The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

Abstract Recent discoveries relating to pathways of anaerobic electron transport in the Rhodospirillaceae are reviewed. The main emphasis is on the organism Rhodobacter capsulatus ** but comparisons are made with Rhodobacter sphaeroides ** f. sp. denitrificans and Rhodopseudomonas palustris . The known electron acceptors for anaerobic respiration in Rhodobacter capsulatus are trimethylamine- N -oxide (TMAO), dimethyl sulphoxide (DMSO), nitrate and nitrous oxide. In each case respiration generates a proton electrochemical gradient and in some cases can support growth on non-fermentable carbon sources. However, the principal objective of this review is to discuss the possibility that, apart from a role in energy conservation, anaerobic respiration in the photosynthetic bacteria may have a special function in maintaining redox balance during photosynthetic metabolism. Thus the electron acceptors mentioned above may serve as auxiliary oxidants: (a) to maintain an optimal redox poise of the photosynthetic electron transport chain; (b) to provide a sink for electrons during phototrophic growth on highly reduced carbon substrates.
Molecular properties of the nitrate reductase, nitrous oxide reductase and a single enzyme responsible for reduction of TMAO and DMSO are discussed. These enzymes are all located in the periplasm. Electrons destined for all three enzymes can originate from the rotenone-sensitive NADH dehydrogenase but do not proceed through the antimycin- and myxothiazol-sensitive cytochrome b/c₁ complex. It is likely, therefor, that the pathways of anaerobic respiration overlap with the cyclic photosynthetic electron transport chain only at the level of the ubiquinone pool. Redox components which might be involved in the terminal branches of anaerobic respiration are discussed.     

An 18 amino acid amphiphilic helix forms the membrane-anchoring domain of the Escherichia coli penicillin-binding protein 5

Small (10 residue) C-terminal deletions of PBP5 cause release of this Inner membrane protein into the periplasm, indicating disruption of the membrane binding domain. To define the extent of the membrane anchoring domain, oligonucleotide-directed mutagenesis was used to introduce both single amino acid changes and novel restriction sites into the DN A, allowing subsequent construction of precise internal deletions. The 10 C-terminal amino acid residues possess very weak membrane anchoring potential. By extending the sequence to 18 residues membrane binding equivalent to that of authentic PBP5 was achieved. A proline substitution in this region, breaking a potential α-helix, also disrupts the membrane binding domain. These results are discussed with respect to the amphi-philicity of the C-terminal sequence when arranged in an α-helix.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

Heterochromatin differentiation and phylogenetic relationship of the A genomes in diploid and polyploid wheats

Summary Heterochromatin differentiation, including band size, sites, and Giemsa staining intensity, was analyzed by the HKG (HCl-KOH-Giemsa) banding technique in the A genomes of 21 diploid (Triticum urartu, T. boeoticum and T. monococcum), 13 tetraploid (T. araraticum, T. timopheevi, T. dicoccoides and T. turgidum var. Dicoccon, Polonicum), and 7 cultivars of hexaploid (T. aestivum) wheats from different germplasm collections. Among wild and cultivated diploid taxa, heterochromatin was located mainly at centromeric regions, but the size and staining intensity were distinct and some accessions' genomes had interstitial and telomeric bands. Among wild and cultivated polyploid wheats, heterochromatin exhibited bifurcated differentiation. Heterochromatinization occurred in chromosomes 4A^t and 7A^t and in smaller amounts in 2A^t, 3A^t, 5A^t, and 6A^t within the genomes of the tetraploid Timopheevi group (T. araraticum, and T. timopheevi) and vice versa within those of the Emmer group (T. dicoccoides and T. turgidum). Similar divergence patterns occurred among chromosome 4A^a and 7A^a of cultivars of hexaploid wheat (T. aestivum). These dynamic processes could be related to geographic distribution and to natural and artifical selection. Comparison of the A genomes of diploid wheats with those of polyploid wheats shows that the A genomes in existing diploid wheats could not be the direct donors of those in polyploid wheats, but that the extant taxa of diploids and polyploids probably have a common origin and share a common A-genomelike ancestor.Contribution of the College of Agricultural Sciences, Texas Tech Univ. Journal No. T-4-233.     

The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.     

Inhibition of Ca2+-induced cytosolic enzyme efflux from skeletal muscle by vitamin E and related compounds.

1. Efflux of an intracellular enzyme (creatine kinase) from normal rat skeletal muscles was induced by treatment with the Ca2+ ionophore A23187. Addition of alpha-tocopherol (230 microM) to the incubation medium was found to significantly diminish this efflux, and this effect was mimicked by alpha-tocopherol acetate, phytol and isophytol, but not by Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). 2. Analysis of muscle cation content has shown that these protective effects of alpha-tocopherol etc. are not due to an inhibition of the Ca2+ accumulating effects of the ionophore. 3. Non-enzymic lipid peroxidation of skeletal-muscle homogenates was found to be inhibited by alpha-tocopherol and Trolox C, partially inhibited by phytol and isophytol, but unaffected by alpha-tocopherol acetate. 4. The activity of lipoxygenase enzymes was partially inhibited by alpha-tocopherol, phytol and isophytol, but not by alpha-tocopherol acetate or Trolox C. 5. Prostaglandin E2 efflux from isolated skeletal muscles was stimulated by treatment with the Ca2+ ionophore, but this was unaffected by alpha-tocopherol treatment.     

Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue

In a preliminary paper (Teasdale et al. 1987) comparing the oestrogen receptor (ER) content of breast cancers by the biochemical dextran coated charcoal (DCC) method and by two histochemical methods, peroxidase immunocytochemistry (ERICA) and immunogold-silver staining (IGSS), it was indicated that ERICA is more sensitive than DCC and that IGSS is as specific as ERICA but less sensitive. This paper describes the comparison of the above three assay methods with two other biochemical methods, iso-electric focusing (IEF) and an enzyme immuno-assay (EIA) on a larger number of cancers. All methods gave statistically comparable results except that IGSS remained less sensitive than the rest. Various modifications to IGSS showed that an immunogold streptavidin enhancement method (IG-SAM) produced sensitivity and specificity equal to that of ERICA. Since IGSS and its modifications are the only methods which can be used on archival paraffin-embedded cancers and IG-SAM gives results highly comparable to ERICA, retrospective studies can be performed on patients whose outcome and response to various treatments are known. Most recent studies have shown that ER positive results can be obtained from 10-year-old paraffin blocks.     

In-vivo effects of ivermection onRhipicephalus appendiculatus: the influence of tick feeding patterns and drug pharmacokinetics

Sublethal effects seen amongstRhipicephalus appendiculatus feeding on ivermectin-treated rabbits were diverse and dependent both on drug dose, pharmacokinetics and tick feeding patterns: changes in drug formulation, the time of infestation relative to treatment, and the tick instar used, profoundly influenced acaricidal activity. Death was a sequel to paralysis only if tick feeding was interrupted for sufficient time to produce irreversible dehydration. Concurrent pharmacokinetic investigations revealed that, for the larvae ofR. appendiculatus, the mean critical lethal dose of ivermectin imbibed over a 5-day engorgement period was 3500 g/kg. This quantity of ivermectin was achieved in the blood-meals of larvae feeding on rabbits treated subcutaneously with a single dose of Ivomec injection (MSD)^*800 g/kg, provided infestation took place within 24 h of treatment. At lower drug doses, or if larval infestations were delayed for>24 h post-treatment, the quantity of circulating ivermectin (and thus imbibed by the tick larvae) fell below 3500 g/kg and an increasing percentage of larvae successfully engorged and detached. More than 90% of such larvae moulted to the nymphal stage. Nymphae and larvae exhibited similar susceptibility to ivermectin on treated rabbits which could be explained by similar feeding patterns. However, adult female and male ticks were markedly less susceptible and interpretation of ivermectin-induced effects was more complex.