A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.     

Exploration of highly selective fluorogenic ‘on–off' chemosensor for H2PO4− ions: ICT‐based sensing and ATPase activity profiling

Abstract In this study, the recognition contour of Chemosensor 1 was investigated using semiaqueous methanol (X_H, mole fraction = 0.31) for a range of anions and bioactive species. Host–receptor signalling based on the internal charge transfer mechanism for Chemosensor 1 was explored and reported. Structure of Chemosensor 1 and its plausible anion coordination based on hydrogen bonding is complemented with density functional theory. Consequently, we investigated the applicability of the synthesized probe in blood plasma, urine, tap water samples, and for monitoring of ATP in lysosomes by apyrase enzyme.     

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

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Melanosomal proteins promote melanin polymerization

In melanocytes, enzymes involved in the generation of melanin monomers are present and active in coated vesicles which are known to be acidic. Melanin polymerization however, occurs only in melanosomes. In vitro, it is not possible to generate melanin at the acidic pH of melanosomes using 3,4-dihydroxyphenylalanine (DOPA) and tyrosinase alone whereas melanin readily forms at higher pH with these reagents. Dimerization and elongation of the melanin polymer is known to require deprotonation. We have hypothesized that the amino acid side chains of melanosomal proteins act as proton acceptors to initiate polymerization and that the protonated basic groups serve to attract the negatively charged oligomers thus aiding polymerization and binding to proteins. We show that basic model proteins and basic premelanosomal proteins promote polymerization at an acidic pH and that positively charged surfaces allow binding of the growing melanin polymer. With progressive polymerization and exhaustion of the proton abstracting ability of melanosomal proteins, melanosomal pH drops further, which, we argue, is an additional controlling step that limits tyrosinase activity and melanin polymerization.     

Transfecting pDNA to E. coli DH5α using bovine serum albumin nanoparticles as a delivery vehicle

We describe the formulation of bovine serum albumin nanoparticles (BSA‐NPs) by the coacervation method using surfactants. Plasmids (pUC18, pUC18egfp and pBBR1MCS‐2) isolated from E. coli were incorporated into the BSA matrix by incubating in albumin solution prior to formulation of NPs. Plasmid incorporation was calculated by % yield, entrapment efficiency, DNA loading capacity and release of entrapped DNA by comparing with blank NPs. BSA‐DNA binding studies were carried out by using fluorescence spectroscopy and Fourier Transform Infra Red Spectroscopy (FT‐IR). The surface charge distribution of the NPs loaded with plasmid was calculated using zeta potential. The photoluminescence of BSA‐NPs was quenched when loaded with pDNA, confirming the interaction of DNA with BSA. Altogether, these results provide evidences for the excellent DNA carrying efficiency of BSA‐NPs without loss of plasmid's integrity. The NPs were used to transfect E. coli DH5α strain lacking ampicillin resistance. They, however, showed ampicillin resistance subsequent to transfection with plasmid encoding ampicillin resistance gene. Effect of transfection was confirmed by confocal microscopy and by the isolation of the plasmid by agarose gel electrophoresis from the transfected bacterial culture. This study clearly demonstrates the efficacy of BSA‐NPs as delivery vehicle for pDNA transfection. Copyright © 2014 John Wiley & Sons, Ltd.     

Chondroprotective potential of root extracts of <Emphasis Type="Italic">Withania somnifera</Emphasis> in osteoarthritis

This is the first report describing two novel chondroprotective activities of aqueous extracts of Withania somnifera root powder. First, these extracts had a statistically significant, short-term chondroprotective effect on damaged human osteoarthritic cartilage matrix in 50% of the patients tested. Second, these extracts caused a significant and reproducible inhibition of the gelatinase activity of collagenase type 2 enzyme in vitro.     

Evidence for a Critical Role of Catecholamines for Cardiomyocyte Lineage Commitment in Murine Embryonic Stem Cells

Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and β-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and β-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.