Capsidiol: Its role in the resistance of Capsicum annuum to Phytophthora capsici

Inoculation of the stems of three Capsicum annuum L. cultivars showing different degrees of sensitivity to the fungal pathogen Phytophthora capsici , resulted in a hypersensitive reaction being expressed along the stems. One of the peppers (cv. Smith-5) showed resistance by total inhibition of fungal growth. Capsidiol, a phytoalexin, which accumulates in the area of necrosis appears to be involved in this resistance. Capsidiol accumulation was analyzed by gas chromatography and was correlated with the restricted growth of P. capsici , in vivo and in vitro, confirming the former's fungistatic and fungitoxic properties. The capacity to inhibit pathogenic growth was evident only when capsidiol production exceeded 1 204 μg ml^-1, a level reached in the resistant variety after 6 days of incubation. Experiments on induced resistance showed that a second inoculation of the stems of the three cultivars also resulted in necrosis and in an accumulation of capsidiol, although to a lesser extent than in the first inoculation. The greater accumulation of capsidiol in the stems of cv. Smith-5 is in accordance with the resistance shown by this cultivar to P. capsici , and confirms the implication of capsidiol in the disease resistance of this cultivar to fungal pathogens. Capsidiol has a fungistatic character at a mean concentration of 3.75 mM, and is fungitoxic at levels above 5 mM. This level must be exceeded and all the growing hyphae must be affected for capsidiol to qualify from being fungistatic to being fungitoxic.     

Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii

Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min⁻¹ mg⁻¹ protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min⁻¹ mg⁻¹ protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F₄₂₀ showed only low activity as electron acceptor.     

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics

Abstract The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S -transferase, UDP-glucuronosyltransferase, UDP-glucosyltransferase, and N -acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min⁻¹ mg⁻¹), UDP-glucosyltransferase (0.27 nmol min⁻¹ mg⁻¹) and glutathione 5-transferase (20.8 nmol min⁻¹ mg⁻¹). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min⁻¹ mg⁻¹) and N -demethylation (0.17 nmol min⁻¹~' mg⁻¹) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min⁻¹ mg⁻¹) and the microsomes (0.13 nmol min⁻¹ mg⁻¹). N -Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120

Abstract Whole cells of the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120 exhibited K _m values for l -glutamine and l -glutamate of 33 μM and 0.5 mM, respectively. V _max of uptake was ca. 30 nmol mg⁻¹ (chlorophyll) min⁻¹ for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is involved in glutamine and glutamate uptake by this cyanobacterium.     

Pyruvate Carboxylase Activity in Primary Cultures of Astrocytes and Neurons

Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min⁻¹· mg⁻¹ protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min⁻¹· mg⁻¹). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.     

Isolation and characterization of two distinct membrane fractions from the Gram-positive nucleotide producer Brevibacterium ammoniagenes ATCC 6872

Abstract Bouyant density gradient centifugation of 'crude membranes' of the coryneform bacterium Brevibacterium ammoniagenes yielded two distinct membrane fractions which differed significantly in equilibrium densities (1.15 and 1.18 g/cm³), NADH dehydrogenase activity (0.29 and 0.09 μmol·min⁻¹·mg⁻¹), protein composition and association of ribosomes. As determined by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proteins unique to either the free membrane (FM) fraction or the denser ribosome-containing complexed membrane (CM) fraction were identified.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.