Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.     

Localization and verification by synthesis of five antigenic sites of bovine serum albumin.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Isolation and Molecular Identification of Fungi with Magnesite Enrichment Potential from KÜMAŞ Quarries in Turkey

Abstract

Magnesite is an important raw material used in various industrial applications, especially the production of high-temperature resistant materials. Due to its high reactant nature, magnesite ore is not found in pure form and it contains a great variety of pollutants such as calcium compounds, which restrict its use when exceeding 1% of the ore. Thus, the development of efficient strategies for the removal of pollutants remains a crucial step for magnesite utilization. In this regard, our present work was conducted to isolate and identify active fungal strains that remove calcium pollutants without changing the main magnesium content of the ore. For this aim, magnesite ore samples were collected from two quarries (Turanoca?? and Ortaocak) of KÜMA? Magnesite Inc. and fungal isolation studies were done by using the ore’s flora. Active isolates were chosen according to their CaCO₃ and MgCO₃ dissolving capabilities and identified by using conventional light microscopy and molecular characterization techniques. 71 fungal isolates were obtained from the isolation step and 14 of them were chosen as active isolates that solve calcium compounds while not affecting the magnesium component. The data of the microscopic examination and 18S rDNA gene sequence analysis showed that 14 active strains with magnesite enrichment potential grouped in Aspergillus alliaceus (3), Aspergillus flavus (2), Aspergillus leporis (1), Aspergillus nomius (1), Fusarium tricinctum (2), Penicillium chrysogenum (1) and Penicillium sp. (4).     

Molecular Characterization of Chitinase Genes from a Local Isolate of Serratia marcescens and Their Contribution to the Insecticidal Activity of Bacillus thuringiensis Strains

The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine–Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.     

Reconstructing changes in the genotype,phenotype, and climatic niche of an introduced species

An introduced species must contend with enormous environmental variation in its introduced range. In this study, we use niche models and ordination analyses to reconstruct changes in genotype, phenotype, and climatic niche of Johnsongrass Sorghum halepense, which is regarded as one of the world's most threatening invasive plants. In the United States, Johnsongrass has rapidly evolved within‐ and among‐population genetic diversity; our results show that genetic differentiation in expanding Johnsongrass populations has resulted in phenotypic variation that is consistent with habitat and climatic variation encountered during its expansion. Moreover, Johnsongrass expanded from agricultural to non‐agricultural habitat, and now, despite occupying overlapping ranges, extant agricultural and non‐agricultural populations are genetically and phenotypically distinct and manifest different plastic responses when encountering environmental variation. Non‐agricultural accessions are broadly distributed in climatic and geographic space and their fitness traits demonstrate plastic responses to common garden conditions that are consistent with local specialization. In contrast, agricultural accessions demonstrate ‘general purpose’ plastic responses and have more restricted climatic niches and geographic distributions. They also grow much larger than non‐agricultural accessions. If these differences are adaptive, our results suggest that adaptation to local habitat variation plays a crucial role in the ecology of this invader. Further, its success relates to its ability to succeed on dual fronts, by responding simultaneously to habitat and climate variability and by capitalizing on differential responses to these factors during its range expansion.     

Allyl isothiocyanate attenuates oxidative stress and inflammation by modulating Nrf2/HO-1 and NF-κB pathways in traumatic brain injury in mice

Molecular Biology Reports - Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in young adults and children in the industrialized countries; however, there are presently...     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Oxidative injury due to chronic nitric oxide synthase inhibition in rat: effect of regular exercise on the heart

Many individuals with cardiac diseases undergo periodic physical conditioning with or without medication. Therefore, this study investigated the interaction of physical training and chronic nitric oxide synthase (NOS) inhibitor (nitro-L-arginine methyl ester, L-NAME) treatment on blood pressure (BP), heart rate (HR) and cardiac oxidant/antioxidant systems in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control (SC), (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, s.c. for 8 weeks) and (4) ET+L-NAME. BP and HR were monitored with tail-cuff method. The animals were sacrificed 24 h after last treatments and hearts were isolated and analyzed. Physical conditioning significantly increased respiratory exchange ratio (RER), cardiac nitric oxide (NO) levels, NOS activity and endothelial (eNOS) and inducible (iNOS) protein expression. Training significantly enhanced cardiac glutathione (GSH) levels, GSH/GSSG ratio and up-regulation of cardiac copper/zinc-superoxide dismutase (CuZn-SOD), manganese (Mn)-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) activity and protein expression. Training also caused depletion of cardiac malondialdehyde (MDA) and protein carbonyls. Chronic L-NAME administration resulted in depletion of cardiac NO level, NOS activity, eNOS, nNOS and iNOS protein expression, GSH/GSSG ratio and down-regulation of cardiac CuZn-SOD, Mn-SOD, CAT, GSH-PX, glutathione-S-transferase (GST) activity and protein expression. Chronic L-NAME administration enhanced cardiac xanthine oxidase (XO) activity, MDA levels and protein carbonyls. These biochemical changes were accompanied by increases in BP and HR after L-NAME administration. Interaction of training and NOS inhibitor treatment resulted in normalization of BP, HR and up-regulation of cardiac antioxidant defense system. The data suggest that physical conditioning attenuated the oxidative injury caused by chronic NOS inhibition by up-regulating the cardiac antioxidant defense system and lowering the BP and HR in rats.     

Vasodilatory activity in horsefly and deerfly salivary glands

Salivary gland extract (SGE) of four horsefly species (Hybomitra bimaculata Macquart, Hybomitra ciureai Séguy, Tabanus bromius L., Tabanus glaucopis Meigen) and one deerfly species (Chrysops relictus Meigen) (Diptera: Tabanidae) were shown to contain vasodilatory activity. Aliquots equivalent to 1, 5 and 10 pairs of salivary glands (SG) relaxed rat femoral artery (with intact endothelium) pre-constricted with phenylephrine. Vasodilatory activity was dose-dependent. SGE of one horsefly species (Haematopota pluvialis L.) did not induce relaxation. The kinetics of vasodilation induced by SGE of four horsefly species differed from the deerfly. These results indicate that tabanid species may produce more than one type of vasodilator to aid blood feeding.