Ascorbic acid (ASA) deficiency causes a decrease in hepat-ic concentration of cytochrome P-450 and a decrease in hepatic activity of drug-metabolizing enzymes in rats unable to synthesize AsA (ODS rats). To study the mechanism of the decrease in hepatic concentration of cytochrome P-450 isozymes by AsA deficiency, we chose the xenobiotics-inducible cytochrome P-450 and performed the experiments indicated below. AsA-deficient rats were fed polychlorinated biphenyls (PCB) which markedly induce both CYP1A subfamily and several isozymes in CYP2B subfamily. First, we assayed the activities of two drug-metabolizing enzymes so that one could be functionally distinguished from another. AsA deficiency significantly reduced the hepatic activity of aminopyrine-N-demethylase in ODS rats with and without dietary PCB, but had no effect on benzo(a)pyrene hydroxylase activity. Secondly, quantitative immunoblot analyses demonstrated that the levels of CYP2B1/2B2 and CYP 1 A 1 in the AsA-deficiency rats fed PCB were approximately 60 and 80% lower than those found in rats fed AsA-supplemented diet. The degree of reduction in CYP2B1/2B2 was greater than CYP 1 A 1. Thirdly, AsA deficiency caused a decrease in hepatic abundance of CYP2B 1 /2B2 mRNA, whereas it had no effect on the levels of CYP 1 A 1 and 1A2 mRNA. These results indicated that dietary AsA selectively affects the levels of CYP2B 1 /2B2 mRNA among cytochrome P-450 induced by PCB and plays important roles for optimum induction of drug-inducible cytochrome P-450. We concluded that AsA deficiency decreases specific Proms of drug-inducible cytochrome P-450, especially CYP2B1/2B2 and that the reduction of CYP2B1/2B2 mRNA level in AsA-deficient rats caused a decrease in cytochrome P-450 concentration and hepatic activity of drug-metabolizing enzymes.
Rats were fed either a low vitamin E (VE) diet (-VE), a basal VE diet (+ VE, 4.5 mg%), or a high VE diet (+ VE, 45 mg%) for 7 weeks. VE content, oxidative DNA damage and lipid peroxide levels in their livers were measured. When purified lard was used as a dietary fat, VE content decreased in the low VE group to one-thirtieth of that in the basal VE group; in the high VE group it increased to 4.5-fold that in the basal VE group. Corresponding to the VE levels, lipid peroxide levels increased to 2.7-fold in the low VE group and decreased to two-thirds in the high VE group. The level of 8-hydroxydeoxyguanosine (8-OHdG) in DNA, a marker of oxidative DNA damage, was about 0.6 per 105 deoxyguanosine in the basal VE group and comparable values were found in the low VE and the high VE groups. When either soybean oil or safflower oil was used as a dietary fat, VE content and lipid peroxide levels in the liver were also markedly changed in both the low VE group and the high VE group. 8-OHdG levels in DNA of the low VE with safflower oil group tended to be higher than that of the basal and high VE groups. However, no significant difference was observed among them. These results suggest that the change in VE has little influence on the level of oxidative DNA damage in the liver.
The substances responsible for regulating cytosolic aspar-tate aminotransferase (AspATc) activity in the cytosolic fraction of rat liver were examined. AspATc was removed from the cytosolic fraction by passing the fraction through an affinity column to which anti-AspATc antiserum was conjugated. The unbound fraction from the column was found to decrease the activity of the purified AspATc. A fraction containing compounds of less than MW 1, 000 was obtained by filtering the cytosolic fraction through a YM 2 membrane. This YM 2 filtrate decreased the activity of the purified enzyme; however, the enzymic activity was protected partially by the addition of 2-oxoglutarate or pyridoxal phosphate (PLP). The YM 2 filtrate also decreased the isoelectric points (pls) of the purified enzyme. Influences of glucose and fructose on AspATc were examined, and fructose was found to decrease the enzymic activity and the pls. Fructose was more effective on apoen-zyme than holoenzyme, suggesting that fructose may bind to the Lys258 residue of AspATc which is the binding site of PLP. The effects of various amino acids including substrates on the enzymic activity were also examined. Some amino acids were found to decrease the enzymic activity to various extents, though the pls were unaltered. These results suggest that under physiological conditions, AspATc activity is modified by various low molecular substances in various ways.
Vitamin B12 production by Gram-negative facultative an-aerobic intestinal bacteria, members of the family Enterobacteriaceae, was examined. Klebsiella pneumoniae IFO13541 was the most effective strain with regard to such production. The growth of the strain and its production of vitamin B12 depended exclusively on the concentration of yeast extract added to the medium. The yeast extract components required for the stimulation of bacterial growth and or vitamin B12 production were identified as aspartic acid and pyrroloquinoline quinone (PQQ) and the relationship between vitamin B12 production and these two components was examined. The metabolism of aspartic acid in this process was also investigated; the major metabolites were alanine, glutam-ic acid, and valine. The formation of alanine depended on dehydrogenase, the activity of which was greatly increased with increasing PQQ concen-tration.
The combined effects of dietary protein (casein, whey protein, or soy protein) and fat (safflower oil, perilla seed oil, or their mixed oil; linoleic acid/a-linolenic acid = 5.0) on several lipid parameters were studied in rats. Serum cholesterol and triglyceride levels were lower in perilla seed oil groups irrespective of the protein sources. Liver triglyceride level, and fecal excretion of steroids were influenced mainly by protein sources, particularly isolated soy protein lowered liver tri-glyceride level, and increased the fecal steroid excretion. The fatty acid composition of liver phosphatidylcholine was influenced by dietary fat sources. The proportion of arachidonic acid was significantly lower in rats fed perilla seed oil than in those fed safflower oil or mixed oil, while the proportion of eicosapentaenoic acid was higher in rats fed perilla seed oil than in those fed safflower oil. Arachidonic acid/linoleic acid ratio was significantly lower in perilla seed oil groups, and tended to be higher in casein groups. Eicosapentaenoic acid/arachidonic acid ratio was in-fluenced by both dietary protein and fat, and protein-fat interaction was observed. Eicosapentaenoic acid/arachidonic acid ratio was higher in perilla seed oil groups, and isolated soy protein in perilla seed oil groups lowered the ratio in comparison with other protein sources in perilla seed oil groups.
Rhizomes of Curcuma xanthorrhiza Roxb. (C. xanthor-rhiza), a medicinal plant in Indonesia, has been shown to exert diverse physiological functions. Hitherto, a little attention has been paid to its effect on immune functions. This study was carried out to determine the effect of this medicinal plant on mitogenic response of splenic lymphocytes in rats and population of splenic lymphocytes and macrophages and peripheral blood macrophages in mice. Mitogenic responses of spleno-cytes to phytohemagglutinin, concanavalin A, and pokeweed mitogens were examined in rat fed C. xanthorrhiza for 3 weeks. The medicinal plant increased the blastogenesis to these mitogens. Flow cytometric analysis was carried out for mice fed the medicinal plant for 3 to 5 weeks. C. xanthorrhiza increased the proportion of the splenic T cells throughout the experimental period, but exerted a variable effect on B cells and T cell subsets, that is, elevations of B cells at 3 weeks and of Th cells at 4 weeks without any elevation of Ts cells. The effect of this medicinal plant on a proportion of macrophages from the spleen and peripheral blood was not consistent. Thus, the present study suggests that C. xanthorrhiza contains some principle(s) activating T and B cell-mediated immune functions.
Female Wistar rats were separated into 9 groups, and 9 different synthetic diets (each diet contains different level of protein and calcium) were given to each group. After 5 weeks of these dietary regimens, all rats were sacrificed and calcium, magnesium, and phospho-rus levels in plasma and various tissues were determined. In calcium-deficient groups, calcium, magnesium, and phosphorus level in bone decreased, plasma calcium level decreased and there was a tendency that magnesium levels in brain and liver and phosphorus level in brain in-creased. When comparison was made among the calcium-deficient groups, calcium, magnesium, and phosphorus levels in bone were higher in low protein/calcium-deficient group than higher protein/calcium-deficient groups. It is probable that protein deficiency inhibits calcium depletion and consequently the influence of calcium deficiency is less significant in the condition of deficiency in both protein and calcium.
In this study, the differences between the pattern of stained proteins from genetically obese Zucker rats and those from lean Zucker rats were analyzed using SDS-polyacrylamide gel electrophoresis. The level of the 120 kDa protein in the liver cytosol fraction of the obese rats was several times higher than that found in the lean rats. This protein was not present in the mitochondrial fractions of either strain. The level of the 120 kDa protein was decreased drastically during a 72-h fast in the obese Zucker rats. An increase in the level of this protein was induced in the lean Zucker rats through a 48-h fast followed by refeeding with a high-carbohydrate diet. However, when the lean Zucker rats were refed with a high-fat diet following the fast, no significant change was observed in the level of the 120 kDa protein. These observations strongly suggest that the elevated levels of the 120 kDa protein seen in the liver cytosol fraction of obese Zucker rats may be responsible for the observed in-creases in lipogenesis and pathogenesis of obesity in these rats.
To provide insight into the intracellular translocation of lactase-phlorizin hydrolase, an immunoelectron microscopy was per-formed on rapidly embedded Lowicryl K4M sections of rat jejunum. Lactase-phlorizin hydrolase immunoreactivity was detected not only in the microvillous membranes and in the smooth apical vesicles, but also in the lateral membranes, suggesting an alternative route for intracellular transport of lactase-phlorizin hydrolase via the lateral membranes to the microvilli.
An experiment was designed to evaluate the bioavailabili-ty of purple layer (Asakusa-Nori, Porphyra tenera Kjellman) magne-sium (Mg) in Mg-scarcity Fischer 344 male rats from serum Mg level, kidney calcification and bone Mg contents. Male rats of 4 weeks of age were divided into four groups of six rats. The four groups were con-trol (20SC), Mg-restricted (-Mg20SC), -Mg20SC plus purple layer (-Mg20SCP), and 20SC plus purple layer (20SCP) group respectively. To-Mg20SC, 1/10 Mg of the 20SC diet was added.-Mg20SCP diet was prepared to contain equal amount of Mg as in the 20SC diet with purple layer as a Mg source. 20SCP diet was designed to contain double amount of Mg. After a 3-week experimental period, rats were decapi-tated. Blood serum, right kidney, and right femur were collected and Mg, calcium (Ca), phosphorus (P) were determined. Serum Mg concentra-tion of the-Mg20SC was 1/3 of the 20SC, indicating apparent hypomag-nesemia. Serum P also showed lowered concentration. On the other hand, the serum Ca indicated higher value than the other groups, indicat-ing hypercalcemia. Addition of purple layer to-Mg20SC diet resulted in a normal serum Mg, Ca, and P level. The Mg-scarcity (-Mg20SC) rats accumulated much amount of kidney Ca. Whereas, there was no signifi-cant difference in kidney Ca between control (20SC) group and purple layer-supplemented (-Mg20SCP) rat group. The-Mg20SC rats showed lowered ash content and reduced Mg and P concentrations in the femur. Purple layer supplementation increased the ash, Mg, and P. All of the results indicated that the purple layer Mg was used as a Mg source.
In order to obtain probes suitable for the determination of the major soybean allergen, Gly m Bd 30 K, in soybean-related processed foods, we have prepared two monoclonal antibodies, F5 of IgG2a and H6 of IgM, by the fusion of P3U1 myeloma cells with the spleen cells of BALB/c mice immunized with the reductively carboxymethylated aller-gen (RCM-allergen). The two monoclonal antibodies were shown to be specific to the intact allergen as well as the RCM-allergen and to recognize distinct epitopes on the allergen. Of the monoclonal antibodies, F5 was conjugated with peroxidase. H6 and the labeled F5 were applied as the fixing antibody and the labeled first antibody, respectively, for a direct sandwich enzyme-linked immunosorbent assay of the allergen. When the allergen was extracted from the soybean-related foods with Tris-HCl buffer containing sodium dodecylsulfate and mercaptoethanol, the aller-gen, Gly m Bd 30 K, was shown to be measured in a range of 5-500 ng in this assay.
Intestinal mucosa of rats was prepared by squeezing the frozen and thawed intestine. The method was much easier compared to the conventional method in which intestine was cut longitudinally or everted and mucosa was scraped. Jejunal mucosal weight prepared by the two methods was not different, but ileal mucosal weight prepared by squeezing was significantly heavier than that by scraped. No significant difference was observed between activity of some brush border enzymes (arylamidase, aminopeptidase, alkaline phosphatase, and sucrase) in mucosa prepared by squeezing and that by conventional scraping method in jejunum. Activity of brush border aminopeptidase and sucrase in ileal mucosa prepared by squeezing was significantly higher than that prepared by scraping.
The following study was undertaken to determine whether dietary supplementation with glutamine can be used to modulate the immune response following challenge with methicillin-resistant Staphylococcus aureus (MRSA) organisms in mice. Thirty BALB/c female mice were randomized into 3 groups: group A (n=10) were fed 20% casein diet (control), whereas the mice in Groups B (n=10) and C (n=10) were given 20% casein diet supplemented with 2 and 4% glutamine, respectively. The diets were made isonitrogenous by glycine and alanine supplementation. On the 10th day on these treatments, each mouse was challenged intravenously with 2×108 colony-forming units (CFU)/ml of MRSA organisms and mortality was noted for 20 days. The survival rate in Group A (20%) tended to be lower than the rates in Group B (40%), and Group C (70%). CFU values of spleen and kidney of the surviving mice 20 days post challenge were not different among the three groups (p<0.05). The present results suggest that dietary glutamine supplementation may be effective as a nutritional immunomodulator for the recovery from MRSA infection.
Responses of the activities of liver microsomal mixed-function oxidases (MFOs) induced by phenobarbital (PB) to dietary linoleic acid (LA) levels were investigated in rats. Diets varied in LA content (0, 1.8, 4.1, 7.0, or 13.8 energy %) were fed to rats for 16 days. The activities of MFOs did not change significantly with increasing the dietary LA level, where they reached a plateau even at 1.8 energy%, although the fatty acid composition of liver microsomal lipids reflected well that of dietary lipids. Accordingly, 2 energy% of LA as essential fatty acid seemed to be sufficient to supply the dietary requirement as assessed by the activities of PB-induced MFOs.