Effect of propionate pathway metabolites on the oxidative activity of liver mitochondria under normal conditions and in vitamin B12 deficiency]

We performed a comparative investigation in vivo and in vitro of the propionate and methylmalonate effect on oxidative activity of liver mitochondria in control and vitamin B12-deficient rats and found that efficiency of the effects were less pronounced in vitamin B12-deficient rats. It is also shown that the rates of respiration and phosphorylation decreased in liver mitochondria of vitamin B12-deficient rats.     

Content of free and bound thiamine diphosphate in the liver hyaloplasm of vitamine B1 deficient rats

The amount of free and protein-bound thiamin diphosphate (TDP) in the liver hyaloplasm of B1 vitamin deficient rats has been measured. In the norm the content of protein-bound TDP remains stable (4.5--4.7 micrograms/g tissue) and does not grow upon thiamin injections. The level of the free coenzyme varies appreciably: in the B1-avitaminotic state the content of free TDP decreases, and in the B1-saturated condition it may exceed the norm 4 times. In the liver this enzyme occurs only as a holoenzyme. In case of B1 vitamin deficiency in the diet the transketolase apoform cannot be detected in the liver. A new model for rapid generation of B1-avitaminosis characterized by a significantly lower level of free and bound TDP is described.     

Lipoamide dehydrogenase in vitamin B2-deficient rat liver

The liver and the kidney of rats fed a B₂-deficient diet showed a decrease in lipoamide dehydrogenase (LADase) activity, 65% and 80% of those of rats fed on a B₂-supplemented diet, respectively, but the heart showed no decrease. The liver of B₂-deficient rats showed also a little decrease in the activity of glutathione reductase, while there were no differences in the activities of GOT and LDH in the liver between the B₂-deficient and B₂-supplemented rats. The activity of LADase in the cytosolic fraction of the liver, which was about one-tenth of that of mitochondrial fraction, was decreased in the B₂-deficient rats in a degree almost equal to the decrease in the activity in the mitochondrial fraction. The results obtained with single radial immunodiffusion test indicated that the decrease in the activity of LADase in B₂-deficient rats resulted from the decrease in the amount of the enzyme protein and that not only the livers of B₂-supplemented rats, but also of B₂-deficient rats contained no apoenzyme of LADase.     

Certain characteristics of the rat liver transketolase

Modification of the method for transketolase purification in the rat liver was used to reveal the existence of two molecular forms of this enzyme. One of the forms does not react to development of avitaminosis in animals caused by oxythiamine in different doses. The method is developed for isolation of the basic transketolase form in the rat liver with the 50-80% yield of activity. Km values of two sites for coenzyme binding on protein do not depend on the extent of holoenzyme reconstruction from apoenzyme.     

Antivitamin activity of oxythiamine disulfide nicotinate]

The B1-antivitamin activity of oxythiamine disulphide nicotinate has been determined in experiments on albino mice and it is shown that in the liver this derivative exerts the equal action while in the blood and heart--a more profound and prolonged inhibitory action on the transketolase activity in comparison with oxythiamine disulphide. Like the initial compound oxythiamine disulphide nicotinate does not penetrate through hemato-encephalic barrier and does not inhibit the brain transketolase.     

Uptake and processing of serine: pyruvate aminotransferase precursor by rat liver mitochondria in vitro and in vivo

Processing and uptake of the precursor of serine: pyruvate aminotransferase [EC 2.6.1.51] by mitochondria were studied in vitro and in vivo. Serine: pyruvate aminotransferase was synthesized mainly on free ribosomes as judged by immunoprecipitation of puromycin-labeled nascent peptides prepared from free and bound ribosomes. The precursor of rat liver serine:pyruvate aminotransferase (pSPT) synthesized in vitro was post-translationally processed to an apparently mature form by isolated rat liver mitochondria. Available evidence indicated that the processed product was localized in the matrix of mitochondria. Mature serine:pyruvate aminotransferase did not inhibit the in vitro processing, suggesting that the extra peptide was necessary for the mitochondrial uptake of the precursor. In the livers of rats fed a vitamin B6-deficient high-protein diet, the induction by glucagon of serine:pyruvate aminotransferase occurred and most of the induced enzyme existed in mitochondria as the apo-form, suggesting that pSPT was taken up by mitochondria and processed in the apo-form under the conditions employed. In the in vitro system, on the other hand, the processing of pSPT proceeded both in the absence and presence of pyridoxal 5'-phosphate. Should the precursor also bind the prosthetic molecule, therefore, it would be transported into mitochondria in both the apo- and holo-forms. When isolated rat hepatocytes were labeled with [35S]methionine, labeled pSPT appeared in the cytosolic fraction and was transported rapidly into mitochondria in association with the processing. This uptake and processing were inhibited by a fluorescent laser dye, rhodamine 123, and the precursor accumulated in the cytosol in the presence of the dye.     

Deleted in Liver Cancer 2 (DLC2) Was Dispensable for Development and Its Deficiency Did Not Aggravate Hepatocarcinogenesis

DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.     

Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

Rejection of mouse cardiac allografts by costimulation in trans

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.     

Western blotting assay of transketolase concentration in human hemolysates

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.     

In vitro apolipoprotein B mRNA editing activity can be modulated by fasting and refeeding rats with a high carbohydrate diet.

Apolipoprotein B mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the metabolic modulation of apo B mRNA editing activity can be assayed in vitro using rat liver extracts. The editing activity in extracts from 48h-fasted rats was suppressed relative to that of normal chow-fed rats. Refeeding with a high-sucrose fat-free chow for 48h stimulated liver in vitro editing activity to approximately three times that of control liver extracts. The physical properties of editosomes assembled in extracts from fasted/refed rats differed from those assembled in control or fasted rat liver extracts. Polypeptide analysis revealed quantitative alterations of several proteins in each treatment group suggesting a complex regulatory process. The data corroborate those from in vivo studies and suggest the potential of the in vitro system in studying factors responsible for metabolic regulation of apo B mRNA editing.     

Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Inhibition of rat liver tryptophan pyrrolase activity and elevation of brain tryptophan concentration by administration of DL-alpha-amino-beta-pyridinepropanoic acid (pyridylalanine) analogs

Single doses of DL-alpha-amino-beta-(2-pyridine)propanoic acid (2-PA, 100 mg/kg) significantly decreased the holoenzyme and apoenzyme activities of rat liver tryptophan pyrrolase (TP) and increased brain tryptophan, serotonin (5-HT) and 5-hydroxyindole-3-ylacetic acid concentrations. 2-PA had no inhibitory effect on either of the enzyme activities in vitro, but its expected metabolites were effective. Single doses of DL-alpha-amino-beta-(3-pyridine)propanoic acid (3-PA, 100 mg/kg) decreased only the holoenzyme activity and elevated brain tryptophan and its metabolites levels in rats. 3-PA and its metabolite, 3-pyridylpyruvate, inhibited only the holoenzyme activity in vitro. DL-alpha-Amino-beta-(4-pyridine)propanoic acid (4-PA) caused significant changes in liver TP (holo- and apoenzyme forms) activity and brain tryptophan concentration only after repeated administration (100 mg/kg/day). 4-PA was a weak inhibitor of the holoenzyme, but its metabolites apparently inhibited the holo- and apoenzyme activities in vitro. These findings suggest that PA analogs (and/or their metabolites) increased brain tryptophan (and hence 5-HT synthesis) by directly inhibiting liver TP activity.     

Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization.

Apo-(acetyl-CoA carboxylase) completely free from the holoenzyme was prepared from biotin-deficient rat adipose tissue by using affinity chromatography. The apoenzyme does not aggregate under conditions favouring the transition of the holoenzyme to the polymeric form. Such transition is possible after the conversion of the apoenzyme into the holoenzyme in vitro, thus demonstrating the requirement of the prosthetic biotinyl group for enzyme activation.     

Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]

The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC 2.7.6.2), pyruvate dehydrogenase (EC 1.2.4.1) and transketolase (EC 2.2.1.1) with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.     

Production and purification ofPenicillium citreoviride toxin and its effect on tpp-dependent liver transketolase

The production isolation and purification of a yellow mycotoxin fromPenicillium citreoviride NRRL 2579 in different culture media was described. When injected subcutaneously to albino rats it alters the kinetic pattern of transketolase (EC 2.2.1.1) in liver in vivo in a competitive manner. In vitro, the inhibition is noncompetitive in nature. However, addition of thiamine diphosphate (TPP) to the m vitro system relieved the inhibitory effect. These findings suggested a relationship between citreoviridininduced beriberi and the probable antithiamine effect of the toxin.     

Metabolism of transketolase coenzyme in the rat liver

Kinetic analysis permitted to determine two sites of hydroxythiamine diphosphate binding in apotransketolase. The Ki values for these sites differed significantly: (7-22) X 10(-9) M and (13.0-19.7) X 10(-8) M. The rate of thiamine diphosphate turnover within holotransketolase in rat liver tissue was studied by the radioisotope method, using [14C]thiamine as a labeled precursor. The absolute values of half-substitution time and the rate constant of coenzyme degradation in the transketolase molecule are close to those for the protein moiety of the enzyme and are 153 hours and 0.108 days-1, respectively. In vivo rat liver transketolase exists in a substituted alpha-carbanion form. Within the holoenzyme molecule substitution of thiamine diphosphate for hydroxythiamine diphosphate does not influence the formation of an intermediate alpha-carbanion form of the enzyme.