Oxidation of pipecolic acid in soils and in rhizosphere soil of different plants

Soil respiration was proportional to its total carbon content. Maximum respiratory activity occurred in garden soil, followed in descending order by chernozem soil, brown soil, and sand. The oxidation of pipecolic acid, as studied by the Warburg manometric technique, in different rhizosphere soils from four crops 7, 13 and 20 days after planting as well as from one crop grown in different soils, was consistently in all cases faster than that by the corresponding non-rhizosphere soils. The curves of the rate of oxygen consumption during pipecolic acid oxidation, by garden soil (whether rhizosphere or non-rhizosphere soil) as well as by chernozem rhizosphere soil of different plants at the three stages of plant growth studied contained two peaks (two phases), whereas in non-rhizosphere chernozem soil as well as in brown soil and sand (whether affected or not affected by plant roots) only one peak was attained in the curves of the rate of oxygen uptake. The rapidity with which pipecolic acid was oxidized in the rhizosphere soil differed from plant to plant and at different phases of plant growth, and also with the type of soil used for plant growing. The extent of pipecolic acid oxidation after the first and second (if it occurred) phases did not differ in the different soils, both rhizosphere and non-rhizosphere soil, but the rate of oxygen uptake was higher in rhizosphere than in the corresponding non-rhizosphere soil. During the first phase, oxygen uptake accounted for slightly less than one-third of the total amount of oxygen required for complete oxidation of the added pipecolic acid. About two-thirds of that total amount were taken up during the both phases of oxidation.     

Oxidation of aromatic compounds in soil

The oxidation ofp-hydroxybenzoic acid, quinic acid, vanillin and coumarin in soil was studied. With vanillin, and particularly with coumarin, the lag phase for oxygen consumption was longer and the rate of oxygen consumption attained more than one peak. In soil preincubated with the relevant substrate, the second dose of the same substrate was oxidized more rapidly. If the soil was preincubated with glucose, the lag phase was also shortened and oxygen consumption was raised with all aromatic substrates.     

Utilization of glucose and cellobiose by the microflora of soil enriched with cellulose

The ability of soil microflora to utilize glucose or celloboise was found to depend on previous incubation of the soil with glucose, celloboise or cellulose. Glucose was utilized more rapidly than cellobiose in soil preincubated with glucose or cellobiose. The opposite situation was observed in soil preincubated with cellulose. In the presence of a mixture of both sugars the rate of utilization of one of them was decreased by the second and this decrease could be characterized as competitive inhibition. Glucose accumulated in the medium during utilization of cellobiose alone in soil preincubated with cellulose. This phenomenon was not observed during the utilization of cellobiose in soil preincubated with glucose or cellobiose.     

Effect of glucose on decomposition of vanillin by soil microorganisms

Oxygen consumption for the oxidation of vanillin in a soil suspension and in structural chernozem samples was accelerated in glucose-treated variants. The effect was observed on adding glucose and vanillin simultaneously and after 16 hours' preincubation of the soil with glucose. Glucose degradation was accompanied by an increase in the proportion of bacteria capable of utilizing vanillin as the sole carbon source, as well as by a general increase in the number of microorganisms. With some of these bacterial strains, in a given pH range, glucose induced the ability to oxidize vanillic acid, or at least shortened the lag phase of oxygen consumption for oxidation of this intermediate product of vanillin decomposition. Glutamic, malic, succinic and pyruvic acid and glycine, ribose and fructose were found to have a similar effect to glucose on the oxidation of vanillin in washed bacterial cell suspensions and on the incidence of vanillin decomposers in soil.     

Intake of Hydrolyzed Casein is Associated with Reduced Body Fat Accretion and Enhanced Phase II Metabolism in Obesity Prone C57BL/6J Mice

The amount and form of dietary casein have been shown to affect energy metabolism and lipid accumulation in mice, but the underlying mechanisms are not fully understood. We investigated 48 hrs urinary metabolome, hepatic lipid composition and gene expression in male C57BL/6J mice fed Western diets with 16 or 32 energy% protein in the form of extensively hydrolyzed or intact casein. LC-MS based metabolomics revealed a very strong impact of casein form on the urinary metabolome. Evaluation of the discriminatory metabolites using tandem mass spectrometry indicated that intake of extensively hydrolyzed casein modulated Phase II metabolism associated with an elevated urinary excretion of glucuronic acid- and sulphate conjugated molecules, whereas glycine conjugated molecules were more abundant in urine from mice fed the intact casein diets. Despite the differences in the urinary metabolome, we observed no differences in hepatic expression of genes involved in Phase II metabolism, but it was observed that expression of Abcc3 encoding ATP binding cassette c3 (transporter of glucuronic acid conjugates) was increased in livers of mice fed hydrolyzed casein. As glucuronic acid is derived from glucose and sulphate is derived from cysteine, our metabolomic data provided evidence for changes in carbohydrate and amino acid metabolism and we propose that this modulation of metabolism was associated with the reduced glucose and lipid levels observed in mice fed the extensively hydrolyzed casein diets.     

Effect of exposure to diabetic and fasted plasma on glucose oxidation in rat aorta

Glucose metabolism is depressed in aortic intima-media of fasted and diabetic rats. The aim of this study was to elucidate the influence of diabetic and fasted plasma on glucose oxidation in rat aorta. Male Sprague-Dawley rats weighing about 200 g were used. Diabetes was induced by streptozotocin (65 mg/kg) and the rats were used after a diabetes duration of two weeks. Fasted rats were used after food deprivation for 3 days. Aortic intima-media was preincubated in plasma for 120 or 240 min. During a further incubation for 2 hours in Krebs-Henseleit bicarbonate buffer the oxidation of 14C-glucose to 14CO2 was measured. Preincubation of normal aorta in diabetic or fasted rat plasma and diabetic human plasma significantly depressed the subsequently determined glucose oxidation in comparison to aorta preincubated in normal plasma. Preincubation of aorta from diabetic or fasted rats in normal rat plasma enhanced the glucose oxidation compared with the glucose oxidation in aorta of diabetic or fasted rats after preincubation in the corresponding plasma. These results suggest that diabetic and fasted plasma contains factor(s) which in vitro depress glucose oxidation in vascular smooth muscle and, thus, may be of importance for the lowered glucose oxidation found in vascular smooth muscle preparations obtained from diabetic or fasted animals.     

Studies on the respiratory metabolism of isolated human adipose cells

1. Some metabolic characteristics of fat cells isolated from 50 patients were examined. 2. The respiratory activity of human fat cells was of the same order of magnitude as cells obtained from the rat, whether the comparison was based on the molar triglyceride content of cells or the nitrogen content of tissue. 3. There was no difference in the respiratory metabolism of fat cells isolated from three separate age groups: 20-40, 40-60 and 60-80 years. 4. The respiratory activity of human adipose cells was partially inhibited by malonate (0.1m), arsenite (1mm) and iodoacetate (1mm). 5. Oxygen consumption by human adipose cells could not be stimulated by adding glucose or glucose plus palmitic acid to the medium. However, stimulation of oxygen uptake with these substrates was observed after the tissue had been preincubated for 5hr. in a bicarbonate buffer. 6. After the tissue had been preincubated for 5hr., there was a linear relationship between the oxygen uptake by isolated cells and the amount of oleic acid added to the incubation medium. Stimulation of oxygen uptake by oleic acid (0.95mumole/ml.) was dependent on the presence of glucose (5.6mm) in the incubation medium. 7. Of the total [1-(14)C]palmitic acid metabolized by human adipose cells, over 99.8% of the radioactivity was recovered in the neutral-lipid fraction and less than 0.2% in carbon dioxide. Under conditions where oxygen uptake is stimulated by [(14)C]-palmitic acid, the radioactivity of the 1,2-diglyceride pool increased 20-fold.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase.

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.     

Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures

Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply.     

Presence and oxidation of amino acids in rhizosphere and non-rhizosphere soil

Manometric studies were carried out on the respiratory activity of different rhizosphere and non-rhizosphere soils to follow quantitatively the over-all microbial activity in the rhizosphere soil as affected by the species and growth phase of plant. Oxygen consumption was distinctly greater in rhizosphere soils as compared to that in non-rhizosphere soils. The difference between oxygen consumption by rhizosphere and non-rhizosphere soils changed with the course of plant growth and it was not the same in different plants. This difference in oxygen consumption might be a measure of the amount of available oxidizable substrate in the rhizosphere. A rhizosphere sample had greater diversity as well as higher concentration of free amino acids than a non-rhizosphere sample of the same soil. Bacterial counts pointed to preferential stimulation in the rhizosphere of bacteria requiring individual amino acids. amino acids, such as glycine, alanine, asparsic acid of tyrosine were oxidized more rapidly in rhizosphere than in non-rhizosphere soil, but the extsent of oxidation for each of the amino acids studied did not differ. The amount of oxygen consumed during oxidation of alanine, aspartic acid or tyrosine was about one-half of the total amount necessary for complete oxidation. With glycine a higher extent of oxidation (60%) was observed. These extents of oxidation of glycine and aspartic acid did not change on investigation at two different phases of plant growth.     

Accumulation and metabolism of pipecolic acid in the brain and other organs of the mouse

The long-term accumulation of pipecolic acid, as well as its disappearance following exogenous administration was studied in brain and other organs of the mouse. Mice were pulse-injected intraperitoneally or intravenously with 1Ci[³H]D,l-pipecolic acid (6.9 nmol/mouse=2.9 g/kg). The total radioactivity retained in tissues was measured in brain, liver, and kidney, as well as in plasma during the period 1 min to 24 hr. TLC separation of DNP-derivatives was performed. Three features of the pattern of retention of pipecolic acid are most salient; first the rapid accumulation in brain, second the rapid secretion of this compound in the urine, and third the long-lasting steady levels of radioactivity maintained in brain.Sixty minutes after i.v. injection, the brain/plasma ratio is approximately 0.2 and approaches unity only at 5 hr. Following intraperitoneal injection the percent recovered as pipecolic acid in brain is 78% at 30 min and 71% at 120 min, suggesting a slow metabolic activity. Liver shows a different trend than brain with a slower accumulation and a faster disappearance. Kidney shows a pattern similar to plasma with a rapid secretion of radioactivity into urine which correlates well with the exponential decrease in plasma and urine. The administration of probenecid significantly increases radioactivity due to pipecolic acid in brain, liver, and urine. Formation of -aminoadipic acid, a known metabolite of pipecolic acid, can be demonstrated in kidney 30 min after intraperitoneal injection. The present data together with results obtained previously with intracarotid injections suggest that pipecolic acid is taken up in the mouse brain from the circulation. Most of the pipecolic acid taken up is rapidly removed through the circulation and secreted in the urine; however, a small part is retained and probably metabolized by brain and kidney.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.     

Role of calcium in acetylcholine-induced desensitization in dog thyroid slices

Incubation of dog thyroid slices with 1 microM acetylcholine (ACH) for 3 h followed by a second 2-h incubation without it induces a diminution of stimulation of glucose oxidation by ACH during a third incubation of 45 min. Using a calcium-free medium during all incubations prevents the desensitization and reduces, but does not abolish, ACH stimulation of glucose oxidation. EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] (2 mM) added to the calcium-free medium in all incubations prevents both refractoriness and stimulation of glucose oxidation induced by ACH. Calcium depletion during the first incubation only, achieved by using EGTA and a calcium-free medium, also prevents refractoriness but not the augmentation of glucose oxidation caused by ACH. Incubation of thyroid slices with 1 microM ionophore A23817 during the 3-h first incubation decreases the stimulation of glucose oxidation induced by its readdition or by 1 microM ACH added for the first time in the third incubation. Ionophore-induced desensitization is not related to a cholinergic muscarinic receptor effect. Initial incubation of dog thyroid slices with 1 microM ACH diminishes the subsequent stimulation of glucose oxidation by 0.5 microM ionophore. However, the ACH-induced desensitization to ionophore can be overcome by a 10-fold increase in the amount of ionophore in the third incubation. Ionophore (1 microM) in the first incubation also induces refractoriness to thyroid-stimulating hormone (TSH) (10 mU/ml)-stimulated glucose oxidation in the third incubation. In contrast, initial incubation of thyroid slices with TSH (25 mU/ml) does not affect the stimulation of glucose oxidation by 0.5 microM ionophore added during the third incubation. These results suggest that increased intracellular calcium plays a major role in, or even mediates, ACH-induced desensitization in the thyroid gland.     

On the ability of galactose to influence hexose and amino acid uptake by isolated rat brain capillaries

The uptake of glucose, 2-deoxyglucose, proline, methionine, and alpha-methylamino isobutyric acid was studied in brain capillaries preincubated with 50 mM galactose either in vitro or isolated from galactose-fed rats. The uptake was not decreased in both cases. The linear rate of glucose oxidation by capillaries was also not altered by preincubation with galactose.     

Course of minimal oxygen consumption in the young rat during protein energy malnutrition followed by return to a balanced diet

Growth and resting oxygen consumption (VO2 minimal) were studied on two strains of six post-weaning male rats during 60 days. The first strain was fed with a balanced diet (15% casein) for 60 days, the second received a 2% casein diet for 30 days, and then the balanced diet for the remaining 30 days. The 2% casein diet completely stopped the body weight and VO2 minimal increases. When return to a balanced diet, these parameters show an evolution similar to that of controls of the same body weight. The VO2 minimal had the same kind of evolution as body weight and was well correlated with growth speed. The protein restriction involves a strong and temporary reduction of the VO2 minimal, which will correspond to a nutritional stress.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Biological decomposition of fulvic acid preparations

Decomposition of preparations of various fractions of fulvio acids in pure cultures of bacteria and in the soil was investigated. In the soils enriched with fulvic acids the amount of bacteria increased, oxygen consumption and formation of carbon dioxide followed a typical sigmoid curve. The above measurements indicated that mineralization occurred after a very short or negligible lag phase. During the decomposition of fulvic acids the ability of microorganisms to oxidize aromatic compounds,e.g. vanillio andphydroxybenzoic acid, increased. The presence of aromatic structures in the used preparations of fulvic acids was demonstrated on the basis of their IR spectra and according to the results of Chromatographic analyses of their hydrolysates. The results indicated a relationship between metabolism offulvio acids and aromatic oompounds. In samples of the soil preincubated with glucose the fulvic acids decomposed more rapidly than in untreated samples.     

Amino Acid Requirements and Proteolytic Activity of Streptococcus sanguis

The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences.