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.     

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.     

Melanocyte–keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes

Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte–keratinocyte co‐cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a ‘ligand‐receptor’ type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.     

Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.     

Mass transfer studies of cell permeabilization and recovery of alkaline phosphatase from Escherichia coli by reverse micellar solutions

Transfer of alkaline phosphatase (AP) directly from Escherichia coli cells into reverse micellar solutions (RMS) was studied by varying the water content, pH, and ionic strength of RMS. Prior to the mass transfer studies, the optimum conditions for the activity of alkaline phosphatase in reverse micellar solutions were determined. The maximum enzyme activity could be detected at higher pH and water content, indicating the ionization of p-nitrophenol to be crucial in the enzyme activity. The transfer of AP from E. coli followed a two-step process with most of the recovery being achieved in the first 3-10 min beyond which it slowed. A model suggesting separate locations for the enzyme in the cell wall has been proposed.     

The close relationship between estimated divergent selection and observed differentiation supports the selective origin of a marine snail hybrid zone

To study the role of divergent selection in the differentiation of the two morphs in a hybrid zone of the intertidal snail Littorina saxatilis, we compared the strength of the divergent selection acting on a series of shell characters (as estimated by the viability of snails in a reciprocal transplant experiment) with the contribution of these characters to the phenotypic differences between the morphs. We found a close correlation between selection and differentiation, which suggests a cause-effect relationship, i.e. that all present differentiation is the result of past divergent selection. In addition, divergent selection was a very important component of the total natural selection acting on shell measures. These novel results support previous evidence, based on allozyme analysis, of a parapatric origin for this hybrid zone. We discuss possible limitations of this interpretation and the circumstances under which allopatric differentiation would produce the same results. Phenotypic analysis of divergent selection may be a useful method of investigating the evolutionary mechanisms involved in differentiation processes.     

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.