Production,cross reactivity,and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase

Eighteen IgGl monoclonal antibodies (blabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10⁸ M^–1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the blabs (N° 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other blabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.Abbreviations ELISA enzyme linked immunosorbent assay - GGT gamma-glutamyltransferase - Mab monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Type A behavior and job performance: some suggestive findings

This study examined Type A and Type B differences in job performance, psychosomatic complaints, and career progression among white-collar employees (N = 218) in a field setting. Significant differences were found between Type A's and Type B's in quality of performance, effort exerted at the job, and psychosomatic complaints. Employees' cultural background and sex moderated some of the relationships observed in the study. Implications of the findings for future research on the topic are discussed.     

Effect of exogeneous methyl oleate on the time course of some parameters of Streptomyces hygroscopicus NRRL B-1865 culture

Addition of methyl oleate to a Streptomyces hygroscopicus NRRL B-1865 culture modified the metabolic properties of this strain. This addition decreased the pH of the medium, increased the valine uptake of the cells and reduced their consumption of glucose until the beginning of antibiotic biosynthesis, which was delayed. At the same time, an increase in growth (× 1.8) and a marked improvement in antibiotic production (× 20) could be observed. The use of labelled methyl oleate showed that methyl oleate was not a precursor of antibiotics produced by S. hygroscopicus NRRL B-1865. It is suggested that methyl oleate addition may cause some alteration in membrane permeability, inducing an increase in H⁺ extrusion and stimulating the accumulation of branched amino acids, known to be direct precursors of polyether antibiotics. Correspondence to: L. David     

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.     

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., G?tz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.     

Positive residues involved in the voltage-gating of the mitochondrial porin-channel are localized in the external moiety of the pore.

The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.     

Rhinitis,Ocular, Throat and Dermal Symptoms,Headache and Tiredness among Students in Schools from Johor Bahru,Malaysia: Associations with Fungal DNA and Mycotoxins in Classroom Dust

There are few studies on rhinitis and sick building syndrome (SBS) among students in tropical countries. We studied associations between levels of five fungal DNA sequences, two mycotoxins (sterigmatocystin and verrucarol) and cat allergen (Fel d 1) levels in schools and rhinitis and other weekly SBS symptoms in the students. Fungal DNA was measured by quantitative PCR and cat allergen by ELISA. Pupils (N = 462) from eight randomly selected schools in Johor Bahru, Malaysia participated (96%). Dust samples were collected by cotton swabs and Petri dishes exposed for one week. None of the schools had a mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures and indoor CO₂ levels were low (mean 492 ppm; range 380–690 ppm). Weekly nasal symptoms (rhinitis) (18.8%), ocular (11.6%), throat (11.1%), dermal symptoms, headache (20.6%) and tiredness (22.1%) were common. Total fungal DNA in swab samples was associated with rhinitis (p = 0.02), ocular symptoms (p = 0.009) and tiredness (p = 0.001). There were positive associations between Aspergillus versicolor DNA in Petri dish samples, ocular symptoms (p = 0.02) and tiredness (p = 0.001). The level of the mycotoxin verrucarol (produced by Stachybotrys chartarum) in swab samples was positively associated with tiredness (p = 0.04). Streptomyces DNA in swab samples (p = 0.03) and Petri dish samples (p = 0.03) were negatively associated with tiredness. In conclusion, total fungal contamination, measured as total fungal DNA) in the classrooms, Aspergillus versicolor and verrucarol can be risk factors for rhinitis and SBS symptoms among students in the tropical country Malaysia.     

Acetophenone and stilbene derivatives from Gnetum ula

The isolation of bergenin, 2-hydroxy-4-benzyloxyacetophenone and the related dimer and stilbene from Gnetum ula is reported.     

Sequential Application of Ligand and Structure Based Modeling Approaches to Index Chemicals for Their hH4R Antagonism

The human histamine H₄ receptor (hH₄R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH₄R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH₄R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH₄R bioactivity. An application of the prediction model on external test set composed of more than 160 hH₄R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼4000 chemicals highly indexed as H₄R antagonists'' candidates. Next, a series of 3D models of hH₄R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH₄R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.