Regulation of Brucella abortus catalase.

All aerobic organisms have mechanisms that protect against oxidative compounds. Catalase, peroxidase, superoxide dismutase, glutathione, and thioredoxin are widely distributed in many taxa and constitute elements of a nearly ubiquitous antioxidant metabolic strategy. Interestingly, the regulatory mechanisms that control these elements are rather different depending on the nature of the oxidative stress and the organism. Catalase is well documented to play an important role in protecting cells from oxidative stress. In particular, pathogenic bacteria seem to use this enzyme as a defensive tool against attack by the host. To investigate the significance of catalase in hostile environments, we made catalase deletion mutations in two different B. abortus strains and used two-dimensional gel analysis, survival tests, and adaptation experiments to explore the behavior and role of catalase under several oxidative stress conditions. These studies show that B. abortus strains that do not express catalase activity exhibit increased sensitivity to hydrogen peroxide. We also demonstrate that catalase expression is regulated in this species, and that preexposure to a sublethal concentration of hydrogen peroxide allows B. abortus to adapt so as to survive subsequent exposure to higher concentrations of hydrogen peroxide.

Sites of volatile anesthetic action on kainate (Glutamate receptor 6) receptors.

Molecular mechanisms of anesthetic action on neurotransmitter receptors are poorly understood. The major excitatory neurotransmitter in the central nervous system is glutamate, and recent studies found that volatile anesthetics inhibit the function of the alpha-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate receptors (e.g. glutamate receptor 3 (GluR3)), but enhance kainate (GluR6) receptor function. We used this dissimilar pharmacology to identify sites of anesthetic action on the kainate GluR6 receptor by constructing chimeric GluR3/GluR6 receptors. Results with chimeric receptors implicated a transmembrane region (TM4) of GluR6 in the action of halothane. Site-directed mutagenesis subsequently showed that a specific amino acid, glycine 819 in TM4, is important for enhancement of receptor function by halothane (0. 2-2 mM). Mutations of Gly-819 also markedly decreased the response to isoflurane (0.2-2 mM), enflurane (0.2-2 mM), and 1-chloro-1,2, 2-trifluorocyclobutane (0.2-2 mM). The nonanesthetics 1, 2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane had no effect on the functions of either wild-type GluR6 or receptors mutated at Gly-819. Ethanol and pentobarbital inhibited the function of both wild-type and mutant receptors. These results suggest that a specific amino acid, Gly-819, is critical for the action of volatile anesthetics, but not of ethanol or pentobarbital, on the GluR6 receptor.

Selective actions of a detergent on ligand-gated ion channels expressed in Xenopus oocytes.

Cytoclean a commercially available detergent, has selective actions on ligand-gated ion channels. Cytoclean (0.0005-0.01% v/v) potentiated 50 microM glycine responses in oocytes expressing alpha-2 glycine receptors by 23 +/- 7% to 342 +/- 43%. Cytoclean is composed of five components dissolved in water, but only one reagent, Bio-Soft D-62, modulated responses of oocytes expressing alpha-2 glycine receptors. Bio-Soft D-62 (0.00005-0.001% w/v), potentiated 50 microM glycine responses by 13 +/- 1% to 474 +/- 50%. Bio-Soft D-62 is composed of linear alkylbenzene sulfonate (> 95% C12 chain). The effects of Cytoclean or Bio-Soft D-62 were examined on alpha-1 beta-2 and alpha-1 beta-2 gamma-2L gamma-aminobutyric acidA, gamma-aminobutyric acid rho 1, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, kainate and 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes. Enhancement of gamma-aminobutyric acidA receptor function ranged from approximately 21% to 458% with Cytoclean (0.0001-0.01%), respectively. Bio-Soft D-62 (0.001%) inhibited GABA rho 1 receptor function by approximately 72%. Cytoclean had no effect on 5-hydroxytryptamine3 or GluR6 function, but Cytoclean (0.005% and 0.01%) inhibited GluR3-mediated currents by approximately 21% and approximately 41%, respectively. These results suggest that trace amounts of Cytoclean, such as amounts adhering to glassware, may modulate ion channel function and potentially confound experimental results.

Brucella abortus arginase and ornithine cyclodeaminase genes are similar to Ti plasmid arginase and ornithine cyclodeaminase.

Brucella abortus arginase and ornithine cyclodeaminase genes have been cloned and sequenced. These gene sequences are located in the same operon and occur in the same order as the homologous genes in Agrobacterium tumefaciens Ti C58 plasmid. The nucleotide sequences of the two genes have 72% and 65% identity to the respective Ti plasmid genes. Both genes are present in a single copy, and expression of arginase is regulated in response to arginine.

Role of immune responses to a GroEL heat shock protein in preventing brucellosis in mice vaccinated with Brucella abortus strain RB51.

Resistance to infection with virulent Brucella abortus strain 2308 and antibody and lymphocyte proliferative responses to a recombinant 60 kDa B. abortus GroEL heat shock protein were measured in mice vaccinated with attenuated B. abortus strain RB51. Mice at 12-20 weeks after vaccination with 5 x 10(8) colony forming units (CFU) of strain RB51 had increased resistance to infection with strain 2308 and increased antibody and lymphocyte proliferative responses to GroEL following challenge infection with 2308. However, these mice at 12-20 weeks after vaccination did not have greater resistance to infection than mice vaccinated with 5 x 10(6) CFU of strain RB51, which had no increased antibody or lymphocyte proliferative response to GroEL. These results indicate that mice vaccinated with strain RB51 can have antibody and cell-mediated immune responses to GroEL during infection with virulent strain 2308, although neither response appeared to have an essential role in vaccine-induced immunity to brucellosis.

Actions of long chain alcohols on GABAA and glutamate receptors: relation to in vivo effects.

1. The effects of n-alcohols on GABAA and glutamate receptor systems were examined, and in vitro effectiveness was compared with in vivo effects in mice and tadpoles. We expressed GABAA, NMDA, AMPA, or kainate receptors in Xenopus oocytes and examined the actions of n-alcohols on receptor function using two-electrode voltage clamp recording. 2. The function of GABAA receptors composed of alpha 1 beta 1 or alpha 1 beta 1 gamma 2L subunits was potentiated by all of the n-alcohols studied (butanol-dodecanol). 3. In contrast to GABAA receptors, glutamate receptors expressed from mouse cortical mRNA or from cRNAs encoding AMPA (GluR3)- or kainate (GluR6)-selective subunits were much less sensitive to longer chain alcohols. In general, octanol and decanol were either without effect or high concentrations were required to produce inhibition. 4. In contrast to the lack of behavioural effects by long chain alcohols reported previously, decanol produced loss of righting reflex in short- and long-sleep mice, indicating that the in vivo effects of decanol may be due in part to actions at GABAA receptors. Furthermore, butanol, hexanol, octanol, and decanol produce similar potentiation of GABAA receptor function at concentrations required to cause loss of righting reflex in tadpoles, an in vivo model where alcohol distribution is not a compromising factor. 5. Thus, the in vivo effects of long chain alcohols are not likely to be due to their actions on NMDA, AMPA, or kainate receptors, but may be due instead to potentiation of GABAA receptor function.

Anomalous phylogenies based on bacterial catalase gene sequences.

Phylogenies based on nine prokaryotic catalase sequences demonstrate no relationship to phylogenies based on rDNA sequences or other known criteria. When this observation is considered together with the monophyletic relationship observed for eukaryotic catalase sequences, it seems likely that the catalase gene sequence has migrated repeatedly from eukaryotes to prokaryotes.

Anesthetics produce subunit-selective actions on glutamate receptors.

We assessed the involvement of specific glutamate receptors in the action of anesthetics. In addition to the clinical anesthetics enflurane, isoflurane and halothane, we tested novel halogenated compounds, which are anesthetic or nonanesthetic in vivo, on glutamate receptor (GluR) subunits. these volatile compounds as well as pentobarbital and phenobarbital were tested on kainate-induced currents in Xenopus oocytes expressing GluR1, GluR3, GluR2+3 or GluR6 subunits. The anesthetic 1-chloro-1,2,2-triflurocyclobutane (F3) weakly inhibited kainate responses in oocytes expressing GluR3 receptors but not oocytes expressing GluR1 or GluR2+3 receptors. Surprisingly, F3 potentiated kainate responses in oocytes expressing the kainate-selective receptor GluR6. The nonanesthetics 2,3-chlorooctafluorobutane (F8) and 1,2-dichlorohexafluorocyclobutane (F6) did not affect GluR3 or GluR6 kainate responses. Isoflurane weakly inhibited although enflurane and halothane modestly inhibited kainate responses in oocytes expressing GluR1, GluR3 or GluR2+3 receptors. As with F3, isoflurane, enflurane and halothane potentiated kainate-induced currents of GluR6 receptors. The respective inhibitory and potentiating effects of halothane on GluR3 and GluR6 receptors were enhanced by increasing duration of halothane exposure. In contrast to the opposing action of volatile anesthetics, pentobarbital and phenobarbital inhibited GluR3 and GluR6 kainate responses and had a much greater effect on GluR6 receptors. These results provide novel evidence that anesthetics produce selective actions on glutamate receptors, suggesting that subunit composition may determine the role of glutamate receptors in anesthesia.

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition.

Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C.

Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl-currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses.

Brucella abortus catalase is a periplasmic protein lacking a standard signal sequence.

A periplasmic catalase has been purified and cloned from Brucella abortus. The functional enzyme is a tetramer with a subunit molecular weight of 55,000. All evidence indicates that a typical N-terminal signal sequence is not associated with the export of this protein to the periplasm.

Ethanol inhibits the function of 5-hydroxytryptamine type 1c and muscarinic M1 G protein-linked receptors in Xenopus oocytes expressing brain mRNA: role of protein kinase C.

Effects of ethanol on the function of Ca(2+)-activated Cl- channels activated by G protein-coupled serotonin (5-hydroxytryptamine, (5-HT)1c) and muscarinic M1 cholinergic receptors were studied in Xenopus oocytes expressing mouse whole-brain mRNA. Ethanol (25-200 mM) inhibited currents evoked by both 5-HT and acetylcholine (ACh), in a concentration-dependent manner. The maximal effect was obtained with 150 mM ethanol, which produced 65 and 49% inhibition of 5-HT and ACh responses, respectively. In the presence of 100 mM ethanol, the EC50 values for both 5-HT and ACh were increased about 4-fold. In contrast, in oocytes expressing rat cerebellar mRNA, metabotropic glutamate receptor responses were much less sensitive to ethanol. To examine potential postreceptor sites for ethanol inhibition, guanosine-5'-O-(3-thio)triphosphate and myo-inositol-1,4,5-trisphosphate were injected intracellularly. Ethanol (100 mM) did not significantly inhibit the currents produced by either guanosine-5'-O-(3-thio)triphosphate or myo-inositol-1,4,5-trisphosphate. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate markedly inhibited 5-HT-induced responses. Both the PKC inhibitor peptide and staurosporine prevented ethanol inhibition of 5-HT-induced responses. Moreover, ethanol, similarly to phorbol-12-myristate-13-acetate and opposite to PKC inhibitors, enhanced the rate of Ca(2+)-activated Cl- current desensitization induced by repeated applications of 5-HT. These results indicate that certain types of receptor-G protein interactions are more susceptible than others to uncoupling by ethanol and that ethanol inhibition of 5-HT1c receptors requires PKC-mediated phosphorylation. We suggest that ethanol may activate PKC, which phosphorylates the receptors, resulting in inhibition of the responses.

Activation of protein kinase C inhibits kainate-induced currents in oocytes expressing glutamate receptor subunits.

The effect of protein kinase C (PKC) activation on maximal kainate (KA)-induced currents was studied in Xenopus oocytes expressing the glutamate receptor (GluR) subunits GluR3, GluR1 + 3, GluR2 + 3, and GluR6. The PKC activator phorbol 12-myristate 13-acetate (PMA) inhibited peak KA responses in a time-dependent manner. The magnitude of inhibition was greatest in GluR6-expressing oocytes. Desensitizing KA currents characterized by a peak, transient current followed by a slower, desensitizing current were observed in oocytes expressing GluR3 and GluR1 + 3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMA's inhibition of peak current amplitude. PMA-mediated inhibition of both desensitization and peak current amplitude was prevented by intracellular injection of the protein kinase C (PKC) inhibitor peptide. These results suggest that the function of GluRs is regulated by PKC-dependent phosphorylation.

GABAA and glutamate receptor subunit mRNAs in cortex of mice chemically kindled with FG 7142.

Messenger RNA (mRNA) for several subunits of the GABAA receptor was measured in the cortex of mice chemically kindled with FG 7142. At 10 days after the final FG 7142 injection, beta 2 and gamma 2S subunit mRNA were significantly increased. At 31 days, alpha 1, alpha 3, beta 2, and gamma 2L mRNA were elevated. In contrast, levels of mRNA for four subunits of the glutamate receptor in the cortex of FG 7142-kindled mice killed at 31 days were not significantly increased. Previous investigations have shown a reduction in GABA-gated chloride channel function and density in mice kindled with FG 7142, and the increases in subunit mRNA found in the present studies may be a response to these decreases. These results indicate that chemical kindling produces long-lasting changes in expression of genes coding for specific neurotransmitter receptor subunits.

Periplasmic location of Brucella abortus Cu/Zn superoxide dismutase.

Two types of superoxide dismutase (SOD) have been found in Brucella abortus, a cytosolic Mn-SOD and a Cu/Zn-SOD of unknown location. We sought to determine the subcellular location of Cu/Zn-SOD in B. abortus ST 19. We report a modified spheroplasting procedure for the release of periplasmic contents from B. abortus cells using a dipolar ionic detergent, Zwittergent 316. This detergent, used in place of EDTA, destabilizes the outer membrane sufficiently to allow penetration of lysozyme and the subsequent selective release of periplasmic proteins by osmotic shock. Cytoplasmic cross-contamination of periplasmic fractions was assessed by assaying for malate dehydrogenase activity. Cyanide-sensitive and cyanide-insensitive SOD activity was measured by both the xanthine oxidase-cytochrome c method and a hematoxylin assay. Results suggest that B. abortus Cu/Zn-SOD activity is periplasmic. This zwittergent-lysozyme extraction procedure may be applicable to the separation, isolation and characterization of many other periplasmic proteins of B. abortus and other Gram-negative organisms especially when cytosolic contamination is undesirable.

Oral immunization of mice and swine with an attenuated Salmonella choleraesuis [delta cya-12 delta(crp-cdt)19] mutant containing a recombinant plasmid.

Salmonella choleraesuis chi 3781, an attenuated [delta cya-12 delta(crp-cdt)19] mutant, was electroporated with the plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Recombinant S. choleraesuis chi 3781 stably maintained the pBA31-R7 plasmid and continued to express the cloned protein following recovery of the organism from orally inoculated animals. Unlike previous studies using S. typhimurium chi 4064(pBA31-R7), S. choleraesuis chi 3781(pBA31-R7) was able to colonize both the gut mucosa and deep tissues of both BALB/cByJ mice and crossbred swine. Orally inoculated mice developed serum antibodies to both the cloned 31-kDa protein (rBCSP31) and to S. choleraesuis chi 3781 endotoxin. These mice also developed a local intestinal antibody response to Salmonella endotoxin but not to rBCSP31. Similarly, mice inoculated with recombinant S. choleraesuis chi 3781 did not develop a delayed-type hypersensitivity (DTH) footpad response following injection with rBCSP31; however, these mice did respond to S. choleraesuis chi 3781 soluble antigen. Conversely, orally inoculated swine did not develop significant serum or intestinal antibody responses to cloned protein or Salmonella endotoxin, but DTH responses to both cloned protein and S. choleraesuis chi 3781 soluble antigen were strongly positive. The cell-mediated nature of these DTH responses was confirmed by histological examination. Results suggest that S. choleraesuis chi 3781 may be a suitable choice for further studies of vaccine efficacy in swine, especially for diseases which require cell-mediated immunity for resolution.

Comparison of ethanol sensitivity of rat brain kainate, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

The effect of ethanol (EtOH) on kainate (KA), DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate (NMDA) receptor-operated channels was examined electrophysiologically in Xenopus laevis oocytes expressing mRNA from rat hippocampus and cerebellum. EtOH (50, 100 mM) inhibited KA-induced currents but did not alter the EC50 for KA (approximately 78 microM). For a series of n-alcohols, potency for inhibition of KA responses was related to chain length. 6,7-dinitroquinoxaline-2,3-dione inhibited maximum KA responses with an IC50 of approximately 1 microM; EtOH (50, 100 mM) did not alter the IC50 for 6,7-dinitroquinoxaline-2,3-dione but did not produce further inhibition of KA-induced currents. Despite the apparent noncompetitive inhibition produced by EtOH on KA receptor-mediated responses, the EtOH inhibition increased as the KA concentration decreased in hippocampal and cerebellar mRNA expressing oocytes. This differential inhibition was not due to the different current amplitudes stimulated by low vs. high KA concentrations. In contrast, oocytes expressing NMDA channels demonstrated a constant percent inhibition by EtOH in the presence of 25 to 200 microM NMDA. Altering the extracellular Ca++ concentration did not affect the ability of EtOH to inhibit NMDA responses. Maximal NMDA-stimulated currents were inhibited by 100 mM EtOH to a lesser extent (31%) in oocytes injected with rat cerebellar mRNA than oocytes expressing rat hippocampal mRNA (47%), suggesting brain regional differences in NMDA channel inhibition by EtOH.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of maternal lineage on milk yield traits of dairy cattle.

Maternal lineage effects on milk yield traits, considered indicative of cytoplasmic inheritance, were evaluated with animal models. Cattle were from a selection experiment begun in 1968. Maternal pedigrees were traced to the first female member in the Holstein-Friesian Herdbook; purchased cows entering the herd, considered foundation females, were assigned to maternal lineage groups. All models accounted for year-season of calving, parity, and selection lines. Maternal lineage effects were included in a repeated records model with cow effects and preadjustment for sire and maternal grandsire transmitting abilities. Maternal lineage accounted for 5.2, 4.1, and 10.5% of phenotypic variation of preadjusted records of milk yield, fat yield, and fat percentage, respectively. Maternal lineage was evaluated as a fixed effect in an animal model including random animal and permanent environmental effects. Maternal lineage significantly affected fat percentage but not milk yield. Maternal genetic (nuclear) effects and their covariance with additive animal effects did not significantly account for additional variation nor did they influence maternal lineage estimates. Maternal lineage affected calculated net energy of milk but was not important for SNF yield or concentration. Maternal lineage influenced fat percentage, energy concentration, and, to a lesser extent, fat yield in milk of dairy cattle.

Acute and chronic ethanol exposure alters the function of hippocampal kainate receptors expressed in Xenopus oocytes.

The effects of acute and extended ethanol exposure on N-methyl-D-aspartate- and kainate-induced currents were examined electrophysiologically in Xenopus oocytes expressing rat hippocampal mRNA. Ethanol inhibited responses stimulated by low and high concentrations of N-methyl-D-aspartate to a similar degree. However, responses produced by low or high concentrations of kainate were differentially inhibited by ethanol. Low kainate concentration responses were much more sensitive to ethanol than high kainate concentrations (e.g., 50 mM ethanol inhibited 12.5 microM kainate responses by 45% compared to 15% inhibition of 400 microM kainate responses). In oocytes cultured in 100 mM ethanol for 1-5 days, the ethanol inhibition of maximum N-methyl-D-aspartate and kainate responses was not different from that in non-ethanol-exposed oocytes. Ethanol treatment, however, selectively decreased the ethanol sensitivity of low kainate concentration responses. Currents stimulated by N-methyl-D-aspartate or kainate were not different between control and ethanol-treated oocytes, indicating that ethanol exposure did not interfere with channel expression. The selective actions of acute and extended ethanol exposure on low kainate responses may indicate selective actions of ethanol on subtypes of kainate receptors expressed in oocytes.

Cloning and nucleotide sequence of the Brucella abortus groE operon.

The cloning and sequencing of the Brucella abortus groES and groEL genes are reported. The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon. Putative promoter and terminator sequences are also identified. The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.