Expression of glutathione transferase isoenzymes in the porcine ovary in relationship to follicular maturation and luteinization

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Immunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione-Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.     

Wild-type and mutant alleles of the Aspergillus nidulans developmental regulator gene brlA: correlation of variant sites with protein function

The DNA sequences of two wild-type and eleven mutant alleles of the developmental regulator gene brlA from Aspergillus nidulans, which encodes a zinc-finger protein, were characterized. Variant sites were located on rescued plasmids or PCR products based either on their meiotic map position or the use of denaturing gradient gel electrophoresis. Mutations in three null mutants, one of which is partially suppressible, encode premature stop codons. Two environmentally sensitive mutants were characterised by substitution of leucines required for stabilisation of α-helices in each of the two putative zinc-finger domains. A third zinc-finger substitution is predicted to disrupt recognition of a guanine residue in the DNA target. The mutations in four other leaky mutants map C-terminal to the zinc fingers; one minimally leaky mutant has a premature stop codon, which results in the removal of the last 38 residues of the protein product. Received: 16 February 1999 / Accepted: 22 July 1999     

Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity

Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.     

Biofilm formation by Helicobacter pylori

Helicobacter pylori NCTC 11637 produces a water-insoluble biofilm when grown under defined conditions with a high carbon:nitrogen ratio in continuous culture and in 10% strength Brucella broth supplemented with 3 g l-1 glucose. Biofilm accumulated at the air/liquid interface of the culture. Light microscopy of frozen sections of the biofilm material showed few bacterial cells in the mass of the biofilm. The material stained with periodic acid Schiff's reagent. Fucose, glucose, galactose, and glycero-manno-heptose, N-acetylglucosamine and N-acetylmuramic acid were identified in partially purified and in crude material, using gas chromatography and mass spectrometry. The sugar composition strongly indicates the presence of a polysaccharide as a component of the biofilm material. Antibodies (IgG) to partially purified material were found in both sero-positive and sero-negative individuals. Treatment of the biofilm material with periodic acid reduced or abolished immunoreactivity. Treatment with 5 mol l-1 urea at 100 degrees C and with phenol did not remove antigenic recognition by patient sera. The production of a water-insoluble biofilm by H. pylori may be important in enhancing resistance to host defence factors and antibiotics, and in microenvironmental pH homeostasis facilitating the growth and survival of H. pylori in vivo.     

Genetic steps of mammalian homologous repair with distinct mutagenic consequences

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.     

Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitor-challenged cassava (Manihot esculenta) suspension cells and leaves

BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.     

Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes: effect on cell growth and oxygen uptake

The hemoglobins found in unicellular organisms show a greater chemical reactivity, protect cells against oxidative stress and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than VHb counterparts.