Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. AfterN-acetylation, the oligosaccharides were labelled with a UV-absorbing compound,p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz¹H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc1-3Man_7–9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man_5–9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man1-6(±GlcNAc1-4)(Man1-3)Man1-4GlcNAc1-4(±Fuc1-6)GlcNAc. The glucosylated oligosaccharides, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂, have not previously been reported in mature glycoproteins from any source.Abbreviations IgG, IgM, IgD, IgE, and IgA immunoglobulin G, M, D, E, and A, respectively - IgY egg-yolk antibody - ABEE p-aminobenzoic acid ethyl ester - HPLC high performance liquid chromatography - FAB-MS fast atom bombardment mass spectrometry - Hex hexose - HexNAc N-acetylhexosamine - hCG human chorionic gonadotropsin     

Dimethylsulfoxide and sodium butyrate enhance the production of recombinant cyclooxygenase 2 in stably transformed Drosophila melanogaster S2 cells

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni²⁺-affinity fractionation to a specific activity of 24 800 U mg^–1 protein. The peak level of recombinant Cox 2 production was 1.6 g (10⁷ cells)^–1, seven days after induction with 0.5 mM CuSO₄. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.     

Energetics of the growth of Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus on ammonium chloride and dinitrogen

Methanobacterium thermoautotrophicum was grown in continuous culture in a fermenter gassed with H₂ and CO₂ as sole carbon and energy sources, and in a medium which contained either NH₄Cl or gaseous N₂ as nitrogen source. Growth was possible with N₂. Steady states were obtained at various gas flow rates with NH₄Cl and with and the maintenance coefficient varied with the gas input and with the nitrogen source. Growth of Methanococcus thermolithotrophicus in continuous culture in a fermenter gassed with H₂, CO₂ as nitrogen, carbon and energy sources was also examined.Abbreviations molecular growth yield (g dry weight of cells per mol of CH₄ evolved) - growth rate (h^-1) - D dilution rate (h^-1) - rate (h_-1); relation of Neijssel and Tempest and of Stouthamer and Bettenhaussen - energy     

Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml^–1. A maximum production of 4.6 g recombinant tumstatin (10⁷ cells)^–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO₄.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells

Fumonisins B₁ and B₂ and AAL toxin are a series of structurally related mycotoxins. Fumonisins B₁ and B₂, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC₅₀ values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 g/ml for fumonisins B₁, B₂ and AAL toxin, respectively in 100 l cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC₅₀ = 2.5, 2 and 5 g/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B₁ and B₂ does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.Abbreviations AAL toxin Alternaria alternata f. sp. lycopersici toxin - IC₅₀ concentration giving 50% inhibition of cell proliferation     

Flight of the honey bee VI: energetics of wind tunnel exhaustion flights at defined fuel content,speed adaptation and aerodynamics

To gain information on extended flight energetics, quasi-natural flight conditions imitating steady horizontal flight were set by combining the tetheredflight wind-tunnel method with the exhaustion-flight method. The bees were suspended from a two-component aerodynamic balance at different, near optimum body angle of attack and were allowed to choose their own speed: their body mass and body weight was determined before and after a flight; their speed, lift, wingbeat frequency and total flight time were measured throughout a flight. These values were used to determine thrust, resultant aerodynamic force (magnitude and tilting angle), Reynolds number, total flight distance and total flight impulse. Flights in which lift was body weight were mostly obtained. Bees, flown to complete exhausion, were refed with 5, 10, 15 or 20 l of a 1.28-mol·l^-1 glucose solution (energy content w=18.5, 37.0, 55.5 or 74.0 J) and again flown to complete exhaustion at an ambient temperature of 25±1.5°C by a flight of known duration such that the calculation of absolute and relative metabolic power was possible. Mean body mass after exhaustion was 76.49±3.52 mg. During long term flights of 7.47–31.30 min similar changes in flight velocity, lift, thrust, aerodynamic force, wingbeat frequency and tilting angle took place, independent of the volume of feeding solution. After increasing rapidly within 15 s a more or less steady phase of 60–80% of total flight time, showing only a slight decrease, was followed by a steeper, more irregular decrease, finally reaching 0 within 20–30 s. In steady phases lift was nearly equal to resultant aerodynamic force; tilting angle was 79.8±4.0°, thrust to lift radio did not vary, thrust was 18.0±7.4% of lift, lift was somewhat higher/equal/lower than body mass in 61.3%, 16.1%, 22.6% of all totally analysable flights (n=31). The following parameters were varied as functions of volume of feeding solution (5–20 l in steps of 5 l) and energy content. (18.5–74.0 J in steps of 18.5 J): total flight time, velocity, total flight distance, mean lift, thrust, mean resultant aerodynamic force, tilting angle, total flight impulse, wingbeat frequency, metabolic power and metabolic power related to body mass, the latter related to empty, full and mean (=100 mg) body mass. The following positive correlations were found: L=1.069·10^-9 f ^2.538; R=1.629·10^-9 f ^2.464; P _m=7.079·10^-8 f ^2.456; P _m=0.008v+0.008; P _m=18.996L+0.022; P _m=19.782R+0.021; P _m=82.143T+0.028; P _m=1.245·bm _f ^1.424 ; P _{mrel e}=6.471·bm _f ^1.040 ; =83.248+0.385. The following negative correlations were found: V=3.939–0.032; T=1.324·10^-4–0.038·10^-4. Statistically significant correlations were not found in T(f), L(), R(), f(), P _m(bm _e), P _{m rel e}(bm _e), P _{m rel f}(bm _e), P _{m rel f}(bm _f).Abbreviations A(m²) frontal area - bl(m) body length - bm(mg) body mass - c(mol·1^-1) glucose concentration of feeding solution - c _D (dimensionless) drag coefficient, related to A - D(N) drag - F _w(N) body weight - F _wp weight of paper fragment lost at flight start - f wingbeat frequency (s^-1) - g(=9.81 m·s^-2) gravitational acceleration - I(Ns)=R(t) dt total impulse of a flight - L(N) lift vertical sustaining force component - P _m(J·s^-1=W) metabolic power - P_{m ret} (W·g^-1) metabolic power, related to body mass - R(N) resultant aerodynamic force - Re v·bl·v ^-1 (dimensionless) Reynolds number, related to body length - s(m) v(t) dt virtual flight distance of a flight - s(km) total virtual flight distance - T (N) thrust horizontal force component of horizontal flight - T _a (°C) ambient temperature - t(s) time - t _tot (s or min) total flight time - v(m·s^-1) flight velocity - v(l) volume of feeding solution - W (J) energy and energy content of V - ( °) body angle of attack between body longitudinal axis and flow direction - ( °) tilting angle ( 90°) between R and the horizont in horizontal flight v(=1.53·10^-5m²·s^-1 for air at 25°) kinematic viscosity - (=1.2 kg·m^-3 at 25°C) air density     

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper

A potent toxin with phospholipase A₂ (PLA₂) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD₅₀ i.p.) of 0.1 mg/kg body weight, was the most toxic PLA₂ so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA₂ toxin (RVV-PFIIc) was shown to be different from other PLA₂s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA₂s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA₂ activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA₂ isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Delta endotoxin inhibits Rb+ uptake,lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells

Summary Delta endotoxin, a 68 kilodalton protein isolated fromBacillus thuringiensis spp.Kurstaki, is a potent entomocidal agent that alters a K⁺ current across midgut tissue of many phytophagous insects. This toxin completely inhibited the vanadate-sensitive⁸⁶Rb⁺ uptake and mimicked the vanadate-induced decrease in cytosolic pH in a cell line (CHE) originating fromManduca sexta embryonic tissue. The toxin also inhibited a K⁺-sensitive-ATPase in the plasma membranes isolated from these cells. Using the K⁺-sensitive-ATPase substratp-nitrophenyl phosphate, delta endotoxin was found to have aK _i of 0.4 m. These data suggest that the toxin inhibits a K⁺-ATPase responsible for⁸⁶Pb⁺ uptake in the CHE cells. The relationship between the toxin inhibition of K⁺-ATPase and toxin-altered K⁺ current is discussed.     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices

The adenosine A₁ receptor selective agonist, N ⁶-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [³H]inositol phosphates ([³H]InsPs), but not the total levels of membrane [³H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 g/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [³H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A₁ receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the G_i/G_o family.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.