The in vitro antioxidant capacities of Polianthes tuberosa L. flower extracts

This study was designed to examine the chemical compositions of scent volatiles and antioxidant activities of Polianthes tuberosa L. flower extract in six different solvents. The main constituents of the volatile components were benzyl benzoate, methyl 2-amino benzoate, methyl isoeugenol, isoeugenol, benzyl salicylate, methyl salicylate, geraniol and 1,8-cineole. Total phenolic content of floral extracts in water, methanol, ethanol, ethyl acetate, hexane and dichloromethane were found to be 0.094, 0.18, 0.14, 0.007, 0.004 and 0.110 mg gallic acid equivalent/mg fresh weight, respectively. The methanol soluble fraction showed highest values of antioxidant activity through DPPH and ABTS assays. Methanol extract effectively inhibits the non site-specific DNA strand breakage caused by Fenton’s reagents. Dichloromethane and aqueous fractions also exhibited high antioxidant capacities. Aqueous extract showed highest value in FRAP assay.     

Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein

The unresolved mechanism by which a single strand of DNA recognizes homology in duplex DNA is central to understanding genetic recombination and repair of double-strand breaks. Using stopped-flow fluorescence we monitored strand exchange catalyzed by E. coli RecA protein, measuring simultaneously the rate of exchange of A:T base pairs and the rates of formation and dissociation of the three-stranded intermediates called synaptic complexes. The rate of exchange of A:T base pairs was indistinguishable from the rate of formation of synaptic complexes, whereas the rate of displacement of a single strand from complexes was five to ten times slower. This physical evidence shows that a subset of bases exchanges at a rate that is fast enough to account for recognition of homology. Together, several studies suggest that a mechanism governed by the dynamic structure of DNA and catalyzed by diverse enzymes underlies both recognition of homology and initiation of strand exchange.     

Investigation of human serum albumin (HSA) binding specificity of certain photosensitizers related to pyropheophorbide-a and bacteriopurpurinimide by circular dichroism spectroscopy and its correlation with in vivo photosensitizing efficacy

A series of pyropheophorbide-a and bacteriopurpurinimides were investigated to understand the correlation between HSA (site II) binding affinity and in vivo photosensitizing activity. In our study, photosensitizers that bound to site II of HSA produced a significant difference in the circular dichroism spectra of the corresponding complexes, especially at Soret band region of the photosensitizers. Our results suggest that CD spectroscopy of the photosensitizer-HSA complexes could be a valuable tool in screening new photosensitizers before evaluating them for in vivo efficacy.     

Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

COX-2 in inflammation and resolution

Aspirin and the other NSAIDs have popularized the notion of inhibiting prostaglandins as a common anti-inflammatory strategy based on the erroneous premise that all eicosanoids are, within the context of inflammation, generally detrimental. However, our fascination with aspirin and the emergence of COX-2 has shown a more affable side to lipid mediators based on our increasing interest in the endogenous control of acute inflammation and in factors that mediate its resolution. Epilipoxins, for instance, are produced from aspirin's acetylation of COX-2 and together with Resolvins and COX-2-derived prostaglandins of the D(2) and J(2) series represent an increasingly important family of immunoregulatory lipid mediators with strong implications for disease control and drug discovery.     

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

Polymorphism in the intermediates and products of amyloid assembly

Amyloid formation reactions exhibit two classes of polymorphisms: the metastable intermediates commonly observed during amyloid formation and the range of conformationally distinct mature fibrils often seen at the reaction endpoint. Although recent data suggest that spherical oligomers and protofibrils in most cases are not obligate intermediates of amyloid assembly, oligomeric states might sometimes serve as on-pathway intermediates. Mature amyloid polymorphs self-propagate as a result of the normally very high fidelity of amyloid elongation, giving rise to strain behavior and species barriers in prion phenomena. Oligomers, protofibrils and various polymorphic forms of mature amyloid fibrils seem to be distinguished by differences in atomic structure that give rise to differences in observed morphologies.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.