Joint action of l-leucine and sodium phosphate buffer solutions as feeding stimulants for protein-deprived females of the house fly Musca domestica

ABSTRACT. Potentiation in joint action was demonstrated between solutions of L-leucine and sodium phosphate buffer (pH 6.3) as feeding stimulants for protein-deprived females of the house fly, Musca domestica L. Both components alone elicited feeding. In two-choice feeding tests, mixtures consisting of equi-stimulating concentrations of the two components were taken in greater quantities than either component alone at twice the concentration in the mixture.
The presence of 1×10^-1 M phosphate buffer markedly lowered the threshold for detection of L-leucine. The presence of phosphate buffer strengthened the preferences shown by flies given choices of concentrations of L-leucine differing by a factor of 2 and enabled them to display preferences at lower concentrations.
The presence of 1×10^-3 M L-leucine increased, somewhat, the ability of flies to detect low concentrations of phosphate buffer. Its presence had relatively little effect on the strength of preference shown between two-fold differences in concentration of phosphate buffer when the higher concentration was 6.3×10^-3 M or less, but markedly strengthened the preferences when the higher concentration was 2.5×10^-2M or greater. Leucine increased the optimal concentration of phosphate buffer by a factor of more than 2 and converted 2×10^-1 M phosphate buffer from a mild feeding deterrent to a powerful feeding stimulant.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

The effect of calcium concentration on the toxicity of copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water

Yolk-sac fry of brown trout were exposed to three levels of single trace metals (Cu, 20,40,80 nmol 1^-1; Pb, 12·5,25,50 nmol 1^-1; Zn, 75,150,300 nmol 1^-1) typical of concentrations reported for acid soft waters, in flowing, artificial, soft water media maintained at pH 4·5 and [Ca] of 20 or 200 μmol 1^-1for 30 days.
Mortalities were high in fry subjected to all levels of the three trace metals at [Ca] 20 μmol 1^-1, with 80% of the total deaths occurring between days 11 and 15 of the experiment. 25% mortality occurred at low [Ca] and pH 4·5 in the absence of trace metals, with only one death recorded at [Ca] 200 μmol1^-1'(Cu, 80 nmol 1^-1). At high [Ca] all three levels of Cu and Pb impaired net Na and K uptake; Cu was the only metal to reduce the uptake of Ca. The Zn treatments had no significant effect on mineral uptake. Calcification of centra was reduced by all three Cu treatments at [Ca] 200 μmol 1^-1. The lowest Zn concentration (75 nmol 1^-1) was the only other treatment to impair skeletal development. In the absence of trace metals, low [Ca] significantly reduced Ca, Na and K uptake, skeletal calcification and dry mass at pH 4·5.
The deleterious effects of low Cu, Pb and Zn concentrations at low pH and low [Ca], and the ameliorative effect of higher ambient [Ca], are discussed in relation to fishery status in soft, acid waters.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Methods to extract NAD+-malate dehydrogenase efficiently from white spruce needles

The spectrophotometrically-determined activity of NAD⁺-malate dehydrogenase (MDH, EC 1.1.1.37) from white spruce [ Picea glauca (Moench) Voss] needles was assayed with NADH and oxaloacetate. Activity was very low when extracted with only acetate buffer (pH 5.4), phosphate buffer (pH 6.8), or Tris-HCl buffer (pH 8.0). However, activity increased from 1 to over 200 μmol (g dry weight)^-1 min^-1 with the addition of polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) and the detergents, Tween 80, Tergitol 15-S-9 and Triton X-100. Best activity was observed when extracted in a buffer at pH 6.8 and with 1% (v/v) for the three detergents and PEG, and 6% (w/v) for PVP.
MDH activity decreased with age of the needles on the tree. Six-year-old needles contained only about one-fifth of the activity of current year, fully-expanded needles. The main decrease in enzyme activity was observed in one-year-old needles. Protein content obtained from needles extracted with just phosphate buffer (pH 6.8) was very low, but increased greatly when the above chemicals were added to the buffer. In contrast with needles, extracts of vegetative buds contained much higher levels of MDH and protein when extracted with only phosphate buffer (pH 6.8). Although MDH activity in needle extracts declined with storage of the extracts at 4°C in the dark for 6 days, the decrease was least for buffers containing a combination of different protective agents.     

Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan

A new moderately halophilic Micrococcus sp. 4, isolated from salt-pan water from India, produced extracellular amylase when cultivated aerobically in medium containing wheat bran, peptone, beef extract and sodium chloride. Other salts, such as sodium nitrate, potassium nitrate and sodium sulphate, were also found to be suitable for growth and enzyme production. Maximum amylase activity (1.2 IU ml^-1) was secreted in the presence of 1 mol 1^-1 sodium chloride. The enzyme requires the presence of either sodium chloride, potassium chloride, sodium nitrate, sodium citrate or sodium acetate for its activity. Maximum activity was found in the presence of 1 mol 1^-1 sodium chloride. The pH and temperature optima for enzyme activity were 7.5 and 50°C, respectively.     

Oxidation of Arsenite by a Soil Isolate of Alcaligenes

A strain of Alcaligenes , isolated from soil and grown in nutrient broth in the presence of arsenite, possessed the ability to oxidize arsenite to arsenate. Washed cell suspensions consumed one-half mol of oxygen/mol of arsenite and produced arsenate. The optimum pH for arsenite oxidation was 7.0. The K_m for arsenite was 1.5 × 10^-4 M and V _max was 6.7 μl of oxygen/min. The arsenite-oxidizing enzyme system was induced by growth in arsenite. Response of the arsenite-oxidizing enzyme system to respiratory inhibitors suggested that electrons resulting from arsenite oxidation by an oxido-reductase with a bound flavin are transferred via cytochrome c and cytochrome oxidase to oxygen. The presence of the cytochromes in crude extract was confirmed by spectral measurements.     

Partial purification and biochemical properties of acid and alkaline phosphatases from Myxococcus coralloides D

F. GONZÁLEZ, M.E. FÁREZ-VIDAL, J.M. ARIAS AND E. MONTOYA. 1994. Acid phosphatase and alkaline phosphatase from vegetative cells of Myxococcus coralloides D were purified by two chromatographic steps. The molecular weights were estimated by gel filtration and SDS-PAGE. Optimum pH, stability, optimum temperature and thermal inactivation studies were made for both enzymes. EDTA and other chelating agents inhibited alkaline but not acid activity. Mg²⁺ activated the alkaline phosphatase, while the acid phosphatase was inhibited by fluoride. Both enzymes degraded a number of phosphomonoesters, but were unable to hydrolyse either polyphosphates or cAMP. The K _m values of the acid and alkaline phosphatases for p -nitrophenylphosphate were 5.0 times 10^-3 mol ***l^-1 and 1.5 times 10^-3 mol l^-1, respectively.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

THE IRREVERSIBLE INHIBITION OF BRAIN L-GLUTAMATE-I-DECARBOXYLASE BY (2RS,3E)-2-METHYL-3,4-DIDEHYDROGLUTAMIC ACID

Abstract— Incubation of chick embryo brain l -glutamate-1-dccarboxylase (GAD, EC 4.1.1.15) with (2RS,3E)-2-methyl-3,4-didehydroglutamic acid (MDG), a substrate analog of l -glutamic acid, results in a time-dependent irreversible inhibition of the enzymic activity. In the presence of 2.0 ± 10^-3 m inhibitor the half-life for inactivation is 11.6min. The inhibitor is a substrate for GAD and requires turnover prior to inactivating the enzyme and is therefore another example of the k _cat class of inactivator. The measured K _l is 6.6 ± 10^-4 m and the k _cat for its turnover is 1.01 ± 10^-3 s-¹ at 37°C (pH 7.2). The inhibitor has no effect on the apoenzymc or the holoenzyme treated with 1.0 ± 10^-3 m hydrazinc. Both l -and d -glutamate, but not mercaptoethanol, reduce the rate of enzymie inactivation by the inhibitor. The exceedingly high specificity implicit in the design of this inhibitor should render it useful in studies designed to uncover the physiological role of GABA.     

Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate

Barley plants were grown in nutrient solutions, which were maintained at either 0 (-P) or 15 μ M orthophosphate (+P). After 11 days phosphate influx into the intact roots of the -P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the -P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)^-1h^-1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r²= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of -P plants was rapidly reduced by the provision of 15 μ M orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)^-1 h^-1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)^-1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.     

Thermal resistance of partially purified proteinase of Pseudomonas fluorescens P-26

Heat resistance of the Pseudomonas fluorescens P-26 proteinase in terms of D -value was studied in whole milk, skim milk, whey and 0.05 mol 1^-1 phosphate buffer at 72.5, 130, 135, 140, 145 and 150°C subsequent to its partial purification through (NH₄)₂ SO₄ precipitation (45–65% saturation) and solvent fractionation with 1.0 to 2.0 volumes of isopropanol. The D -value was maximum for the proteinase at all temperatures when determined in whole milk ( D ₁₅₀= 0.088).     

The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal

Rose Bengal was cytotoxic to the following bacteria at the concentrations given in parentheses (highest concentrations of dye in mol/1 at which growth occurred on nutrient medium): Brochothrix thermosphacta and Deinococcus radiodurans (1 times 10^-6 or less); Streptococcus, Micrococcus, Staphylococcus, Bacillus, Arthrobacter and Kurthia spp. (1 times 10^-5–1 x 10^-4), and Pseudomonas spp. and Enterobacteriaceae (5 times 10^-3–1 x 10^-2 or greater). These organisms were killed rapidly when suspended in illuminated (170 μE/m²/s) solutions of Rose Bengal (1 times 10^-4 mol/1) providing oxygen was present. Singlet oxygen was identified as the lethal agent, because the rate of killing was increased by dissolving the dye in deuterium oxide while the organisms were protected against photoinactivation by L-histidine or crocetin. Yeasts from chilled foods were killed in illuminated solutions of Rose Bengal but a light intensity of 315 μE/m²/s was needed for a death rate comparable with that of bacteria. The yeasts present in a range of chilled meat and dairy products failed to form colonies on Rose Bengal (5 times 10^-5 mol/1) media exposed continuously to modest illumination (55–80 μE/m²/s).     

Purification and characterization of diacetyl reductase from Kluyveromyces marxianus

Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K _m and V _max values for diacetyl were 2.5 mmol 1^-1 and 0.026 mmol 1^-1 min^-1, respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa.     

Nutrient reserves of Salicornia europaea seeds

Seeds of Salicornia europaea L. were analyzed for their nutrient reserves. The content of potassium and sodium was 216 and 39 mmol (kg dry seeds)^-1, respectively. Calcium and magnesium accounted for 30 and 138 mmol (kg dry seeds)^-1, respectively. Whereas most of the alkali metals were water soluble, the alkaline earth metals were mostly acid soluble. The acid-soluble calcium plus magnesium corresponded well with the acid-soluble phosphate. Chloride was accumulated to a level equivalent to that of sodium. Carbonate was present at a concentration of 9 mmol (kg dry seeds)^-1. Carbohydrates accounted for 93 g (kg dry seeds)^-1, nearly half of which was derived from sucrose. Fructose and glucose were present only in traces. Total nitrogen was determined to be 55 g (kg dry seeds)^-1, 16% of which was diethylether soluble. The remaining nitrogen was separated into 39 g (kg dry seeds)^-1 ethanol-insoluble and 8 g (kg dry seeds)^-1 ethanol-soluble nitrogen. About 10% of the ethanol-soluble nitrogen were derived from amino acids. Total lipid content was about 280 g (kg dry seeds)^-1. The alcoholic component of the storage lipids was glycerol and the glycerides were calculated from gas chromatography to be 66% of the total lipids. About 90% of the fatty acids consisted of unsaturated acids, linoleic and oleic acid, the majority (77%) of which was linoleic acid.     

β-Amylase from Clostridium thermocellum SS8 - a thermophilic, anaerobic, cellulolytic bacterium

The extracellular amylase produced by Clostridium thermocellum strain SS8 on starch was characterized as a β-amylase based on blue value reduction test and the production of maltose from starch. The enzyme had a temperature and pH optima of 60°C and 6.0, respectively. Of the metal ions tested, Ca^{2 +} and Mg^{2 +} had little effect on enzyme activity, but their presence increased its thermal stability. Ca^{2 +} displayed a higher stabilizing effect and at 10 mmol 1^-1 Ca^{2 +}, the enzyme retained 86% activity even after exposure at 70°C for 30 min. The amylase was induced on starch or maltose but was repressed strongly by glucose.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol 1^-1 phosphate, and this appeared to be related to decreased rates of O₂ consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol 1^-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol 1^-1 may not be appropriate in CTC and INT reduction assays.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.