Effects of deuterium oxide on folate metabolism in Lactobacillus casei.

Various folate coenzymes and their polyglutamyl derivatives involved in 1-carbon metabolism are modulated as a result of altered physiological states and also vary with respect to growth conditions. We studied the metabolic changes in folic acid and their conjugated polyglutamyl derivatives in Lactobacillus casei cells grown in the presence of D20. A 40% decrease in methyltetrahydrofolyl polyglutamate derivatives was observed in the cells grown in media prepared with D20-depleted water (D20 content, 8-10 ppm). Chromatographic analysis of folates showed significant alterations in the formyl- and methyltetrahydrofolate derivatives and their polyglutamylation profiles. Higher amounts of oxidized folates were also present in the cells grown in D20-depleted conditions. No significant changes were observed in folates and their polyglutamate derivatives when the cells were grown in the presence of 300, 450 and 600 ppm D20. The altered folate homoestasis is attributed to changes in the metabolic adaptation of cells to D20-depleted environment.

Catalytic Aerobic Oxidation of Cycloalkanes with Nanostructured Amorphous Metals and Alloys.

Under mild conditions (40 atm O(2), 28 degrees C, 10-15 h), an efficient aerobic oxidation of cycloalkanes to cycloalkanols can be achieved using nanostructured amorphous metals such as Fe and Co and an amorphous alloy like Fe(20)Ni(80) as catalysts. For example, cyclohexane is oxidized to cyclohexanol with 32-41 % conversion, while 1-adamantanol is formed from adamantane with 52-57 % conversion.

Atherogenic diet alters folate enzymes in mice: implications of folate deficient homocysteinemia.

The two key enzymes, methylenetetrahydrofolate reductase and methionine synthase involved in methionine synthesis from homocysteine were studied in atherogenic diet fed mice. Methylenetetrahydrofolate reductase activity was elevated while methionine synthase was impaired in atherogenic diet fed group. Impaired methionine synthase activity would adversely affect the methionine synthesis from homocysteine, resulting in a rise in the homocysteine levels, which are atherogenic. This is reflected by the increased levels of very low density and low density lipoprotein cholesterol values and a higher ratio for total cholesterol to high density lipoprotein cholesterol.

Deranged folic acid metabolism in diabetes-induced rats.

Metabolic changes in the folic acid and their conjugated polyglutamyl derivatives have been studied in streptozotocin-induced diabetic rats. Conjugated folates which constituted about 70% in normal rat liver get rapidly hydrolysed to simple unconjugated forms as the diabetes progressed leaving behind only 2% after 25 days of the onset of diabetes. Chromatographic analysis shows significant alterations in the formyl- and methyltetrahydrofolate derivatives and their polyglutamylation profiles. A 7-8 fold higher urinary excretion of mainly methyltetrahydrofolate is also observed in diabetic rats.

Evidence for the induction and release of pancreatic folylpolyglutamate hydrolase by the ingestion of folyl polyglutamates in the rat.

Pteroyl polyglutamate hydrolase (folyl conjugase), which hydrolyses the dietary polyglutamyl folates into simple forms prior to absorption, has been shown to be induced in rat pancreas in response to dietary polyglutamyl folates but not by ingestion of synthetic unconjugated folates. Folate absorption, as measured by the rise in serum folate levels after ingestion, of dietary conjugated folates is impaired in pancreatectomised animals whereas absorption of synthetic simple folate is not. A severe build-up of folyl conjugase is observed in the lumen of control but not in pancreatectomised animals after dietary folate ingestion. These results taken together would suggest that dietary folates are hydrolysed to monoglutamyl forms suitable for absorption in the lumen; the hydrolysis is catalysed by a luminal folyl conjugase of pancreatic origin induced by dietary conjugated folates.

Role of methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured L1210 cells.

The effect of low methotrexate levels on methylenetetrahydrofolate and four other reduced folate pools in cultured L1210 cells has been examined over a 48-h period. Media folate levels and methotrexate were used to alter intracellular levels of reduced folates, and the distribution among individual reduced folates, so that they could be evaluated in terms of their effects on thymidylate synthesis and cell proliferation. Over the media folate concentration range of 0.25-50 microM, growth rate and thymidylate synthesis remained essentially unchanged while total intracellular reduced folates, determined from the summation of the five individual pools measured, increased approximately 25-fold. The 5-methyltetrahydrofolate and 10-formyltetrahydrofolate pools accounted for over 90% of the total reduced folate at the highest media folate level, while low media folate resulted in a much more equal distribution among the five reduced folates examined. Methotrexate, over the concentration range of 0.25-30 nM, caused extensive growth and intracellular thymidylate synthesis inhibition at media folate levels used in RPMI 1640 media (2.5 microM) and lower. However, growth inhibition was much less at the highest media folate level used, and thymidylate synthesis was not inhibited to a statistically significant extent. Intracellular reduced folates also responded differently to methotrexate depending upon the level of media folate. Depletion of the thymidylate synthase substrate, methylenetetrahydrofolate, could not account for diminished growth or thymidylate synthesis inhibition, since at 0.25 and 2.5 microM media folate no depletion occurred in response to methotrexate and only slight depletion was observed at 50 microM media folate. Dihydrofolate showed a tendency to increase at each of the media folate levels used with the least increase at the highest folate level. However, the ratio of dihydrofolate to total reduced folates was quantitatively most consistent with thymidylate synthesis and growth inhibition results.

Mechanisms of cross-resistance to methotrexate and 5-fluorouracil in an A2780 human ovarian carcinoma cell subline resistant to cisplatin.

Some properties of the human ovarian carcinoma line A2780 and a subline three times more resistant than the parent line to cisplatin are compared in this report. The rates of uptake and release of cisplatin were similar in the two cell lines. Resistance to cisplatin was associated with: (a) cross-resistance to 5-fluorouracil and methotrexate; (b) a 2.5-fold increase in thymidylate synthase, as measured by both enzyme activity and the capacity to complex 5-fluorodeoxyuridylate; and (c) an increase in the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate. These data suggest that cross-resistance to 5-fluorouracil and methotrexate in A2780 cells may be a consequence of increases in their respective target enzymes.

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate.

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate is described based on the stable ternary complex formed from methylenetetrahydrofolate, tritiated fluorodeoxyuridylate and thymidylate synthase. Tissue extract 10-formyltetrahydrofolate is deacylated with 10-formyltetrahydrofolate deacylase and the tetrahydrofolate formed is converted to methylenetetrahydrofolate using formaldehyde. Mouse tissue 10-formyltetrahydrofolate levels and their stability to extraction procedures are described.

Effects of methotrexate on folates in Krebs ascites and L1210 murine leukemia cells.

Changes in reduced folates upon exposure of Krebs ascites cells and L1210 murine leukemia cells to methotrexate (MTX) have been measured by stoichiometric entrapment of tissue methylenetetrahydrofolate into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxy-uridine-5'-monophosphate. Tetrahydrofolate and 5-methyltetrahydrofolate were determined after conversion to methylenetetrahydrofolate. In both tumor cell lines, treatment with methotrexate at levels which had little effect on methylenetetrahydrofolate and tetrahydrofolate concentrations resulted in nearly complete elimination of the methyltetrahydrofolate pool. Thus, an initial effect of methotrexate on folate metabolism appears to be on methyltetrahydrofolate.

Noninvolvement of a rat intestinal folate binding protein in physiological folate absorption.

Folic acid and its derivatives are observed to be instantaneously bound to rat intestinal mucosal cell curface binding protein. Various parameters were measured such as the optimal pH for binding (6.5), the saturation kinetics observed for folate binding and the avid affinity with which polyglutamyl folates are preferentially bound to the binder. A comparison of the observed results with the conditions reported as favorable for intestinal folate absorption in the rat precludes the involvement of this folate-binding protein in the physiological absorption of folates.

Folate malabsorption in aged rats related to low levels of pancreatic folyl conjugase.

Folate absorption and transport were studied in young (2 to 3 months) and aged (28 to 30 months) rats. From studies of the rise in serum folate levels after ingestion and transport across everted intestinal segments it was concluded that the absorption of dietary polyglutamyl folates was impaired in aged animals. Simple folates (monoglutamyl pteroates) were utilized equally well in both age groups. Decreased hydrolysis of polyglutamylfolates to simple forms have been observed in the aged group. High folyl conjugase (pteroyl gamma-glutamyl hydrolase) levels are induced in pancreas as a response to dietary folate intake; the conjugase is secreted into the gut lumen where enzyme levels rise sequentially reaching maximum in the midgut region within 30 to 60 min after folate ingestion. The rise in serum folate after folate ingestion parallels the rise in luminal folyl conjugase. Pancreatic and luminal conjugases (but not mucosal conjugase) have similar pH optima. It is suggested that dietary folates are preferentially hydrolyzed to absorbable forms by a luminal folyl conjugase which is of pancreatic origin. The overall enzyme levels are much lower in pancreas of the aged rats, leading to reduced availability of absorbable dietary folates and resulting in folate malabsorption in these animals. It is suggested that the intestinal mucosal conjugase does not play a significant role in the physiological absorption of dietary folates.

An ATPase dependent, radiosensitive acidic microclimate essential for intestinal folate absorption.

1. 5-Methyltetrahydrofolic acid transport was studied across everted sacs of rat jejunal segments from control and whole body X-irradiated (700 rad) rats at 10(-5)M concentrations (at which optimum transport occurs) at various pHs.2. The folate transport from mucosal to serosal compartment was inhibited by about 55% in irradiated rat at the pH of the intestinal chyme (6.5). Extraneous ATP in the incubation system could restore the defective transport of the irradiated intestine.3. The maximum folate transport which occurred at pH 4.0 was not adversely affected by whole body irradiation. An acidic, pH dependent, passive uptake of 5-methyltetrahydrofolic acid was observed.4. The normal absorption barrier of the small bowel was not disrupted by the acidification process as practically no uptake was observed with irradiated segments pretreated at pH 4.0 except in the presence of ATP.5. Leucine and serine transport at a zero concentration gradient indicated active transport mechanisms which were not affected by acidification. Their uptake was additively increased in the presence of glucose and ATP, further indicating that the normal physiology of the intestines was not affected by the acidification process.6. An intestinal mucosal cell surface ATPase was observed which was Mg(2+) dependent. It could hydrolyse solution phase ATP and thus generate the protons necessary for the acidification of a microenvironment where passive uptake of the neutral folate species could occur.7. The ATPase activity was inhibited about 90% by 50 mM-Na azide at pH 6.5. Below this concentration folate transport was also inhibited.8. Na azide did not inhibit folate transport at pH 4.0, suggesting that its inhibition of folate uptake at pH 6.5 is related to its inhibitory effect on ATPase, rather than on folate transport per se. ATPase activity was therefore essential for folate transport at the pH of the intestinal chyme.

Genotypic and environmental effects on the maturity time of autumn cauliflowers.

Twelve genotypes representing a wide range of autumn cauliflower were grown in two seasons from six seedling propagation treatments in three sequential sowings. Genotypes differed in their mean time of maturity and spread of maturity, and in their sensitivity to environment for these characters. Those genotypes derived from self-incompatible stocks showed greater stability than those from self-compatible stocks. In particular, a National Vegetable Research Station breeding line 'KC' revealed outstanding stability and is thus a potential new variety. The best environments for discriminating between genotypes were those which gave the least check to early growth.