Ouabain-binding site of (Na+ + K+)-ATPase in right-side-out vesicles has not an externally accessible SH group

The fluorescing sulfhydryl reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) inactivates purified (Na+ + K+)-ATPase at 20 microM. This inactivation results in a decrease of the ouabain-binding capacity of the enzyme. Treatment of (Na+ + K+)-ATPase, embedded in right-side-out-oriented vesicles, by DACM does not affect ouabain binding to the enzyme. Incorporation of DACM into the alpha subunit of (Na+ + K+)-ATPase embedded in right-side-out vesicles is also not affected by the presence or absence of 100 microM ouabain. It is therefore concluded that a sulfhydryl group does not reside within the ouabain-binding site of (Na+ + K+)-ATPase.     

Evidence that Aspartate Aminotransferase Activity and Ketodicarboxylate Carrier Function Are Essential for Biosynthesis of Transmitter Glutamate

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.     

Killer toxin-like chitinases in filamentous fungi: Structure,regulation and potential roles in fungal biology

Fungal chitinases are hydrolytic enzymes responsible for degradation of chitin. Chitinases are involved in several aspects of fungal biology, including cell wall remodelling during hyphal growth, conidial germination, autolysis, mycoparasitism and nutrient acquisition. They are divided into three distinct phylogenetic groups; A, B and C. Chitinases from the C group show structural similarities with the killer toxin zymocin produced by the yeast Kluyveromyces lactis and it is speculated that they have a similar function in filamentous ascomycetes, by facilitating penetration of toxins into cells of competing individuals. Genome analyses show that certain fungal species with a mycoparasitic lifestyle contain high numbers of killer toxin-like chitinases, compared with specialized saprotrophs and plant pathogens. Recent developments within this research field have revealed considerable variation in the modular structure and regulation of killer toxin-like chitinases, suggesting more diverse roles than merely fungal-fungal interactions. In this review, we summarize the current knowledge about this intriguing class of chitinases, including their modular structure, evolution, gene regulation, and functional analyses in mycoparasitic as well as in saprotrophic species. We also propose important questions for future research.     

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12?836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.     

Characterization of the soluble nanoparticles formed through Coulombic interaction of bovine serum albumin with anionic graft copolymers at low pH

A static light scattering (SLS) study of bovine serum albumin (BSA) mixtures with two anionic graft copolymers of poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulphonate)-graft-poly(N,N-dimethylacrylamide), with a high composition in poly(N,N-dimethylacrylamide) (PDMAM) side chains, revealed the formation of oppositely charged complexes, at pH lower than 4.9, the isoelectric point of BSA. The core-corona nanoparticles formed at pH = 3.00 were characterized. Their molecular weight and radius of gyration were determined by SLS, while their hydrodynamic radius was determined by dynamic light scattering. Small angle neutron scattering measurements were used to determine the radius of the insoluble complexes, comprising the core of the particles. The values obtained indicated that their size and aggregation number of the nanoparticles were smaller when the content of the graft copolymers in neutral PDMAM side chains was higher. Such particles should be interesting drug delivery candidates, if the gastrointestinal tract was to be used.     

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.