Chisae Umezawa

NAD+ biosynthesis and metabolic fluxes of tryptophan in hepatocytes isolated from rats fed a clofibrate-containing diet

Hepatocytes were isolated from rats fed a diet with or without 0.25% clofibrate, and NAD+ synthesis by the hepatocytes was determined using either [carboxyl-14C]nicotinic acid or [5-3H]tryptophan. NAD+ and total pyridine nucleotides synthesized from [14C]nicotinic acid by the clofibrate-treated cells were not significantly different from those synthesized by the control cells when expressed on the basis of nanomoles per hour per milligram of DNA. On the contrary, NAD+ synthesized from [3H]tryptophan was significantly higher in the clofibrate-treated cells (158% of the control cells) on the basis of nanomoles per hour per milligram of DNA. Clofibrate was inhibitory to tryptophan metabolism as a whole, affecting the glutarate pathway more (decreased to 37% of control) than the kynureninase flux (decreased to 64% of control). As a result, the quinolinate-NAD flux, estimated as the difference in the amounts of tryptophan metabolized by the two metabolic pathways, increased in the clofibrate-treated hepatocytes. The increase in quinolinate during the incubation was 8 times more in the clofibrate-treated cells than in the control cells, which confirmed alteration in the metabolic fluxes of tryptophan in the clofibrate-treated cells. Hepatic quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity increased with dietary clofibrate and returned to the control level 1 week after removing clofibrate from the diet. Nicotinate phosphoribosyltransferase (EC 2.4.2.11) and NAD+ glycohydrolase (EC 3.2.2.5) activities remained unchanged with dietary clofibrate.

Protein W, a spore‐specific protein in Myxococcus xanthus, formation of a large electron‐dense particle in a spore

The gene for the major spore‐specific protein, termed protein W, was cloned, and it was found that protein W is composed of 426 amino acid residues including 31% charged (133 residues) and 39% hydrophobic (166 residues) amino acids. In the protein, a motif consisting of five amino acid residues [(V/L/I)–R–E–R–(V/L/I)] is repeated 28 times, and another motif [M–M–(P/G)–Q–G] five times. Protein W is synthesized during a very late stage of development, forming a single, large electron‐dense particle (200–400 nm in diameter) inside a spore. X‐ray microanalysis of the particle revealed that it contains a high amount of phosphate in addition to calcium and magnesium. It is proposed that protein W consisting of highly charged repetitive sequences is a polyphosphate storage protein to store energy in spores. The disruption of the gene for protein W resulted in delayed fruiting body formation and a lower spore yield.

Cloning and nucleotide sequence of the gene for acidocin 8912, a bacteriocin from Lactobacillus acidophilus TK8912

K. KANATANI, T. TAHARA, M. OSHIMURA, K. SANO AND C. UMEZAWA. 1995. Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT, was cloned and determined. It was located on the 14–kb plasmid pLA103 and encoded a 46 amino acid precursor including a 20 amino acid N‐terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.

Community pharmacy practice in Japan—results of a survey

Objective: To survey the present condition of community pharmacies as future sites for pharmacy students’ externship in Japan. Method: A questionnaire consisting of 55 questions was sent to 425 graduates from Kobe Gakuin University, School of Pharmacy, who owned or worked in community pharmacies. Results: Of the 85 responders, about half were owners and half employees of pharmacies. Ninety per cent of pharmacy owners operated three and fewer pharmacies. Fifty per cent of pharmacies only dispensed drugs, 32% handled both OTC drugs and dispensing, and 18% handled only OTC drugs. Among the 44 dispensing pharmacies, 16 were one‐to‐one type pharmacies, 13 were located in front of the big medical institutions, nine dispensed prescriptions from various medical institutions and five were hospital‐owned pharmacies. Forty‐five per cent of pharmacies employed 1–4 part‐time pharmacists and 52% employed 1–4 pharmacist assistants. Thirty‐one per cent of prescriptions came from internal medicine departments and the daily number of prescriptions dispensed by each pharmacy was in the range 10–99 for 41% of the pharmacies and 100–199 for 36% of the pharmacies. The average daily number of prescriptions dispersed by each pharmacist was in the range 30–39 for 29% of pharmacies and in the range 20–29 for 22% of pharmacies. Pharmaceutical information was provided at 73% of pharmacies and patients were counselled orally on their medication at 80% of pharmacies. Patients’ medication histories were recorded at 88% pharmacies. Only 15% of pharmacies conducted patients’ medication counselling at their home, but 34% of pharmacies were planning to start this service. Community pharmacists attended very few professional meetings or continuing education programmes and only 20% of them obtained information through computers. Forty‐seven pharmacists out of the 85 obtained their information from medical representatives of pharmaceutical companies and 32 pharmacists through marketing specialists of wholesalers. Ninety per cent of community pharmacists who responded thought that separation of prescribing and dispensing functions would progress further in the future and 50% of them thought positively about the future social status of pharmacists. Most of our graduates who responded were willing to accept pharmacy students from Kobe Gakuin University as externs at their pharmacies. Conclusion: Despite the low response rate, our survey suggests that community pharmacy in Japan requires substantial improvement. This is to ensure that pharmacists working in that sector can provide the quality information that is required for the optimum management of patients and that the environment is suitable for pharmacy externships.

Effect of Thioridazine or Chlorpromazine on Increased Hepatic NAD+ Level in Rats Fed Clofibrate, a Hypolipidaemic Drug

The effect of the phenothiazines, thioridazine and chlorpromazine, on the increased hepatic NAD+ level of rats fed clofibrate, a hypolipidaemic drug, has been investigated.

Primary Role of NADH Formed by Glucose Dehydrogenase in ATP Provision at the Early Stage of Spore Germination in Bacillus megaterium QM B1551

Metabolic events involved in energy metabolism were studied in order to evaluate the ATP‐forming ability of Bacillus megaterium QM B1551 spores at the very early stage of germination. When heat‐activated spores were germinated on glucose as a sole substrate, its oxidation into gluconate (catalyzed by glucose dehydrogenase, EC 1.1.1.47), the accompanying NADH formation, oxygen uptake, and RNA synthesis were initiated immediately after germination, even when anaerobic breakdown of 3‐phosphoglycerate (an ATP source for spores) and the subsequent glucose metabolism via the phosphorylating pathway were impaired by potassium fluoride (KF). In contrast, fructose metabolism and the accompanying metabolic events did not begin until a few minutes after triggering of germination, and those events were entirely abolished by KF, indicating that fructose metabolism is initiated exclusively via its phosphorylation by the ATP derived from endogenous 3‐phosphoglycerate. Thus those results provided further evidence for our previous proposal (Otani et al (1987) Microbiol. Immunol. 31: 967–974; Sano et al (1988) Biochem. Biophys. Res. Commun. 151: 48–52) that the first molecules of ATP in germinating spores can be efficiently generated via aerobic oxidation of NADH, which is formed by glucose dehydrogenase. Fluorescence monitoring of NADH in germinating spores also supported this conclusion.

Nad Levels In The Rat Primary Cultured Hepatocytes Affected By Peroxisome- Proliferators

The effect of peroxisome-proliferators (PPs) on the NAD level in primary cultured rat hepatocytes was investigated and compared with that in the liver of rat administered with PPs. Various PPs, including fibrates, phthalate esters and steroid, increased NAD level in the cultured hepatocytes as in whole animal with a little exception. The NAD level decreased after once reaching the peak by the addition of most PPs. The gradual decrease of NAD observed in primary cultured hepatocytes, was partially inhibited by the addition of poly(ADP-ribose) polymerase and/or NAD glycohydrolase inhibitors. In the presence of these inhibitors, the increase of NAD by PPs still remained. The mechanism of increasing NAD level by PPs will be due to the stimulation of tryptophan (Trp)-NAD biosynthesis, with the possibility of the transcriptional regulation of genes related to Trp-NAD pathway by PPs. This in vitro system, therefore, can be used to clarify detailed mechanism of the stimulation of hepatic NAD biosynthesis in rats administered PPs.

Characterization of a small cryptic plasmid, pLA105, from Lactobacillus acidophilus TK8912

The complete nucleotide sequence of a cryptic plasmid pLA105 from Lactobacillus acidophilus TK8912 was determined. Sequence analysis revealed that pLA105 has a molecular size of 3,220 bp and contains two open reading frames (ORFA and ORFB) encoding polypeptides of 127 and 123 amino acid residues. The ORFA-encoded protein showed a significant homology (75%) to the replication protein of pediococcal plasmid. However, ORFB exhibit no homology to DNA or protein sequences involved in gram-positive bacterial plasmid replication. The region preceding ORFA contains conspicuous sequences of inverted and direct repeats. The plasmid vector containing this region was able to replicate in the Lactobacillus host when provided with ORFs A and B in trans, suggesting that the repeat region contains the origin sequences essential for pLA105 replication. The constructed plasmid vectors based on the pLA105 replicon are structurally and segregationally stable in the Lactobacillus host.

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.

Effects of peroxisome-proliferators on the TRP-NAD pathway.

The mechanism of the elevation of hepatic NAD level in the rats administered clofibrate and other peroxisome-proliferators were investigated. In the hepatocytes from the clofibrate-fed rats, NAD biosynthesis from Trp, but not from nicotinic acid, was specifically stimulated. The activities of peroxisomal marker enzymes, the peroxisome-proliferator activated receptors (PPAR)-dependent enzymes and key enzymes in the Trp-NAD pathway changed in parallel with the hepatic NAD increase; the activity of quinolinate phosphori-bosyltransferase (QPRT) was increased whereas that of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) was drastically reduced. The results strongly suggest that the hepatic NAD increase might be caused by transcription of genes coding the key enzymes of the Trp-NAD pathway via PPAR.