Effect of thiouracil in modifying folate function in severe vitamin B12 deficiency.

The effects of thiouracil in correcting defects in folic acid function produced by B12 deficiency were studied. Addition of the thyroid inhibitor, thiouracil, to a low methionine diet containing B12, increased the oxidation of [2-14C]histidine to carbon dioxide, and increased liver folate levels. Addition of 10% pectin to the diet accentuated B12 deficiency as evidenced by a greatly decreased rate of histidine oxidation (0.19%) and an increased excretion of methylmalonic acid. Addition of thiouracil to the diet restored folate function as measured by increased histidine oxidation and increased liver folate levels similar to that produced by addition of methionine to a B12-deficient diet. Thiouracil decreased methylmalonate excretion, and increased hepatic levels of B12 in animals on both B12-deficient and -supplemented diets. Hepatic methionine synthase was increased by thiouracil, which may be the result of the elevated B12 levels. S-Adenosylmethionine and the enzyme methionine adenosyltransferase were also increased by thiouracil. Thus it is possible that the effect of thiouracil in increasing folate function consists both in the effect of thiouracil in decreasing levels of methylenetetrahydrofolate reductase, and also in its action in increasing S-adenosylmethionine which exerts a feedback inhibition of this enzyme.

Catabolism of arginine by the mixed bacteria in human salivary sediment under conditions of low and high glucose concentration.

The catabolism of glucose by the oral mixed bacteria results in a lowering of the pH whereas arginine degradation favours a rise. In the mouth, low and high levels of glucose cause different plaque pH conditions which, in turn, might affect the rate and mode of degradation of arginine. This possibility was examined in the suspended salivary-sediment system where these pH conditions can be simulated. With the pH, the metabolic parameters examined were arginine utilization, ammonia, carbon dioxide and putrescine formation, utilization of glucose and changes in levels of L(+)- and D(-)-lactic acid. At the lower glucose concentration, the pH rapidly fell and then slowly rose whereas, with the higher glucose level, the pH showed a greater fall and no subsequent rise. The more acidic pH conditions favoured by the higher glucose level inhibited arginine degradation and the appearance of its various end-products and intermediates. Arginine degradation with arginine-[U-14C] and paper chromatography-autoradiography showed successive appearance of citrulline, ornithine and putrescine and, depending upon the pH, some succinate. When the pH was held constant at several different values, arginine degradation was optimal when the pH was near neutrality. In supplementary experiments, arginine had little effect on the ability of the oral mixed bacteria to utilize glucose and produce and utilize lactic acid, whereas the arginine peptide, arginylisoleucine and saliva supernatant stimulated these processes. Thus glycolysis enhancement and a more rapid clearance of fermentable carbohydrate by the oral bacteria would accompany pH-rise activity with arginine peptide and saliva but would not accompany pH-rise activity with arginine.

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.

Influence of hypothalamic and ambient temperatures on sleep in kangaroo rats.

Unanesthetized, unrestrained kangaroo rats (Dipodomys) were studied to examine the changes in the frequency and duration of sleep states caused by long-term manipulations of hypothalamic temperature (Thy) at a thermoneutral (30 degrees C) and a low (20 degrees C) ambient temperature (Ta). A cold stimulus present in either the hypothalamus or the skin decreased both the total sleep time (TST) and the ratio of paradoxical sleep (PS) to TST. At a low Ta, TST, but not the PS-to-TST ratio, was increased by raising Thy, indicating that a cold peripheral stimulus could differentially inhibit PS. At a thermoneutral Ta, cooling Thy decreased both TST and the PS/TST. Changes in the amount of PS were due largely to changes in the frequency, but not the duration, of individual episodes of PS, suggesting that the transition to PS is partially dependent on the thermoregulatory conditions existing during slow-wave sleep (SWS). These results are consistent with the recent findings that the thermoregulatory system is functional during SWS but is inhibited or inactivated during PS.

Accelerated mineralization of two organophosphate insecticides in the rhizosphere.

Numerous xenobiotic compounds, including the organophosphate insecticides O, O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate (diazinon) and O, O-diethyl-O-p-nitrophenyl phosphorothioate (parathion), appear to be degraded in the soil environment by an initial cometabolic attack. Comparing the mineralization rates of radiolabeled diazinon and parathion in root-free and in rhizosphere soil, we tested our hypothesis that, because of the presence of root exudates, the rhizosphere is an especially favorable environment for such co-metabolic transformations. The insecticides were added individually at 5 mug/g to sealed flasks containing either soil permeated by the root system of a bush bean plant or identical soil without roots. Periodically, the flask atmospheres were flushed through traps and the evolved (14)CO(2) was quantitated. Bush bean plant roots without associated rhizosphere microorganisms failed to produce a significant amount of (14)CO(2). During 1 month of incubation, rhizosphere flasks mineralized 12.9 and 17.9% of the added diazinon and parathion radiocarbon, respectively, compared to 5.0 and 7.8% by the soil without roots. The mineralization of parathion but not of diazinon was stimulated in a similar manner when soil without roots was repeatedly irrigated with a root exudate produced in aseptic solution culture. Viable counts of microorganisms on soil extract agar were not significantly altered by root permeation or by root exudate treatment of the soil, leaving population selection and/or enhanced cometabolic activity as the most plausible interpretations for the observed stimulatory effects. Rhizosphere interactions may substantially shorten the predicted half-lives of some xenobiotic compounds in soil.

Oxygen toxicity: comparative sensitivities of membrane transport, bioenergetics and synthesis in Escherichia coli.

The oxygen sensitivities of basic cell functions were compared to evaluate their significance as potential causes of the reversible growth inhibition produced in Escherichia coli by exposure to hyperbaric oxygen. Growth and net incorporation of radioactive glucose into cell structure, and specifically in to protein, were completely inhibited in approximately 1/20 of a generation by a gas phase containing 4.2 atmospheres of oxygen. The inhibition occured before there was significant decrement in cellular glucose transport, respiration, or intracellular concentration of adenosine triphosphate. The data indicate that fundamental steps leading to protein biosynthesis from glucose should be examined in the search for specific primary sites of oxygen toxicity.

Some comparative observations on the relative contributions of alternate pathways in the metabolism of glucose by Candida utilis.

Candida utilis was grown in batch, chemostat, and continuously synchronised (phased) culture on a nitrogen-limited glucose mineral salts medium: phosphorus- and carbon-limited phased cultures were also used. The 14CO2 evolved from [G-1-14c]and [G-6-14c] was used, as a simple C1/C6 ratio, to observe the relative changes in EMP and HMP contributions during growth of the cultures. The ratio varied during the cell cycle, and changed with growth rate, and with nutrient limitation. The changes generally indicated that the HMP predominated, most notably in the early part of the batch-growth sequence and early in the cell cycle. The overall results reflected the relative merits of the different cultivation techniques for examining microbial metabolism: the advantage of a greater resolution by the synchronised method, based upon the unit performance rather than the randomised mean performance of the cell population...

Factors influencing the production of cellulases by Sporotrichum thermophile.

Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi.

Compartmentation and control of arginine metabolism in Neurospora.

The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.

Role of arginine deiminase in growth of Mycoplasma hominis.

Arginine has been considered as the major energy source of nonglycolytic arginine-utilizing mycoplasmata. When three strains of Mycoplasma arginini, and one strain each of Mycoplasma arthritidis, Mycoplasma fermentans, Mycoplasma gallinarum, Mycoplasma gallisepticum and Mycoplasma hominis were grown in the medium with high arginine concentration (34 mM) compared with low arginine (4 mM), both the protein content of the organisms and the specific activity of arginine deiminase increased. M. fermentans, the one arginine-utilizing species included in the survey which is also glycolytic, showed an increase in protein content but no increase in specific activity of the enzyme. The glycolytic non-arginine-utilizing M. gallisepticum did not show an increase in either parameter. The Km for arginine deiminase from crude cell extracts was 1.66 X 10(-4)M. The enzyme demonstrated a hyperbolic activation curve subject to substrate inhibition and was not affected by the presence of L-histidine. When mycoplasmic protein and arginine deiminase were determined for M. hominis under aerobic and anaerobic conditions, aerobically grown cells exhibited no detectable enzymatic increases until late in log phase. Higher levels of arginine deiminase were observed earlier in the anaerobic growth cycle. The rate of 14CO2 evolution from [guanido-14C]arginine was not altered in arginine-supplemented cells compared with cells grown in low arginine. In addition, CO2 production did not parallel increased arginine deiminase activity. These observations argue that arginine is used only as an alternate energy source in these organisms.

Failure of complex supplementation of minimal cultures to elicit a shift-up response in Pseudomonas putida.

The addition of complex supplements (particularly amino acids) to cultures of Pseudomonas putida growing on a good carbon source did not result in a substantial increase in the growth rate. Amino acids entered the cells within 30 s of addition and reached significant internal pool concentrations. Endogenous amino acid biosynthesis was quickly inhibited (about 75%), with a substantial sparing of the original carbon source. Within 20 min of supplementation significant respiration of added amino acids was detected, yet the ATP pool size did not increase and the bacteria did not grow faster. The RNA content of P. putida growing in complex medium differed from that of enteric bacteria in that, although it varied with growth rate, it was not substantially larger than the RNA content of bacteria grown in a minimal medium with a good carbon and energy source. The rate of RNA accumulation on shift-up remained substantially unchanged on supplementation if the minimal medium had a carbon source producing fast growth, and did not increase for about 30 min if the carbon source was relatively poor. In other respects RNA synthesis was similar to that of the enteric bacteria, being stringently controlled, inhibited by trimethoprim and continuing in the presence of chloramphenicol. It is proposed that growth of P. putida in complex media is limited by the rate of synthesis of stable RNA.

Effects of temperature and crude oil composition on petroleum biodegradation.

The biodegradability of seven different crude oils was found to be highly dependent on their composition and on incubation temperature. At 20 C lighter oils had greater abiotic losses and were more susceptible to biodegradation than heavier oils. These light crude oils, however, possessed toxic volatile components which evaporated only slowly and inhibited microbial degradation of these oils at 10 C. No volatile toxic fraction was associated with the heavier oils tested. Rates of oil mineralization for the heavier oils were significantly lower at 20 C than for the lighter ones. Similar relative degradation rates were found with a mixed microbial community, using CO2 evolution as the measure, and with a Pseudomonas isolate from the Arctic, using O2 consumption as the measure. The paraffinic, aromatic, and asphaltic fractions were subject to biodegradation. Some preference was shown for paraffin degradation, especially at low temperatures. Branched paraffins, such as pristane, were degraded at both 10 and 20 C. At best, a 20% residue still remained after 42 days of incubation. Oil residues generally had a lower relative percentage of paraffins and higher percentage of asphaltics than fresh or weathered oil.

Presence of nonoxidative enzymes of the pentose phosphate shunt in Tetrahymena.

Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-14C]ribose into CO2 and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.

Sugar metabolism in transketolase mutants of Escherichia coli.

This paper continues the description of transketolase mutants of Escherichia coli; they are absolutely unable to grow on pentoses, but slightly "leaky" with respect to their aromatic requirement (B. L. Josephson and D. G. Fraenkel, 1969). Several experiments have explored the degree of leakiness and shown it to be low. There is little conversion of radioactive xylose to carbon dioxide. The labeling of ribose in cells grown on [1-(14)C]glucose and [2-(14)C]glucose accords with its origin being chiefly by the oxidative pathway. A mutant lacking both transketolase and gluconate-6-phosphate dehydrogenase has been constructed; it requires supplementation with pentose. Pentoses are inhibitory to growth of transketolase mutants, but high levels of pentose phosphates do not accumulate in this situation. Several experimental results are suggestive of regulation of metabolic flow in the oxidative branch of the hexose monophosphate shunt.