The role of vitamin B12 in the metabolism of Euglena gracilis var. bacillaris

1. The concentrations of RNA, DNA and protein are decreased in cells of Euglena gracilis var. bacillaris grown on suboptimum concentrations of vitamin B(12). 2. The addition of vitamin B(12) to deficient cells stimulates the incorporation of [(14)C]formate into the above cell components as well as into thymine of DNA and serine and methionine of protein. 3. In a cell-free system from vitamin B(12)-deficient cells, the incorporation of labelled formate into thymidylate is decreased to a greater extent with uridine than with deoxyuridine as the substrate. 4. The addition of unlabelled glutamate dilutes the radioactivity incorporated into thymine from labelled formate. 5. These results are interpreted to mean that, in DNA synthesis, vitamin B(12) has a greater role in the reduction of ribotides to deoxyribotides than in the reduction of formate to thymine methyl and that the vitamin B(12)-dependent conversion of glutamate into beta-methylaspartate also contributes to thymine synthesis.     

Effect of cobalamin derivatives on DNA synthesis in cells of Propionibacterium freudenreichii subsp. shermanii

The increase of DNA-synthesis rate (according incorporation [8-14C]adenine) in B12-deficient cells Propionibacterium shermanii as a result of different cobalamines adding into the cell suspension including metoxyethyladenile analog of adenozilcobalamin and some components of vitamin B12 molecule has been found. The DNA-synthesis rate in B12-deficient cells is nearly twice lower as compared with one in B12-normal cells. Considerable stimulative effect (80-100%) was provided with coenzyme forms of cobalamin. The data confirm the participation of vitamin B12 in DNA-synthesis in Propionibacterium cells.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Metabolism of thymine nucleotides synthesized via the ''de novo'' mechanism in normal, megaloblastic and methotrexate-treated human cells and in a lymphoblastoid cell line.

Human bone-marrow cells and lymphocytes were incubated with [3H]deoxyuridine (dU) to study the metabolism of thymine nucleotides labelled via the thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) step of the 'de novo' biosynthetic pathway. (1) Continuous labelling with [3H]dU was used to compare incorporation of label into DNA with the specific radioactivities of thymine nucleotides separated by paper chromatography. (2) Cells were also labelled with [3H]dU at 13 degrees C, and 'chased' in unlabelled medium at 37 degrees C in order to quantify the proportion of thymine nucleotides incorporated into DNA and the proportion degraded. Only 40% of labelled thymine nucleotides were incorporated into lymphocyte DNA during a 'chase', whereas 100% were incorporated by MOLT 4 cells (a lymphoblastoid cell line of thymic origin, Thy-ALL line). Unincorporated nucleotides were rapidly degraded in lymphocytes, but degradative activity was very low in MOLT 4 cells. The results described here reinforce our previous conclusions [Taheri, Wickremasinghe & Hoffbrand (1981) Biochem. J. 194, 451-461] that there is a single thymine nucleotide compartment in Thy-ALL cells, but at least two pools in lymphocytes and bone-marrow cells. This compartmentation of nucleotides in human cells is consistent with a model which proposes that deoxyribonucleotides are localized near replication forks by the activity of multienzyme complexes [Mathews, North & Reddy (1978) Adv. Enz. Regul. 17, 133-156]. Our results also suggest that thymine nucleotides derived by the 'de novo' mechanism may be more highly localized than those derived by salvage. In cells from patients with megaloblastic anaemia owing to deficiency of vitamin B12 or folate or in normal cells treated with methotrexate, there was a massive accumulation of labelled dUMP and decreased incorporation of label into DNA. There was no measurable incorporation of labelled deoxyuridine residues into DNA of megaloblastic cells, but deoxyuridine residues were detected in DNA of cells treated with methotrexate.     

Deoxyribonucleoside triphosphate pools in vitamin B-12-deficient Euglenagracilis.

The size of the deoxyribonucleoside triphosphate pools of vitamin B-12-deficient cells of Euglena gracilis, and of vitamin B-12-deficient cells repleted with the vitamin, were measured. We found that the pools were very small, if they exist at all, in deficient cells but expand rapidly with the addition of the vitamin. The sizes of the pools decrease when DNA synthesis is completed, and are very small when the cells begin to divide.     

Ribonucleotide reductase activity in vitamin B12-deficient Euglena gracilis.

The ribonucleotide reductase activities in vitamin B12-sufficient and -deficient cells of Euglena gracilis were measured. We found that the cells progress into vitamin B12 deficiency the enzyme activity increases, reaching a maximum value of 20-fold in advanced deficiency. No signigicant differences in the activities were found to result as a consequence of different growth conditions. We propose that the increased activity in vitamin B12-deficient cells is due to an increase in enzyme protein.     

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.     

Nature of the Repair of Methyl Methanesulfonate-Induced Damage in Bacillus subtilis

A nuclease present in extracts of Bacillus subtilis inserts breaks in deoxyribonucleic acid (DNA) treated with the monofunctional alkylating agent, methyl methanesulfonate (MMS), but the nature of the sites within the alkylated macromolecule at which these breaks occur is not known. DNA extracted from B. subtilis cells that have recovered from MMS damage has lost its susceptibility to enzyme action. The recovery process is accompanied by some DNA breakdown and by the incorporation of thymidine. Some recovery from ultraviolet irradiation (UV) and MMS occurred in organisms starved for thymine or adenine, but UV recovery was stimulated by their addition. It is possible that MMS recovery proceeds by a process of excision and repair similar to, but not identical with, UV repair.     

Effect of the Folic Acid Analogue, Trimethoprim, on Growth, Macromolecular Synthesis, and Incorporation of Exogenous Thymine in Escherichia coli

The effect of trimethoprim [2,4-diamino-5(2',4',5'trimethoxybenzyl)-pyrimidine] in the presence of thymine on Escherichia coli B temperature-sensitive and non-temperature-sensitive Thy(') strains and a phosphodeoxyribomutase-negative mutant was studied. The inhibitory effect of 5 mug of trimethoprim per ml on the growth of E. coli B was not overcome by thymine, thymidine, or thymidylate even in the presence of one-carbon metabolites and related metabolites. Deoxyribonucleic acid (DNA) and protein synthesis were more severely inhibited than ribonucleic acid (RNA) synthesis. The inhibition of DNA synthesis was partially reversed by addition of deoxyadenosine to increase the incorporation of exogenous thymine. By contrast, the inhibition of protein was not reversed even with one-carbon metabolites present, in keeping with the requirement for formylmethionyl-transfer RNA(F) for initiation. However, the inhibition of both DNA and protein synthesis in a phosphodeoxyribomutase-negative strain by 1 mug of trimethoprim per ml with thymine present was partially reversed by deoxyadenosine and one-carbon metabolites, and nearly normal growth occurred. 5-Fluorodeoxyuridine added at the time of addition of trimethoprim prevented the inhibition. Sulfadiazine in the presence of thymine inhibited both Thy(+) and Thy(-) strains whereas trimethoprim (with thymine) did not inhibit Thy(-) organisms. The effect of trimethoprim on the incorporation of labeled thymine into DNA was also studied. These experiments support the concept that trimethoprim in conjunction with the action of thymidylate synthetase inhibits the growth of Thy(+) cells because of a depletion of tetrahydrofolate. DNA synthesis is inhibited initially by a limitation of thymine nucleotide precursor, resulting from the indirect inhibition of thymidylate synthetase and the poor incorporation of exogenous thymine.     

Effects of maternal vitamin B12 deficiency from end of gestation to weaning on the growth and haematological and immunological parameters in mouse dams and offspring

Vitamin B12-deficiency may induce specific symptoms as neurological alterations and unspecific symptoms such as anaemia and growth retardation. In this study, maternal vitamin B12 deficiency from end of gestation to weaning was evaluated in mouse dams, which was provoked by feeding a vitamin B12-deficient diet. The animals were divided into two groups (control and deficient). The control group received the vitamin B12-deficient diet supplemented with commercial vitamin B12. Compared to the control, the vitamin B12-deficient dams and their offspring showed a significant decrease of body weight (by 20 and 39%, respectively), serum vitamin B12 concentration (by 61 and 67%, respectively), haematological values as haematocrit (25 and 26%, respectively), and IgA producer cells (by 36 and 54%, respectively). In both, vitamin B12-deficient mouse dams and their offspring, histological alterations of small intestine were observed, whereas growth retardation occurred in the offspring only. This experimental murine model allows assessing the incidence of maternal cobalamin deficiency in offspring and would be useful for evaluating novel adjuncts such as functional foods to prevent vitamin B12 deficiency.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.     

Replication of the deoxyribonucleic acid of multiple-drug-resistance factor in Escherichia coli.

1. It was shown that a system previously described for labelling R-factor DNA during transfer to an irradiated recipient strain of Escherichia coli did not allow high selectivity in the incorporation of thymine into R-factor DNA. 2. Lack of selectivity was shown to be due to cross-feeding from recipient to donor strain. 3. An improved system using a nalidixic acid-resistant recipient strain is described in which incorporation of thymine into the DNA of donor cells is minimized by addition of nalidixic acid after completion of transfer of the plasmid during conjugation.     

Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR.

The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia.     

Nitrogen Metabolism in Paramecium aurelia

Nitrogen in cell fractions of Paramecium aurelia varied according to the growth medium. Trichloroacetic acid-soluble fractions of cells were chromatographer. Adenine, adenosine, guanine, guanosine, hypoxanthine, aspartic acid, glutamic acid, histidine, lysine, proline, and phenylalanine were identified. Fyrimidines and xanthine, or their respective ribosides and ribotides, were not detected. Ammonia was released into the medium by both actively growing and "resting" cells. Culture fluids of "resting"cells also contained hypoxanthine and lesser amounts of adenine and guanine. Urea, uric acid, creatine, cretonne, and ailantoin were absent.
Pyrimidine nitrogen seems excreted as dihydrouracil. The following enzymes were detected in homogenates and cell-free preparations: nucleotidases, nucleoside hydrolases, and cytidine deaminase. Urease, uricase, adenase, guanase, xanthine oxidase, adenosine deaminase, and 5'-adenylic acid deaminase were not present in this organism.
Purine and pyrimidine incorporation into nucleic acids was investigated by the use of radioactive tracers. Guanosine gives rise to nucleic-acid guanine and adenine; adenosine was precursor to nucleic acid adenine only. Formate was incorporated into purines; glycine was not. P. aurelia can interconvert cytidine and uridine; both give rise to nucleic acid thymine. The methyl group of thymine may be derived from formate.     

DNA synthesis and induced mutations in the presence of 5-bromouracil I. DNA synthesis in the presence of 5-bromouracil

Summary An investigation has been carried out dealing with the incorporation of BU into DNA ofE. coli 15 thy^– under conditions of complete thymine deficiency. It was found that exponentially growing cells can increase their DNA 5-fold upon suspension in BU-supplemented medium. DNA increased in a linear fashion and followed the series ×, 2×, 3×, 4× where × is the amount of DNA initially present. If thy^– cells were starved for 30 minutes before being provided with BU, DNA appeared to increase stepwise although the increase during each period of synthesis was equal only to the amount of DNA initially present. Paper chromatography revealed that BU totally replaced thymine in the newly-synthesized DNA. Equilibrium density gradient techniques and radioactive labeling made it possible to ascertain that the DNA in which BU fully replaced thymine was functional on the primary level, that of priming or taking iart in the synthesis of new DNA. Cellular inhibition as indicated by lethality was described and possible explanations for the inhibition resulting from incroporation of BU into DNA were discussed.     

Vitamin B6-deficient diet plus 4-deoxypyridoxine (4-DPD) reduces the inflammatory response induced by T. spiralis in diaphragm,masseter and heart muscle tissue of mice

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-infl ammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 g/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T. spiralis) per os.Chronic inflammation was caused by infection of treated or untreated mice with T. spiralis parasite. After 14 days post-infection all mice developed a chronic inflammatory response. Mice fed with a B6-deficient diet showed a significant decrease in the number of cysts found in the diaphragm when compared to mice treated with normal diet. In addition, in all mice treated with vitamin B6-deficient diet plus 4-DPD the average body weight was significantly lower, compared to the mice on normal diet in all weeks examined. Moreover, in sections of the diaphragm, masseter and miocardium muscles, the infiltration of inflammatory cells, such as macrophages, lymphocytes, and eosinophils were more intense in untreated mice compared to those fed a vitamin B6-deficient diet.These results show that BALB/c mice infected with T. spiralis and fed a vitamin B6-deficient diet plus the vitamin B6 antagonist, 4-DPD, prolong the time of invasion of the larvae in the muscle cells, influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction compared to infected untreated mice (control)     

Folate (vitamin B9) and vitamin B12 and their function in the maintenance of nuclear and mitochondrial genome integrity

Folate plays a critical role in the prevention of uracil incorporation into DNA and hypomethylation of DNA. This activity is compromised when vitamin B12 concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation, DNA hypomethylation and mitochondrial DNA deletions. In vivo studies show that folate and/or vitamin B12 deficiency and elevated plasma homocysteine (a metabolic indicator of folate deficiency) are significantly correlated with increased micronucleus formation and reduced telomere length respectively. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is greater than 100nmol/L. Intervention studies in humans show (a) that DNA hypomethylation, chromosome breaks, uracil incorporation and micronucleus formation are minimised when red cell folate concentration is greater than 700nmol/L and (b) micronucleus formation is minimised when plasma concentration of vitamin B12 is greater than 300pmol/L and plasma homocysteine is less than 7.5μmol/L. These concentrations are achievable at intake levels at or above current recommended dietary intakes of folate (i.e. >400μg/day) and vitamin B12 (i.e. >2μg/day) depending on an individual's capacity to absorb and metabolise these vitamins which may vary due to genetic and epigenetic differences.     

NUTRITIONAL STUDIES OF THE EDIBLE SEAWEED PORPHYRA TENERA I. THE INFLUENCE OF DIFFERENT B12 ANALOGUES, PLANT HORMONES, PURINES AND PYRIMIDINES ON THE GROWTH OF CONCHOCELIS

The red alga Porphyra tenera has been obtained in axenic cultureby the "dip and drag" technique in an agarized medium containingantibiotics. In the axenic culture, the Conchocelis of P. teneraneeds vitamin B₁₂ for growth. The addition of other vitaminsdoes not increase growth further. The pattern of specificityis similar to that of Escherichia coli 113-3. Factor B, pseudovitaminB₁₂ and Factor Z₂ support as much growth as vitamin B₁₂, while2-methylmercaptoadenine and 5-methyl benzimidazole cobalamineincrease the growth more than B₁₂. All the analogues containingbenzimidazole can replace B₁₂. Kinetin, adenine, indoleaceticacid and especially gibberellic acid increase growth. Threepurines, (xanthine, hypoxanthine and guanine) and three pyrimidines(uracil, methylcytosine and thymine) also promote growth inthe presence of vitamine B₁₂. (Received January 18, 1965; )