Synthesis,characterization and thermoluminescence studies of Mn‐doped ZnS nanoparticles

ZnS:Mn nanoparticles were prepared by a chemical precipitation method and characterized by X‐ray diffraction (XRD), field emission gun scanning electron microscope (FEGSEM), and high resolution transmission electron microscopy (HRTEM). Capping agent (mercaptoethanol) concentrations used were 0 M, 0.005 M, 0.01 M, 0.015 M, 0.025 M, 0.040 M, and 0.060 M, and resulted in nanoparticles sizes of 2.98 nm, 2.9 nm, 2.8 nm, 2.7 nm, 2.61 nm, 2.2 nm and 2.1 nm, respectively. The thermoluminescence (TL) glow curve was recorded by heating the sample exposed to UV‐radiation, at a fixed heating rate 1°C sec^–1. The TL intensity initially increased with temperature, attained a peak value I_m for a particular temperature, and then decreased with further increase in temperature. The peak TL intensity increased with decreasing nanoparticle size, whereas the temperature corresponding to the peak TL intensity decreased slightly with reducing nanocrystal size. As a consequence of increase in surface‐to‐volume ratio and increased carrier recombination rates, the TL intensity increased with decreasing nanoparticle size. It was found that, whereas activation energy slightly decreased with decreasing nanoparticle size, the frequency factor decreased significantly with reduction in nanoparticle size. Copyright © 2015 John Wiley & Sons, Ltd.     

Basal insulin, glucagon, and growth hormone replacement

Conclusions drawn from the pancreatic (or islet) clamp technique (suppression of endogenous insulin, glucagon, and growth hormone secretion with somatostatin and replacement of basal hormone levels by intravenous infusion) are critically dependent on the biological appropriateness of the selected doses of the replaced hormones. To assess the appropriateness of representative doses we infused saline alone, insulin (initially 0.20 mU.kg(-1).min(-1)) alone, glucagon (1.0 ng.kg(-1).min(-1)) alone, and growth hormone (3.0 ng.kg(-1).min(-1)) alone intravenously for 4 h in 13 healthy individuals. That dose of insulin raised plasma insulin concentrations approximately threefold, suppressed glucose production, and drove plasma glucose concentrations down to subphysiological levels (65 +/- 3 mg/dl, P < 0.0001 vs. saline), resulting in nearly complete suppression of insulin secretion (P < 0.0001) and stimulation of glucagon (P = 0.0059) and epinephrine (P = 0.0009) secretion. An insulin dose of 0.15 mU.kg(-1).min(-1) caused similar effects, but a dose of 0.10 mU.kg(-1).min(-1) did not. The glucagon and growth hormone infusions did not alter plasma glucose levels or those of glucoregulatory factors. Thus, insulin "replacement" doses of 0.20 and even 0.15 mU.kg(-1).min(-1) are excessive, and conclusions drawn from the pancreatic clamp technique using such doses may need to be reassessed.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Use of Neurospora spore killer strains to obtain centromere linkage data without dissecting asci

Use of a centromere-linked Spore killer gene Sk reduces manyfold the labor involved in obtaining tetrad data that would otherwise require ordered dissection of intact linear eight-spored asci. Heterozygous crosses are made for Spore killer (SkK X SkS) and for markers to be tested. In such crosses only SkK ascospores survive. The four viable (SkK) and four aborted (SkS) ascospores of each ascus are ejected from the perithecium as a physically disordered group. The four surviving SkK ascospores of individual asci are germinated and scored. SkK segregates from SkS at the first meiotic division. If both marker alleles are represented in the surviving products, they must therefore have segregated from one another at the second division. Four-spore (Fsp) genes have been used to eliminate one postmeiotic nuclear division, so that only two ascospores per ascus need to be scored. The Spore killer method has been useful for mapping closely linked genes in centromere regions, for identifying genes that are far out on chromosome arms, for obtaining information on meiotic crossing-over, and for comparing linkages in different species.     

A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI,the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Background

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca²⁺.

Methodology

Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.

Principal Findings

NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.

Conclusions/Significance

We propose that W542 and Ca²⁺ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca²⁺ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.     

Synthesis,Characterization, and Biological Activities of Pendant Arm‐Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and Cytotoxic Studies

2‐(1H‐Tetrazol‐5‐yl)pyridine ( L ) has been reacted separately with Me₂NCH₂CH₂Cl?HCl and ClCH₂CH₂OH to yield two regioisomers in each case, N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanamine ( L1 )/N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanamine ( L2 ) and 2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanol ( L3 )/2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanol ( L4 ), respectively. These ligands, L1 – L4 , have been coordinated with CuCl₂?H₂O in 1 : 1 composition to furnish the corresponding complexes 1 – 4 . EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar geometry, with nuclear hyperfine spin 3/2. Single X‐ray crystallographic studies of 3 revealed that the Cu center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption; viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double‐stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various conditions, where cleavage of DNA takes place by oxidative free‐radical mechanism (OH ^? ). In vitro anticancer activities of the complexes against MCF‐7 (human breast adenocarcinoma) cells revealed that the complexes inhibit the growth of cancer cells. The IC₅₀ values of the complexes showed that Cu complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin.     

Specific domains of plant defensins differentially disrupt colony initiation,cell fusion and calcium homeostasis in Neurospora crassa

MsDef1 and MtDef4 from Medicago spp. are small cysteine‐rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ‐core motif (GXCX_3–9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ‐core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ‐core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca²⁺ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca²⁺ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca²⁺ were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ‐core motifs play important roles in their different modes of antifungal action.     

Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiol-mediated neuroprotection

In the present study, we tested the hypothesis that 17beta-estradiol (betaE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H(2)O(2))-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both betaE2 and 17alpha-estradiol (alphaE2) significantly protected against H(2)O(2)-induced retinal neuron degeneration; however, progesterone had no effect. betaE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [(32)gammaATP] were used as substrate. Phospho-Akt levels were also transiently increased by betaE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of betaE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of betaE2, suggesting that betaE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that betaE2 and alphaE2, but not progesterone, identified phosphorylated insulin receptor beta-subunit (IRbeta) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRbeta activation of PI3K. Systemic administration of betaE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of betaE2 activated retinal IRbeta, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.