Effects of pH on the activity and structure of choline oxidase from Alcaligenes species

A reversible effect of pH on the ionization of amino-acid residues at the active center of choline oxidase was observed near the optimum pH (8). Inactivation of choline oxidase took place in the pH ranges 3-6 and 9-11, in which irreversible changes in the structure occur leading to the enzyme inactivation. The first order rate constants of the enzyme's inactivation at various pH values were estimated for the irreversible changes. The Arrhenius analysis revealed no significant changes in the activation enthalpy, while an increase in the activation entropy reflected an increase in the conformational freedom.     

Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its role in the pathogenesis of many diseases. Here, we report attempts to improve soluble expression and purification of hexahistidyl-tagged recombinant human IDO from Escherichia coli (EC538, pREP4, and pQE9-IDO). Significant formation of inclusion bodies was noted at the growth temperature of 37 degrees C, with reduced formation at 30 degrees C. The addition of the natural biosynthetic precursor of protoporphrin IX, delta-aminolevulinic acid (ALA), coupled with optimisation of IPTG induction levels during expression at 30 degrees C and purification by nickel-agarose and size exclusion chromatography, resulted in protein with 1 mol of heme/mol of protein and a specific activity of 160 micromol of kynurenine/h/mg of protein (both identical to native human IDO). The protein was homogeneous in terms of electrophoretic analysis. Yields of soluble protein (3-5 mg/L of bacterial culture) and heme content are greater than previously reported.     

Chemical modification of lysine residues in Bacillus licheniformis alpha-amylase: conversion of an endo- to an exo-type enzyme

The lysine residues of Bacillus licheniformis alpha-amylase (BLA) were chemically modified using citraconic anhydride or succinic anhydride. Modification caused fundamental changes in the enzymes specificity, as indicated by a dramatic increase in maltosidase and a reduction in amylase activity. These changes in substrate specificity were found to coincide with a change in the cleavage pattern of the substrates and with a conversion of the native endo- form of the enzyme to a modified exo- form. Progressive increases in the productions of rho-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble starch, respectively, was used as substrate, were observed upon modification. The described changes were affected by the size of incorporated modified reagent: citraconic anhydride was more effective than succinic anhydride. Reasons for the observed changes are discussed and reasons for the effectivenesses of chemical modifications for tailoring enzyme specificities are suggested.     

Localized Surface Plasmon Resonance (LSPR)-Based Nanobiosensor for Methamphetamin Measurement

Aptamers are DNA or RNA single-stranded molecules that bind specifically to target molecules with high affinity. Function of nucleic acid aptamers is based on organized tertiary structure of them that is related to primary sequence, length of nucleic acid molecule, and environmental conditions. Herein, a localized surface plasmon resonance (LSPR) nanobioprobe has been developed based on specific aptamer-conjugated gold nanoparticles for rapid detection of methamphetamine. Detection of methamphetamine was studied via monitoring the gold nanoparticles (GNPs) LSPR band alterations in the presence of different concentrations. The covalent conjugation has been confirmed with FT-IR spectroscopy, and size alterations of gold nanoparticles before and after the conjugation state were monitored using dynamic light scattering (DLS) technique. The results show high affinity of aptamer to methamphetamine. Moreover, the results show conjugated aptamer with GNP in different concentrations of methamphetamine that contribute to color changes that is visible with unaided eye. Also, 14 nm LSPR shift was seen after conjugation of aptamer with GNP. Nanoparticle diameter after conjugation with aptamer was increased from 30 to 91 nm and decreased after incubation with methamphetamine (due to folding) from 91 to 84 nm. Detection limit of this designed nanoprobe is 500 nM. Plasmonic nanoparticle-based nanobioprobe is a new field for development of sensitive detection systems.

     

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.     

Bioassay evaluation of the entomopathogenic fungus,Lecanicillium longisporum (Petch) Zare & Gams against egg and nymphs of Trialeurodes vaporariorum Westwood in laboratory conditions

Abstract

This study was carried out to determine the lethality of the entomopathogenic fungus, Lecanicillium longisporum on eggs, young and old nymphs of the greenhouse whitefly, Trialeurodes vaporariorum. Mortality percentage was significantly differed based on stage of T. vaporariorum and conidial concentrations of L. longisporum. Average of the infection level to insect was very low particularly in egg with only 9.81%, even with higher conidial concentrations (1×10⁷ conidia mL^-1). Whereas, it was higher in 1^st and 2^nd instar (46.56%) and 3^rd and 4^th instars (37.21%). Three parameters were assessed with T. vaporariorum eggs, namely; egg infection, egg hatchability and crawlers emergence. Egg mortality percentages averaged 3.56, 7.14, 9.64, 16.42 and 20.35% with fungal concentrations of 1×10³, 1×10⁴, 1×10⁵, 1×10⁶ and 1×10⁷ conidia mL^-1, respectively. Daily infection percentages were varied depend upon the conidia concentration where the highest infection rate of eggs was occurred with 1×10⁷. Egg hatch was very high and the mortality among the emerged crawlers was neglectable compared with the control. Efficiency of L. longisporum on whitefly nymphs also was varied based on the insect instar and fungal concentration. Mortality percentages were obviously higher in young nymphs (1^st and 2^nd instars) than in older ones (3^rd and 4^th instars). The results indicated that nymphs were highly susceptible to fungal treatment compared with eggs.     

Maternal zinc intake of Wistar rats has a protective effect in the alloxan-induced diabetic offspring

Zinc has a role in the synthesis, storage, and secretion of insulin, and has been suggested to be beneficial when used in the diabetic state. Effect of zinc intake in pregnant rats has been studied here on diabetized offspring. Pregnant rats were divided in two groups; the control group received normal food and water, and the experimental group received zinc sulfate during pregnancy and 3 weeks after offspring birth. Male offspring from the control (C) and experimental (E) groups were divided each in three groups: C1, fed with normal food and water; C2, diabetized with alloxan; C3, received zinc sulfate; E1, fed with normal food and water; E2, diabetized with alloxan; and E3, receiving zinc sulfate. After 30 days, the histological changes of pancreatic tissues were investigated by light microscopy. Body weight, blood glucose, serum insulin levels, food intake, water intake, and urine quantity were also compared between the groups. Water intake and urine quantity were decreased significantly (p?<?0.01and p?<?0.001) in E2 (experimental diabetic group) in comparison with C2 (control diabetic group), but there was no significant difference in the body weight in C2 in comparison with E2, while blood glucose was decreased significantly (p?<?0.001) and blood insulin level was increased significantly (p?<?0.01) in E2 in comparison with C2. Microscopic evaluation of pancreas showed that E2 were protected against alloxan-induced beta-cell degeneration. In conclusion, this work showed that maternal zinc intake may influence subsequent deleterious effects of diabetes on alloxan-diabetized offspring.     

The discovery of novel isoflavone pan peroxisome proliferator-activated receptor agonists

Twenty three dual PPARα and γ molecules of natural product origin, previously reported by our group, were further investigated for pan PPAR transactivation against PPARδ. The in vitro cell toxicity profile, as well as, in silico study of the most active molecules within this new class of pan PPAR agonists are also described. 3′,5′ Dimethoxy-7 hydroxyisoflavone 6, Ψ-baptigenin 7, 4′ fluoro-7 hydroxyisoflavone 8, and 3′ methoxy-7 hydroxyisoflavone 9 were identified as the most potent molecules studied within the set compared to the commercially available pan PPAR agonist, bezafibrate 1. These novel active molecules may thus be useful as future leads in PPAR-related disorders, including type II diabetes mellitus and metabolic syndrome.