Bicyclic peptidomimetic tetrahydrofuro[3,2-b]pyrrol-3-one and hexahydrofuro[3,2-b]pyridine-3-one based scaffolds: synthesis and cysteinyl proteinase inhibition

A stereoselective synthesis of (3aS,6aR)-tetrahydrofuro[3,2-b]pyrrol-3-ones and (3aS,7aR)-hexahydrofuro[3,2-b]pyridine-3-ones has been developed through Fmoc protected scaffolds 12 and 13. A key design element within these novel bicyclic scaffolds, in particular the 5,5-fused system, was the inherent stability of the cis-fused geometry in comparison to that of the corresponding trans-fused. Since the bridgehead stereocentre situated beta to the ketone was of a fixed and stable configuration, the fact that cis ring fusion is both kinetically and thermodynamically stable with respect to trans ring fusion provides chiral stability to the bridgehead stereocentre that is situated alpha to the ketone. To exemplify this principle, building blocks 12 and 13 were designed, prepared and utilised in a solid phase combinatorial synthesis of peptidomimetic inhibitors 10, 45a-e, 11 and 46. Both series were chirally stable with 5,5-series 10 and 45a-e exhibiting potent in vitro activity against a range of CAC1 cysteinyl proteinases. Compound 10, a potent and selective inhibitor of cathepsin K, possessed good primary DMPK properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.     

A halotolerant and thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. degrades hydrocarbons and produces tensio-active emulsifying agent

A Bacillus sp. strain DHT, isolated from oil-contaminated soil, grew and produced biosurfactant when cultured in variety of substrate at salinities of up to 100 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, various pure alkanes and PAHs as a sole carbon and energy source across a wide range of temperature and salinity. Over the range evaluated, the degradation of hydrocarbon and biosurfactant production was not influenced by salinity (0–10% wv⁻¹) and temperature (30–45°C). The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as the best substrate and toluene as the poorest. From 16S rDNA analysis, strain DHT was related to Bacillus licheniformis.     

Development and comparison of ELISA and PCR methods for the early detection of Ganoderma disease of coconut

Abstract

Molecular diagnosis, chemo-diagnosis and physiological parameter have been applied for detecting the Ganoderma disease of coconut. Polyclonal antibodies (PAbs) raised against mycelial protein of Ganoderma, specific mycelial protein (62 kDa) of Ganoderma isolates and basidiocarp protein of Ganoderma were used for detection. All the PAbs could detect Ganoderma in diseased coconut root tissues in early stage of the disease before symptom expression by indirect – ELISA at the antiserum dilution of 1:1000 for mycelial protein, 1:700 for specific protein and 1:3000 for basidiocarp protein. Low cross reactions were observed with saprophytic fungi occurring in coconut roots and also with other basidiomycetous fungi. For polymerase chain reaction tests, the primer was generated from the internal transcribed spacer region one (ITS 1) of rDNA of Ganoderma, which produced a PCR product of 167 bp in size. Utility of this method was confirmed at the field level.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.     

A structural basis for the allosteric regulation of non-hydrolysing UDP-GlcNAc 2-epimerases

The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.