 100 s) confined compression. Real-time birefringence mapping gave the local collagen concentration and orientation along with piston stress. Variations in the retardation allowed material-point tracking and qualitative determination of the strain distribution. RESULTS: Ramp tests showed classical poroelastic behavior: compression near the piston and relaxation to a uniform state. Step tests, however, showed an irreversibly collapsed region near the piston. CONCLUSIONS: Our results suggest that interstitial flow and fibril bending at crosslinks are the dominant mechanical processes during compression, and that fibril bending is reversible before collapse.     

Flux balance analysis of mycolic acid pathway: targets for anti-tubercular drugs

Mycobacterium tuberculosis is the focus of several investigations for design of newer drugs, as tuberculosis remains a major epidemic despite the availability of several drugs and a vaccine. Mycobacteria owe many of their unique qualities to mycolic acids, which are known to be important for their growth, survival, and pathogenicity. Mycolic acid biosynthesis has therefore been the focus of a number of biochemical and genetic studies. It also turns out to be the pathway inhibited by front-line anti-tubercular drugs such as isoniazid and ethionamide. Recent years have seen the emergence of systems-based methodologies that can be used to study microbial metabolism. Here, we seek to apply insights from flux balance analyses of the mycolic acid pathway (MAP) for the identification of anti-tubercular drug targets. We present a comprehensive model of mycolic acid synthesis in the pathogen M. tuberculosis involving 197 metabolites participating in 219 reactions catalysed by 28 proteins. Flux balance analysis (FBA) has been performed on the MAP model, which has provided insights into the metabolic capabilities of the pathway. In silico systematic gene deletions and inhibition of InhA by isoniazid, studied here, provide clues about proteins essential for the pathway and hence lead to a rational identification of possible drug targets. Feasibility studies using sequence analysis of the M. tuberculosis H37Rv and human proteomes indicate that, apart from the known InhA, potential targets for anti-tubercular drug design are AccD3, Fas, FabH, Pks13, DesA1/2, and DesA3. Proteins identified as essential by FBA correlate well with those previously identified experimentally through transposon site hybridisation mutagenesis. This study demonstrates the application of FBA for rational identification of potential anti-tubercular drug targets, which can indeed be a general strategy in drug design. The targets, chosen based on the critical points in the pathway, form a ready shortlist for experimental testing.     

Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases

Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal alphaC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases.