Utilization of serine, leucine, isoleucine, and valine by Thermoanaerobacter brockii in the presence of thiosulfate or Methanobacterium sp. as electron acceptors

Thermoanaerobacter brockii fermented serine to acetate and ethanol. It oxidized leucine to isovalerate, isoleucine to 2-methylbutyrate, and valine to isobutyrate only in the presence of thiosulfate, or when co-cultured with Methanobacterium sp. This oxidative deamination was rendered thermodynamically possible by the ability ofT. brockii to reduce thiosulfate to sulfide or the transfer of reducing equivalents to the hydrogenotrophic methanogen. The results suggest that T. brockii may be of ecological significance in thermal environments in the turnover of amino acids, especially with thiosulfate or H(2)-utilizing methanogens are present.     

Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB₁4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB₁4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Thiosulfate reduction and alanine production in glucose fermentation by members of the genus Coprothermobacter

Coprothermobacter platensis is an anaerobic, proteolytic, thermophilic bacterium, which is phylogenetically related to the genera Fervidobacterium and Thermotoga. The organism was found to reduce thiosulfate to sulfide during growth on carbohydrates and proteinaceous substrates. Growth on glucose was inhibited by hydrogen, but this inhibition was overcome by thiosulfate reduction, stirring, increasing the headspace volume and coculturing with a hydrogen-consuming methanogen. Alanine was detected during glucose fermentation, its formation was influenced by the hydrogen concentration in the gas phase suggesting an electron sink mechanism, as was previously reported for the phylogenetically related Thermotogales and the archaeal hyperthermophile Pyrococcus furiosus.     

Unsaturated Organic Acids as Terminal Electron Acceptors for Reductase Chains of Anaerobic Bacteria

This paper summarizes the current knowledge of unsaturated organic acids in their role as terminal electron acceptors for reductase chains of anaerobic bacteria. The mechanisms and enzyme systems involved in the reduction of fumarate by Escherichia coli, Wolinella succinogenes, and some species of the genus Shewanella are considered. Particular attention is given to reduction of the double bond of the unnatural compound methacrylate by the δ-proteobacterium Geobacter sulfurreducens AM-1. Soluble periplasmic flavocytochromes c, found in bacteria of the genera Shewanella and Geobacter, are involved in the hydrogenation of fumarate (in Shewanella species) and methacrylate (in G. sulfurreducens AM-1). In E. coli and W. succinogenes, fumarate is reduced in cytosol by membrane-bound fumarate reductases. The prospects for research into organic acid reduction at double bonds in bacteria are discussed.     

Dental variation ofProconsulfrom the Tinderet region, Kenya

The morphology of mandibular molars of the twoProconsulspecies,Proconsul majorandProconsul africanus, from the Tinderet region, Kenya was analyzed. While the molar size variability within the TinderetP. majorwas slightly greater than those of local African ape subspecies, the shape variability was comparable. Because the twoProconsulspecies show some differences in cusp areal proportions,P. majoris not just a largerP. africanusin molar morphology. Napak specimens are generally similar to the TinderetP. major.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

Oligochitosan induced Brassica napus L. production of NO and H2O2 and their physiological function

NO (nitric oxide) and H₂O₂ (hydrogen peroxide) are important signaling molecule in plants. Brassica napus L. was used to understand oligochitosan inducing production of NO (nitric oxide) and H₂O₂ (hydrogen peroxide) and their physiological function. The result showed that the production of NO and H₂O₂ in epidermal cells of B. napus L. was induced with oligochitosan by fluorescence microscope. And it was proved that there was an interaction between NO and H₂O₂ with L-NAME (N^G-nitro-l-arg-methyl eater), which is an inhibitor of NOS (NO synthase) in mammalian cells that also inhibits plant NO synthesis, and CAT (catalase), which is an important H₂O₂ scavenger, respectively. It was found that NO and H₂O₂ induced by oligochitosan took part in inducing reduction in stomatal aperture and LEA protein gene expression of leaves of B. napus L. All these results showed that oligochitosan have potential activities of improving resistance to water stress.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

Comparative use of amino acids by three auxotypes of Neisseria gonorrhoeae

Auxotypes of Neisseria gonorrhoeae are usually distinguishable by their particular requirements for growth; these requirements often include amino acids. It is possible that strains needing particular substrates to grow can be distinguished not merely by their growth requirements but also by their metabolism of these particular substrates. In this work amino acid utilization and oxidation studies were performed enabling prototype, pro- and thia-strains to be distinguished. The metabolism study also underlined the importance of proline as an energy source and pointed to the probability of distinct relationships with the metabolism of the key amino acids, glutamic and aspartic acids, for the three auxotypes.It is proposed that the specific amino acid required by the naturally occurring auxotype serves as an energy source at the site of infection and has important implications with respect to particular auxotypes at various sites.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Role of formate and hydrogen in the degradation of propionate and butyrate by defined suspended cocultures of acetogenic and methanogenic bacteria

The butyrate-degradingSyntrophospora bryantii degrades butyrate and a propionate-degrading strain (MPOB) degrades propionate in coculture with the hydrogen- and formate-utilizingMethanospirillum hungatii orMethanobacterium formicicum. However, the substrates are not degraded in constructed cocultures with twoMethanobrevibacter arboriphilus strains which are only able to consume hydrogen. Pure cultures of the acetogenic bacteria form both hydrogen and formate during butyrate oxidation with pentenoate as electron acceptor and during propionate oxidation with fumarate as electron acceptor. Using the highest hydrogen and formate levels which can be reached by the acetogens and the lowest hydrogen and formate levels which can be maintained by the methanogens it appeared that the calculated formate diffusion rates are about 100 times higher than the calculated hydrogen diffusion rates.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.     

Growth,14CO2 fixation,activites of photosystems,ribulose 1,5-bisphosphate carboxylase and nitrate reductase in trees as affected by simulated acid rain

In seedlings of the tropical tree speciesErythrina variegata Lam. andHardwickia binata Roxb. exposed to different acidic mist (H₂SO₄, pH 5, 3 and 2) for 5 d significant reduction in seedling growth, biomass accumulation and¹⁴CO₂ fixation were determined. In isolated chloroplasts a decrease in the activities of photosystem 2 and whole electron transport chain was observed only at pH 3 and 2, but no significant change in photosystem 1 activity was observed. SDS-PAGE analysis of crude leaf extracts of ribulose 1,5-bisphosphate carboxylase (RuBPC) indicated a significant loss of 55 and 15 kDa polypeptides at pH 2 inErythrina. The reduction in the RuBPC activity in seedlings grown under acidic mists correlated well with CO₂ fixation.     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.     

Purification,Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete,Tricholoma matsutake

A new H₂O₂-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H₂O₂ and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V _max, K _m, and k _cat for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50°C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.