Evidence for a second chemotactic system in the cellular slime mold, Polysphondylium.

An unidentified substance(s) in a commercial guanosine 3′,5′-cyclic monophosphoric acid (cyclic 3′,5′-GMP) preparation is effective in attracting the aggregating amoebae of the cellular slime mold, Polysphondylium pallidum. Bacterial extracts (Escherichia coli) and amoeba extracts (P. pallidum) attract both vegetative and aggregating amoebae. A crude enzyme preparation from amoebae is capable of reducing the chemotactic activity of the extracts on aggregating amoebae and eliminating the activity of the unknown substance in the commercial cyclic 3′,5′-GMP preparation. As only the extracts were shown to contain folic acid, and since the enzyme does not reduce folic acid activity, it is suggested that the extracts contain a factor (possibly folic acid) primarily active on vegetative amoebae and an acrasin. The commercial cyclic 3′,5′-GMP preparation contains only an acrasin. The acrasin is heat stable and nondialyzable.     

Correlation between acrasins and spore germination inhibitors in cellular slime molds.

Discadenine,3-(3-amino-3-carboxypropyl)-N6-delta 2-isopentenyladenine, which inhibits spore germination, was previously found in Dictyostelium discoideum. Studies on the distribution of discadenine in different species of cellular slime molds by high-pressure liquid chromatography showed that discadenine is present in D. discoideum, Dictyostelium purpureum, and Dictyostelium mucoroides, but not in Dictyostelium minutum, Polysphondylium violaceum, or Polysphondylium pallidum. Discadenine synthetase, which is involved in biosynthesis of discadenine with N6-delta 2-isopentenyladenine as substrate, was only detected in cells of the former three species. In addition, discadenine inhibited spore germination only in these three species. These results clearly demonstrate that discadenine is produced as an inhibitor of spore germination in the species of cellular slime molds in which the acrasin is cyclic adenosine 5'-monophosphate (AMP). This means that there is a structural and biochemical correlation between the spore germination inhibitor and the acrasin, since 5'-AMP, a direct precursor in discadenine biosynthesis, can be derived from cyclic AMP by hydrolysis with cyclic AMP phosphodiesterase.     

Characterization of the folic acid C9-N10-cleaving enzyme of Dictyostelium minutum V3

Folic acid is a chemoattractant for the slime mold Dictyostelium minutum V3. The activity of extracellular folic acid is regulated by a folic acid C9-N10 splitting enzyme (FAS). The products were identified as pterin-6-aldehyde and p-amino-benzoylglutamic acid. The enzyme was stabilized by EDTA. For the extracellular enzyme, the Km was 10(-7) M, and the optimal pH was 4.0. During starvation, FAS activity was mainly secreted into the medium; after 3 h, a plateau was reached. The membrane-bound activity was constant, but only 12% of the extracellular activity at 3 h. Intracellular activity also increased up to 3 h to a level of 23% of the extracellular FAS. The substrate recognition of FAS was found to be based on 4-O or N3 or both, N5 or N8 or both, N10, and the p-aminobenzoic acid moiety, whereas 2-NH2, N1, and the glutamic acid moiety were not recognized. Other slime mold species were found to secrete FAS with 20-fold or more reduced activity than D. minutum V3.     

Some Amino Acid-Folic Acid Interrelationships in Tetrahymena pyriformis

DL-serine, DL-methionine or DL-serine + DL-methionine in excess inhibited the growth of Tetrahymena pyriformis H. Excess serine was most inhibitory at high concentration of folic acid, whereas the effect of excess methionine or methionine + serine was most pronounced at low levels of folic acid. Inhibition due to excess serine was relieved by raising the level of methionine or by adding pyrimethamine to lower the effective folic acid level, and was intensified by adding Dl.-ethionine or by raising the level of folic acid. Similarly, inhibition due to excess methionine was relieved by supplying more serine or adding DL-ethionine (which reduced the amount of available methionine) and was intensified by adding pyrimethamine. Inhibition by excess methionine + serine was reversed by increasing threonine, provided there was ample guanine present. Low levels of guanine or the presence of 8-azaguanine prevented this reversal. Comparisons are made with the work of others.     

Synthesis and activity of a folate peptide camptothecin prodrug

A folate receptor targeted camptothecin prodrug was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. The conjugate was found to possess high affinity for folate receptor-expressing cells and inhibited cell proliferation in human KB cells with an IC(50) of 10nM. Activity of the prodrug was completely blocked by excess folic acid, demonstrating receptor-mediated uptake.     

Effects of altered maternal folic acid, vitamin B12 and docosahexaenoic acid on placental global DNA methylation patterns in Wistar rats

Potential adverse effects of excess maternal folic acid supplementation on a vegetarian population deficient in vitamin B(12) are poorly understood. We have previously shown in a rat model that maternal folic acid supplementation at marginal protein levels reduces brain omega-3 fatty acid levels in the adult offspring. We have also reported that reduced docosahexaenoic acid (DHA) levels may result in diversion of methyl groups towards DNA in the one carbon metabolic pathway ultimately resulting in DNA methylation. This study was designed to examine the effect of normal and excess folic acid in the absence and presence of vitamin B(12) deficiency on global methylation patterns in the placenta. Further, the effect of maternal omega 3 fatty acid supplementation on the above vitamin B(12) deficient diets was also examined. Our results suggest maternal folic acid supplementation in the absence of vitamin B(12) lowers plasma and placental DHA levels (p<0.05) and reduces global DNA methylation levels (p<0.05). When this group was supplemented with omega 3 fatty acids there was an increase in placental DHA levels and subsequently DNA methylation levels revert back to the levels of the control group. Our results suggest for the first time that DHA plays an important role in one carbon metabolism thereby influencing global DNA methylation in the placenta.     

Regulation of the synthesis and activity of thymidine phosphorylase in Lactobacillus casei

Abstract: Lactobacillus casei cells grown on excess thymine or on folic acid contained low levels of thymidine phosphorylase. On the other hand, thymine starved cells and also cells of a thymidine-monophosphate-kinase-defective mutant grown on excess thymine, possessed derepressed levels. These results suggest that the synthesis of thymidine phosphorylase is regulated by the end product of the thymidine-triphosphate-biosynthetic pathway. L. casei cells lacked 2-deoxyribose-1-phosphate-mutase activity and did not grow on 2-deoxyribose or thymidine as the sole-carbon source. Growth in the presence of thymidine did not result in induction of thymidine-phosphorylase synthesis, probably due to the inability of the cell to convert it to 2-deoxyribose-5-phosphate, which is known to act as an inducer in E. coli cells. Thymidine triphosphate inhibited non-competitively the activity of thymidine phosphorylase. It was also inhibited by dihydrofolic acid.     

Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3H-folic acid at pH 5.5 was (i) Na+-independent; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX); (iii) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS); (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin); (vi) trans-inhibited by MTX and folic acid; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3H-folic acid was (i) Na+-independent; (ii) inhibited by folic acid and MTX, but not by 5-MTHF; (iii) inhibited by SITS, but not by DIDS; (iv) not affected by FCCP; (v) inhibited by monensin (but not by cytochalasin D); (vi) trans-inhibited by folic acid (but not by MTX); and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.     

Differential effects of stimulus termination on excitation and desensitization of folic acid receptors and guanylate cyclase in Dictyostelium discoideum

The response of guanylate cyclase to addition of extracellular stimuli is well documented. Here we report for the first time the response of guanylate cyclase to removal of stimuli. Three methods were employed to terminate rapidly a stimulus of folic acid. (1) Addition of a highly active folate deaminase preparation, or (2) 12-fold dilution of the stimulated cell suspension, or (3) addition of an excess concentration of a non-agonistic derivative of folic acid, i.e., 2-deaminofolic acid, which chases the folate agonist from its cell-surface receptors. Accumulation of cGMP terminated instantaneously upon addition of deaminase, but degradation of the synthesized cGMP was not observed until 10–12 s after stimulation. Also in a cGMP phosphodiesterase-lacking ‘streamer’ mutant an instantaneous termination of further cGMP accumulation was observed upon stimulus removal. This suggests that the termination of cGMP accumulation is due to inactivation of guanylate cyclase instead of a steady state of cGMP synthesis and degradation. Further accumulation of cGMP was approx. 75% reduced upon dilution of a cell suspension after stimulation with both agonists. Stimulation by 300 nM folic acid or by 30 nM N¹⁰-methylfolic acid (a more potent agonist) yielded identical results. However, upon addition of deaminofolic acid the accumulation of cGMP continued normally if the cells had been stimulated with N¹⁰-methylfolic acid, but only slightly in the case of a folic acid stimulus. The effect of stimulus duration on desensitization was monitored; it was observed that 50% desensitization was induced by stimulation for 1 s, while 4 s was sufficient for maximal desensitization. Short stimuli were observed to elicit high levels of desensitization without much excitation of guanylate cyclase. A desensitization-like process was observed at the level of the folate-binding chemotactic receptors as well. Relationships between the cGMP response data and folic acid receptor kinetics are discussed.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Folic Acid as Second Chemotactic Substance in the Cellular Slime Moulds

IT has been known for some time that in certain species of cellular slime moulds acrasin, the substance which attracts the amoebae to central collection points during the aggregation phase, is cyclic AMP^1–4. We were also able to show that E. coli gave off another substance besides cyclic AMP (henceforth referred to as bacterial factor, or BF) which attracted the vegetative amoebae of Dictyostelium discoideum⁵. Here we demonstrate that this second attractant has the properties of folic acid or one of its derivatives. We also show that folic acid and related compounds not only attract the vegetative amoebae of D. discoideum (No. NC-4H) but also the amoebae of six other species (Dictyostelium rosarium No. CC-7; D. mucoroides No. 11; D. purpureum No. 2; D. minutum No. V-3; Polysphondylium violaceum No. 1; P. pallidum No. 2). For the latter three species cyclic AMP is not the aggregative attractant (ref. 6 and J. T. B., E. M. H., S. Noller, F. B. Oleson and A. B. Roberts, in preparation) which raises the interesting question of whether their acrasin might be related to the folates.     

Antiinflammatory activity of Muktashukti bhasma.

Muktashukti bhasma (MSB), an Ayurvedic compound, consisting of pearl, Aloe vera and vinegar, inhibited acute and subacute inflammation in albino rats as induced by subplanter injection of carrageenan, histamine, 5-HT, nystatin and subcutaneous implant of cotton pellets. In all the test procedures the antiinflammatory response of 1000 mg/kg MSB was comparable to the response observed with 300 mg/kg acetylsalicylic acid (ASA). Oral premedication with MSB delayed castor oil-induced diarrhoea in rats, indicating its prostaglandin inhibitory activity. The antiinflammatory activity of the compound is attributed to its ability to cause inhibition of prostaglandins, histamine and 5-HT and also by stabilization of the lysosomal membranes. The antiinflammatory activity of MSB seems one third to half as potent as ASA.     

Cellular aggregation towards steady point sources of attractant.

The aggregation of a layer of chemotactically sensitive cells towards a steady point source of a chemical attractant (acrasin), a phenomenon exhibited, for instance, by the cellular slime mould Dictyostelium minutum, is treated analytically. An acrasin gradient is set up by diffusion from the source, taken to be a continuous one of constant strength. The aggregative movement of the cells in response to the gradient is expressed in terms of chemotactic laws and the random movement taken into account through a diffusion coefficient. Various chemotactic laws are considered [response velocity (1) proportional to the relative acrasin gradient, (2) proportional to the gradient (3) constant or maximal]; the resulting partial differential equations are solved to obtain the time dependence for the number of cells in the aggregate. Each law yields a characteristic time dependence, the dominant term of which is ～ t^a, 0 < α ? 2.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Separation of Folic Acid Reductase from Streptococcus faecium (ATCC 8043)

A strain of Streptococcus faecium (ATCC 8043) which is highly resistant to the antifolic acid compound, amethopterin, was gently ruptured by exposing protoplasts of the organism to a hypotonic solution. The crude lysate resulting there-from was treated by various chemical and physical techniques designed to separate folic acid reductase from dihydrofolic acid reductase. In the process, the enzyme was purified approximately 160-fold; however, throughout the process, the enzyme preparation maintained the ability to reduce folic acid to tetrahydrofolic acid. Attempts to isolate mutants showing a deficiency in either folic acid reductase or dihydrofolic acid reductase were unsuccessful. Based on these results, it is concluded that folic acid is reduced to tetrahydrofolic acid by one enzyme in S. faecium (ATCC 8043). The crude lysate was also subjected to ultracentrifugation. An analysis of the supernatant fluid and the sediment indicated that the reductive activity is located in the soluble fraction of the cell.     

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.     

Folic acid alleviates age-related cognitive decline and inhibits apoptosis of neurocytes in senescence-accelerated mouse prone 8: deoxythymidine triphosphate biosynthesis as a potential mechanism

Disturbed deoxythymidine triphosphate biosynthesis due to the inhibition of thymidylate synthase (TS) can lead to uracil accumulation in DNA, eventually, lead to neurocytes apoptosis and cognitive decline. Folic acid supplementation delayed cognitive decline and neurodegeneration in senescence-accelerated mouse prone 8 (SAMP8). Whether folic acid, one of nutrition factor, the effect on the expression of TS is unknown. The study aimed to determine if folic acid supplementation could alleviate age-related cognitive decline and apoptosis of neurocytes by increasing TS expression in SAMP8 mice. According to folic acid concentration in diet, four-month-old male SAMP8 mice were randomly divided into three different diet groups by baseline body weight in equal numbers. Moreover, to evaluate the role of TS, a TS inhibitor was injected intraperitoneal. Cognitive test, apoptosis rates of neurocytes, expression of TS, relative uracil level in telomere, and telomere length in brain tissue were detected. The results showed that folic acid supplementation decreased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, alleviated telomere length shorting, increased expression of TS, then decreased apoptosis rates of neurocytes, and alleviated cognitive performance in SAMP8 mice. Moreover, at the same concentration of folic acid, TS inhibitor raltitrexed increased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, and exacerbated telomere length shorting, decreased expression of TS, then increased apoptosis rates of neurocytes, and decreased cognitive performance in SAMP8 mice. In conclusion, folic acid supplementation alleviated age-related cognitive decline and inhibited apoptosis of neurocytes by increasing TS expression in SAMP8 mice.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.     

Effect of vitamin C and folic acid on seed vigour response and phenolic-linked antioxidant activity

Folic acid and vitamin C were used in the concentration range of 0-500muM as exogenous growth enhancers to stimulate pea (Pisum sativum) seedling vigour. The results suggest that a concentration of 50muM folic acid and 500muM vitamin C were optimum in maximally enhancing seed vigour and potentially seedling performance according to both agronomic and biochemical seed vigour parameters. Results indicated that germination percentage, shoot weight, shoot height, and root length were enhanced in folic acid and vitamin C treated plants compared to control plants. The levels of enhanced phenolic content in response to folic acid and vitamin C treatments were highest on days 8 and 10. Evaluation of critical biochemical parameters indicated that the average glucose-6-phosphate dehydrogenase (G6PDH) activity and proline content in response to treatments were higher than control and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Key enzymes, guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) were also higher in response to treatments and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Taken together, these studies support the hypothesis that the proline-linked pentose phosphate pathway stimulates phenolic synthesis and related free-radical scavenging antioxidant activity. Further, this proline-linked pentose phosphate pathway stimulation in response to folic acid and vitamin C was also correlated to antioxidant enzyme response indicated by the stimulation of GPX, SOD, and CAT activities. Therefore, this study indicates the enhancement of seed vigour response by folic acid and vitamin C as reflected in both agronomic and biochemical responses, and this occurred through the stimulation of phenolic-linked antioxidant response that is likely positively modulated through the proline-linked pentose phosphate pathway.     

Synthesis and biological evaluation of diethylenetriamine pentaacetic acid-polyethylene glycol-folate: a new folate-derived, (99m)Tc-based radiopharmaceutical

(99m)Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEG-folate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEG-folate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 = 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPA-PEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [(99m)Tc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2 microCi/microg) were achieved. After subcutaneous injection, [(99m)Tc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emission computed tomography following subcutaneous injection of [(99m)Tc] DTPA-PEG-folate. In conclusion, the [(99m)Tc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.