Phase Relations between Host and Grafted SCN Depend on Graft Location in Rats

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third ( n = 24) or lateral ( n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values oc curred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission.     

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.     

Application of vitamin B(12)-targeting site on Lactobacillus helveticus B-1 to vitamin B(12) assay by chemiluminescence method

Lactobacillus helveticus B-1 is assumed to have a vitamin B(12)-targeting (or B(12)-binding) site on the cells, since the binding reaction of vitamin B(12) with L. helveticus B-1 cells proceeded instantly and quantitatively. This reaction is specific to complete B(12) compounds, cobalamins, and can be used for a vitamin B(12) assay method by chemiluminescence. The calibration graph was linear from 0.1 to 10.0 ng/mL. The B(12) contents in oyster and sardine were 75.9 and 39.4 microg/100g, respectively. These values were very close to those obtained using a chemilumi-ADVIA Centaur immunoassay system with intrinsic factor and to those obtained by microbiological assays.     

Plasma Trace Elements, Vitamin B12, Folate, and Homocysteine Levels in Cirrhotic Patients Compared to Healthy Controls

Increased serum homocysteine (Hcy) can induce liver diseases and can play a remarkable role in hepatic disorders. The purpose of the present study therefore was to investigate the relationship between serum vitamin B(12), folate, zinc and copper, cysteine, and Hcy level differences between cirrhotic patients and healthy subjects. We studied 32 cirrhotic patients (12 females and 20 males) aged 45 +/- 11 years and 32 control subjects (12 females and 20 males) aged 39 +/- 9 years. There was an inverse correlation between Hcy and vitamin B(12) in controls (r = -0.442, p < 0.011) but not in cirrhotic patients (r = -0.147, not significant). Also, mean plasma folate was decreased in cirrhotic patients compared to controls (p < 0.001). Copper increased whereas zinc decreased significantly in cirrhotic patients. A positive correlation was seen between the Cu/Zn ratio and Cu in controls (r = 0.690, p < 0.01), but the correlation between the Cu/Zn ratio and Cu was not significant in the cirrhotic group. Negative correlations were seen between plasma concentration of zinc and the Cu/Zn ratio in controls and cirrhotic patients (r = -0.618, p < 0.01 and r = -0.670, p < 0.01, respectively). Cirrhotic patients displayed multiple abnormalities, including changes in cysteine metabolism and in zinc and copper levels. Although hyperhomocysteinemia is known as an atherogenic and thrombogenic risk factor for cardiovascular disease, it might also be a risk factor for cirrhotic patients. Plasma Hcy, vitamin B(12), and folic acid measurement may be useful in the evaluation of cirrhotic patients.     

Decrease of trace elements in erythrocytes and plasma after removal of dental amalgam and other metal alloys

The objective of this study was to determine the concentration changes of 13 elements in erythrocytes and plasma after the removal of dental amalgam, and other metal alloys. Blood samples from 250 patients were collected, separated into erythrocytes and plasma, and analyzed by inductively coupled plasma-mass spectrometry. The 250 patients were divided into 3 groups (Negative, Zero, and Positive) depending on their estimation of quality of life in an earlier study. Magnesium in plasma, selenium and mercury in plasma, and erythrocytes showed decreased concentrations after amalgam removal in all groups (p<0.05). Titanium in plasma, copper in plasma, and erythrocytes and zinc in plasma exhibited decreased concentrations after amalgam removal in the Negative and Positive groups (p<0.05), Silver in plasma and gold in erythrocytes decreased in the Zero and Positive groups after amalgam removal (p<0.05). Copper in erythrocytes and silver and gold in plasma showed higher concentrations after amalgam removal in the Negative compared to the Positive group (p<0.05), suggesting that patients in the Negative group excrete metals slowly. Moreover, the cobalt levels in plasma were lowest in the Negative group and only this group showed a significant increase in vitamin B₁₂ levels in blood after amalgam removal.     

Methionine,folic acid and vitamin B12 in growing-finishing pigs: Impact on growth performance and meat quality

Growth performance, metabolic variables, and meat quality were measured in 78 growing-finishing pigs using supplements of 0 (C), or 0.2% of DL-methionine (M), and three combinations of folic acid [mg/kg] and cyanocobalamin [μg/kg], respectively 0 and 0 (V0), 10 and 25 (V1), and 10 and 150 (V2) in a 2 × 3 factorial arrangement. Feed conversion was lower (p = 0.05) in M than in C pigs during the growing period (0–4 weeks). Both V1 and V2 treatments increased plasma vitamin B₁₂ (p < 0.01) and decreased plasma homocysteine (p < 0.01). Plasma 5-methyl-tetrahydrofolates were the lowest, highest and intermediate in V0, V1 and V2 pigs (p < 0.04), respectively. In V2 meat, folates were 32% higher, vitamin B₁₂, 55% higher and homocysteine, 28% lower than in V0 (p < 0.01). Oxidative stability of the fresh meat was similar among treatments during a storage period of 42 days. Therefore, methionine supplements improved growth performance during the growing period. Vitamin supplements interacted with the methionine cycle pathway, increased vitamin content of pork meat but did not improve oxidative stability of the fresh meat during storage.     

Effect of restricted feed intake and addition of vitamins B2 and B6 and folic acid on the liver concentration of zinc and copper in rats

The aim of the present study was to investigate the influence of nutritional deficiency and dietary addition of vitamins (B₂, B₆, and folate) on hepatic concentration of zinc and copper in rats. The experiment was performed on 260 growing male Wistar rats divided into 13 groups. Animals of 11 groups were fed isocaloric diets (14.7 MJ/kg) in which the 20% of energy was derived from protein. Another two groups of rats were offered diets with 9% or 4.5% of energy originating from protein. Animals of both mentioned groups and of the control group (20% of energy from protein) were offered diets ad libitum. The other 10 groups were offered 50% and 30% of the amount consumed in the control group. Eight groups, from those 10 restricted ones, were differentiated by dietary addition of vitamins B₂ and B₆ and folate (300% addition). Restricted feed intake did not affect the liver zinc concentration but significantly increased the copper concentration. The addition of vitamin B₆ decreased the liver Zn concentration. The highest liver Cu concentration was noted in rats offered restricted diets to only 30% of intake in the control group and high in vitamin B₂ and in rats supplemented with all of studied vitamins together. It suggests that vitamin B₂ had the strongest impact on liver Cu concentration in rats fed restricted diets.     

Effects of nutrition on zinc, folic acid, and vitamin B12 levels during pregnancy

In this article, we report the results of a case control study carried out on 290 Turkish pregnant women at 5–24 wk of gestation to determine their zinc, folic acid, and vitamin B₁₂ levels in relation to their nutritional and socioconomic status. The women were divided into two groups (n=145 each), depending on the stage of gestation. Group I consisted of women in the first trimester of gestation; those in the second trimester were allocated into group II. Twenty-five age-matched, healthy nonpregnant women were selected as controls. The nutritional status of the subjects was determined by means of a survey. Based on this, the women were subdivided into three subgroups: malnourished (PN), moderately nourished (MN), and well nourished (WN). Also from the survey, the socioeconomic status was classified as good (G) or bad (B). A statistically significant decrease on zinc and folic acid was observed in group I women, as their socioeconomic status worsened. In group II, zinc decreased only in the PN and MN subgroups, p<0.001. The folic acid and vitamin B₁₂ levels did not change significantly during the second trimester of gestation in all subgroups. Regarding zinc, nutrition, or dietary habits are more relevant than socioeconomic status, but poor nutrition affects folic acid levels only during the first trimester of gestation.     

Effect of nitrous oxide on nickel deprivation in rats

Because nickel may have a biological function in a pathway in which vitamin B₁₂ is important, an experiment was performed to determine the effects of nitrous oxide exposure in rats deprived of nickel. Exposure to nitrous oxide (N₂O) causes inactivation of cobalamin and a subsequent decrease in the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl CoA mutase. Rats were assigned to dietary groups of 12 in a factorially arranged experiment with dietary variables of nickel (0 or 1 μg/g) and vitamin B₁₂ (0 or 50 ng/g). After 6 wk, one-half of the rats from each dietary group were exposed to 50% N₂O/50% O₂ for 90 min/d for the last 28 d of the experiment. Vitamin B₁₂, N₂O, or their interaction had numerous effects; classical findings included N₂O-induced reduction in plasma vitamin B₁₂ and decreases in the vitamin B₁₂-dependent enzymes. Inactivation of vitamin B₁₂ by N₂O, however, did not exacerbate signs of nickel deprivation, possibly because the rats were able to metabolically compensate to N₂O exposure. Mention of a trademark or proprietary product in this article does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro

The activity of histidase (l-histidine ammonia-lyase EC 4. 3. 1. 3) of rat liver was inhibited by cystine competitively with the substrate histidine. Histidase was inactivated by preincubation with cystine, but the inactivated enzyme was reactivated by the addition of glutathione to the reaction mixture. Cysteine, at a lower level, partly reactivated the enzyme inactivated by cystine, but, at a higher level, did not reactivate it. Cysteine itself acted inhibitory on the enzyme. Glutathione did not reactivate histidase activity inhibited by cysteine. No other amino acid affected histidase activity at the level of 2 mm. Apparent Km of rat liver histidase was 1.8 × l0^?m.

Neither inhibitory nor activating effect on histidase activity was observed by the addition of liver homogenate obtained from rats fed a histidine imbalanced diet to that from those fed a basal diet and vice versa.     

Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).     

Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes

Transcobalamins, vitamin B₁₂ binding proteins, deliver B₁₂ to cell surface receptors which then permit B₁₂ to cross cell membranes for metabolic use. There is little documentation concerning B₁₂ binding proteins in bacteria and protists. We found that prokaryotes and eukaryotes requiring B₁₂, as well as those protists synthesizing B₁₂, also produce several transcobalamins for functionally transporting B₁₂ similar to humans.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.     

Wave Propagation Mediated by GABAB Synapse and Rebound Excitation in an Inhibitory Network: A Reduced Model Approach

A reduction method is used to analyze a spatially structured network model of inhibitory neurons. This network model displays wave propagation of postinhibitory rebound activity, which depends on GABA_B synaptic interactions among the neurons. The reduced model allows explicit solutions for the wavefronts and their velocity as a function of various parameters, such as the synaptic coupling strength. These predictions are shown to agree well with the numerical simulations of the conductance-based biophysical model.     

Characterization of a Vitamin B12 Compound in the Edible Purple Laver,Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B₁₂ compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B₁₂ compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B₁₂, but not to those of vitamin B₁₂ analogues inactive for humans.     

The Aeration-Dependent Effect of Vitamin B12 on DNA Biosynthesis in Methylobacterium dichloromethanicum

The effect of vitamin B₁₂ (cobalamin) on DNA biosynthesis in Methylobacterium dichloromethanicum was studied. When cultivated in media with methanol or dichloromethane, the bacterium produced approximately 10 g corrinoids per gram dry biomass, compared to about 7 g/g when cultivated on ethanol or succinate. Exogenous adenosylcobalamin (AdoCbl) stimulated DNA biosynthesis in M. dichloromethanicum cells grown under poor aeration, the effect being mediated by AdoCbl-dependent ribonucleotide reductase. In vitro studies showed that M. dichloromethanicumalso has AdoCbl-independent ribonucleotide reductase. Under good aeration, exogenous AdoCbl had no effect on DNA biosynthesis, while hydroxyurea suppressed it. These data suggest that AdoCbl-independent ribonucleotide reductase, which is likely to be activated by oxygen, plays an important part in DNA biosynthesis when M. dichloromethanicum is cultured with good aeration, whereas AdoCbl-dependent ribonucleotide reductase is active under conditions of poor aeration.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

四吡咯化合物生物合成研究进展

四吡咯化合物是存在于生物体中一类具有重要功能的化合物,已经广泛应用于农业、食品和医药等领域.由于化学合成法的烦琐流程和高昂成本以及动植物提取法存在品质不均一等缺点,大幅度限制了其工业化生产与相关应用.近年来,合成生物学的快速发展为微生物利用可再生生物质资源高效合成四吡咯化合物提供了新的技术手段.针对四吡咯化合物生物合成...