Crystallization and preliminary x-ray diffraction study of the flavoprotein NADH peroxidase from Streptococcus faecalis 10C1

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.     

Use of a chemiluminescence detector for quantitation of nitric oxide produced in assays of denitrifying enzymes

We have developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen (NOx) analyzer for the quantitation of any nitric oxide present in these gases. The system enabled us to study both the stoichiometry and the kinetics of NO production by a copper-containing nitrite reductase (EC 1.7.99.3) purified from Achromobacter cycloclastes IAM 1013. In addition to its much greater sensitivity in comparison with standard gas chromatographic (GC) techniques, the method offers the advantage that NO, a very reactive free radical, is immediately swept away and quantitated, obviating the necessity for periodic manipulations and disturbances of the reaction mixture characteristic of other GC quantitations. The characteristics of the system are discussed and its utility in studies of the kinetics and stoichiometry of NO production from nitrite is confirmed by comparison with results obtained using manometric and GC techniques.     

One thousand heart attacks in Grampian: the place of cardiopulmonary resuscitation in general practice.

The outcome of 1011 heart attacks in patients under the care of general practitioners who practised cardiopulmonary resuscitation and were equipped with defibrillators is reported. The 28 day mortality was 36% (367 patients), and 59% of deaths occurred outside hospital. The general practitioner was the first medical contact in 92% of heart attacks and was equipped with a defibrillator in 80% of such calls. Fifty six patients had a cardiac arrest in the presence of a general practitioner, and resuscitation was attempted in 47 cases, representing 5% of all calls for heart attacks. Twenty one (45%) resuscitated patients reached hospital alive, and 13 (28%) survived to leave hospital. The opportunities for cardiopulmonary resuscitation in general practice occur sufficiently often to warrant training and equipping general practitioners for advanced life support. The results of resuscitation by general practitioners working alone compare favourably with those of mobile coronary care units based in hospitals.     

Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.     

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.     

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N , N -dimethyl- p -phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf ( p -dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes , and Corynebacterium . Of the various isolates, Alcaligenes DM₄ showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM₄ grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM₄ has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM₄. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.     

Effects of TCDD on the EGF receptor of XB mouse keratinizing epithelial cells

TCDD was found to cause a marked inhibition of 125I-epidermal growth factor (EGF) binding to its receptor on the cell surface of XB mouse keratinizing epithelial cells (XB cells) cultured in vitro. The EC50 concentration was estimated to be on the order of 3 x 10(-11) M 24 hours after TCDD administration. As early as 12 hours after the addition of 10(-9) M of TCDD, XB cells showed signs of a decline in 125I-EGF binding levels. The level of such EGF receptor downregulation reached a maximum at 24 hours, continued until day 2, but completely recovered by day 3. This was accompanied by a rise in protein kinase activities, particularly those of the protein tyrosine kinases during the initial period of 6-24 hours. To test the hypothesis that the EGF receptors of the cells, by showing TCDD-induced symptoms of downregulation, actually are being activated and triggering EGF-like signals, we examined the effects of both TCDD and exogenously added EGF on cell morphology, colony formation degree of keratinization, the pattern of activation of protein kinases and de novo protein synthesis, and EGF receptor phosphorylation. Based on the similarity of cell responses to these between TCDD- and EGF-treated cells, we concluded that TCDD, directly or indirectly, causes activation of the EGF receptor. In contrast, 12-O-tetradencanoylphorbol-13-acetate (TPA), which is known to downregulate EGF receptors by blocking their protein tyrosine kinase, produced dissimilar end results. The balance of evidence support the notion that the action of TCDD in this cell line is tightly coupled to the activation of the EGF receptor and that one of the key consequences of such a biochemical change is that it signals these cells to commit to terminal differentiation.