The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Annual TSP and Trace Metal Distribution in the Urban Atmosphere of Islamabad in Comparison with Mega-Cities of the World

The objective of this work was to assess the current concentrations of total suspended particles (TSP) and trace metals in the urban atmosphere of Islamabad, Pakistan, where local industries, transportation, and urbanization are adversely affecting air quality. Air sampling was done using a high-volume sampler and the metals were estimated in TSP by Flame Atomic Absorption Spectrophotometry. An annual mean TSP of 151.9 μ g/m³ was determined for the period May 2003–April 2004, which exceeded the World Health Organization's (WHO's) primary and secondary standards of air quality. The TSP metal concentrations were compared with those reported in literature for other regions of the world and with air quality standards laid down by regulatory agencies. The levels of Na, Fe, Zn, Pb, Mn, Cr, Co, and Cd for the present study were higher than most of the levels reported for other typical industrial and urban areas of the world. The airborne Ni concentrations were higher than the WHO and the U.S. Environmental Protection Agency's standards. Cluster and Principal Component Analyses of the data were used to identify the sources of the metals and establish their mutual correlations in the local atmosphere.     

Phytotoxin Production in Bursaphelenchus xylophilus-Infected Pinus sylvestris

Our findings suggest that i) phytotoxic materials can be isolated from Bursaphelenchus xylophilus-infected Scots pine, but not from noninfected pines; ii) the phytotoxins cause wilting of 45-day-old and 2-year-old pine seedlings in a dose and species dependent manner; iii) the phytotoxins are produced early in the infection, accumulate or increase with time, and may function to suppress water transport in the tree; and iv) the phytotoxins are lipid materials of low molecular weight which are not acidic.     

In-vitro model systems to study Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design.     

Inherent properties of adenylosuccinate lyase could explain S-Ado/SAICAr ratio due to homozygous R426H and R303C mutations

Adenylosuccinate lyase (ADSL) is a homotetrameric enzyme involved in the de novo purine biosynthesis pathway and purine nucleotide cycle. Missense mutations in the protein lead to ADSL deficiency, an inborn error of purine metabolism characterized by neurological and physiological symptoms. ADSL deficiency is biochemically diagnosed by elevated levels of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado), the dephosphorylated derivatives of the substrates. S-Ado/SAICAr ratios have been associated with three phenotypic groups. Different hypotheses to explain these ratios have been proposed. Recent studies have focused on measuring activity on the substrates independently. However, it is important to examine mixtures of the substrates to determine if mutations affect enzyme activity on both substrates similarly in these conditions. The two substrates may experience an indirect communication due to being acted upon by the same enzyme, altering their activities from the non-competitive case. In this study, we investigate this hidden coupling between the two substrates. We chose two mutations that represent extremes of the phenotype, R426H and R303C. We describe a novel electrochemical-detection method of measuring the kinetic activity of ADSL in solution with its two substrates at varying concentration ratios. Furthermore, we develop an enzyme kinetic model to predict substrate activity from a given ratio of substrate concentrations. Our findings indicate a non-linear dependence of the activities on the substrate ratios due to competitive binding, distinct differences in the behaviors of the different mutations, and S-Ado/SAICAr ratios in patients could be explained by inherent properties of the mutant enzyme.     

Modulation of age‐related changes in oxidative stress markers and energy status in the rat heart and hippocampus: a significant role for ozone therapy

Oxidative stress emerges as a key player in the ageing process. Controlled ozone administration is known to promote an oxidative preconditioning or adaptation to oxidative stress. The present study investigated whether prophylactic ozone administration could interfere with the age‐related changes in the heart and the hippocampus of rats. Four groups of rats, aged about 3 months old, were used. Group 1 (Prophylactic ozone group) received ozone/oxygen mixture by rectal insufflations (0.6 mg/kg) twice/week for the first 3 months, then once/week till the age of 15 months. Group 2 (Oxygen group) received oxygen as vehicle for ozone in a manner similar to group 1. Group 3 (Aged control group) was kept without any treatment until the age of 15 months. A fourth group of rats (Adult control group) was evaluated at 3 months of age to provide baseline data. Ozone alleviated age‐associated redox state imbalance as evidenced by reduction of lipid and protein oxidation markers, lessening of lipofuscin deposition, restoration of glutathione levels in both tissues and normalization of glutathione peroxidase activity in the heart tissue. Ozone also mitigated age‐associated energy failure in the heart and the hippocampus, improved cardiac cytosolic Ca²⁺ homeostasis and restored the attenuated Na⁺, K⁺‐ATPase activity in the hippocampus of aged rats. These data provide new evidence concerning the anti‐ageing potential of prophylactic ozone administration. Copyright © 2012 John Wiley & Sons, Ltd.