Structure and expression of the quinolinate phosphoribosyltransferase (QPT) gene family in Nicotiana.

Synthesis of wound-inducible pyridine alkaloids is characteristic of species in the genus Nicotiana. The enzyme quinolinate phosphoribosyltransferase (QPT) plays a key role in facilitating the availability of precursors for alkaloid synthesis, in addition to its ubiquitous role in enabling NAD(P)(H) synthesis. In a previous study, we reported that Nicotiana tabacum L. var. NC 95 possesses a QPT RFLP pattern similar to its model paternal progenitor species, Nicotiana tomentosiformis Goodsp. Here we show that although some varieties of N. tabacum (e.g. NC 95 and LAFC 53) possess QPT genomic contributions from only its paternal progenitor species, this is not the case for many other N. tabacum varieties (e.g. Xanthi, Samsun, Petite Havana SR1 and SC 58) where genomic QPT sequences from both diploid progenitor species have been retained. We also report that QPT is encoded by duplicate genes (designated QPT1 and QPT2) not only in N. tabacum, but also its model progenitor species Nicotiana sylvestris Speg. and Comes and N. tomentosiformis as well as in the diploid species Nicotiana glauca Graham. Previous studies have demonstrated that the N. tabacum QPT2 gene encodes a functional enzyme via complementation of a nadC(-)Escherichia coli mutant. Using a similar experimental approach here, we demonstrate that the N. tabacum QPT1 gene also encodes a functional QPT protein. We observe too that QPT2 is the predominate transcript present in both alkaloid and non-alkaloid synthesising tissues in N. tabacum and that promoter regions of both QPT1 and QPT2 are able to produce GUS activity in reproductive tissues. In N. tabacum and in several other Nicotiana species tested, QPT2 transcript levels increase following wounding or methyl jasmonate treatment whilst QPT1 transcript levels remain largely unaltered by these treatments. Together with conclusions from recently published studies involving functional interaction of MYC2-bHLH and specific ERF-type and transcription factors with QPT2-promoter sequences from N. tabacum, our results suggest that whilst both members of the QPT gene family can contribute to the transcript pool in both alkaloid producing and non-producing tissues, it is QPT2 that is regulated in association with inducible defensive pyridine alkaloid synthesis in species across the genus Nicotiana.

Effects of acid suppression on microbial flora of upper gut.

Decreased acid secretion, due to therapy or disease, predisposes to increased bacterial counts in gastric juice. As bacterial numbers increase, the number of nitrate-reducing strains and the concentration of luminal nitrite usually also increase. However, there is controversy (mainly because of assay problems) about whether decreased acid increases generation of N-nitroso compounds: these may be produced by acid or by bacterial catalysis, and the relative contributions of each are still uncertain. Other potentially important factors include ascorbate secretion (can prevent nitrite conversion to nitroso compounds) and the particular spectrum of nitroso compounds produced. Nitrosation of several histamine H2-receptor antagonists has been demonstrated experimentally, but under conditions that are very unlikely to be encountered clinically. Some acid suppressant therapies have been claimed to aid eradication of Helicobacter pylori, but more work is needed to evaluate this. If ulcer treatment regimens do not also address eradication of H. pylori (when present), gastritis will progress, and the recently documented association between H. pylori and gastric carcinoma needs to be considered. Enteric flora probably also increase if acid secretion is markedly reduced: this does not appear to have nutritional consequences but probably reduces the resistance to occasional infections, of which cholera is the best documented.

Mesalazine: a global safety evaluation.

The potential benefit of sulphasalazine in inflammatory bowel disease is limited by the wide variety of side effects that occur in about one-third of treated patients. Most of the side effects of sulphasalazine are due to the sulphapyridine moiety. Claversal has the advantage of delivering the active ingredient of sulphasalazine--mesalazine--without the undesirable effects of sulphapyridine. Four international multicentre trials involving 932 patients with ulcerative colitis or Crohn's disease compared 0.75 g/day or 1.5 g/day mesalazine, 1.5 g/day or 3.0 g/day sulphasalazine, or placebo. Forty-seven (14%) of 331 patients receiving mesalazine reported adverse events, whereas 33 (23%) of 144 patients receiving sulphasalazine and 23 (19%) of 123 patients receiving placebo reported adverse effects. When lower doses of both mesalazine (0.75 g/day) and sulphasalazine (1.5 to 2.0 g/day) were evaluated in a maintenance trial, the percentage of adverse events was similar for both drugs--14% and 12%, respectively. In these trials the incidence of adverse effects was similar with both doses of mesalazine; however, doubling the sulphasalazine dose resulted in a twofold increase in adverse effects. In contrast, mesalazine appeared not to induce dose-related effects, suggesting that patients may be able to tolerate even higher mesalazine doses than those studied. The withdrawal rate owing to adverse events in all four controlled trials was similar for patients treated with mesalazine (6%) and sulphasalazine (8%). Results of an open postmarketing sampling trial of just over 1700 patients in Germany showed a low overall incidence of adverse effects (3%) with mesalazine and none of the rare, more serious effects, such as hepatoxicity, agranulocytosis, or pulmonary complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the nitrosamine hypothesis of gastric carcinogenesis in precancerous conditions.

A 24 hour gastric aspiration study was carried out on nine Polya gastrectomy, eight pernicious anaemia, and nine matched control subjects. Intragastric pH, bacteria, nitrite, and N-nitroso compounds were assessed half hourly whilst ambulant and hourly when in bed. Both total and nitrate reducing bacterial counts were positively related to pH (chi 2 = 279.3; p less than 0.001), as was nitrite concentration (F = 19.1; p less than 0.0001). By contrast, total (F = 40.6; p less than 0.0001) and stable (F = 257.4; p less than 0.0001) N-nitroso compound concentrations were negatively related to pH. Clear differences in these gastric juice factors were not apparent between matched control and either pernicious anaemia, or Polya gastrectomy because the Polya gastrectomy and matched control groups were heterogeneous for gastric acidity. Thus, although eight of eight pernicious anaemia subjects were hypoacidic (defined as intragastric pH greater than 4 for greater than 50% of both daytime and night time periods), only five of nine Polya gastrectomy and two of nine matched control subjects were hypoacidic. When subjects were rearranged into hypoacidic (n = 15) and acidic (n = 11) groups, bacterial counts (p less than 0.01) and nitrite concentrations (p less than 0.01) were higher, whereas N-nitroso compounds tended to be lower (NS) in the hypoacidic group. These data suggest that, although hypoacidity predisposes to bacterial overgrowth and nitrite generation, it does not enhance nitrosation. Instead, this is maximal at low pH, suggesting chemical rather than bacterial nitrosation, contrary to the nitrosamine hypothesis of gastric carcinogenesis.

Toxicology of cimetidine.

Cimetidine has been shown to have low acute toxicity in dogs and rodents. Repeated-dose studies of up to 24 months' duration in rodents at doses up to 950 mg day-1 kg-1 showed few adverse effects. Liver weight was consistently increased at the highest dose and testis, prostate and seminal vesicle weights were reduced in a dose- and time-related fashion. Cimetidine was not carcinogenic in the rat. In tests of up to 1 year's duration in dogs two animals receiving 504 mg day-1 kg-1 had to be killed before the end of the study. They had degenerative changes in the liver and renal tubular nephrosis. These and other dogs at 504 mg day-1 kg-1 had elevated serum transaminases. No such changes were seen at 366 mg day-1 kg-1 or less. Prostate weights were reduced in a dose- and time-related fashion. In a 7-year study in dogs, specifically designed for the purpose, no changes of the stomach mucosa were seen during regular biopsy. Although shown to be a mild anti-androgen, cimetidine produced no significant adverse effects in reproductive studies. The large body of evidence that cimetidine is not a risk for gastric cancer is reviewed. Over 30 million patients have so far been treated with cimetidine and the prediction from the animal studies that it would be an extremely safe therapeutic agent has been borne out in practice.

Intragastric N-nitrosation is unlikely to be responsible for gastric carcinoma developing after operations for duodenal ulcer.

Three groups of patients studied after operations which had cured their duodenal ulcer were compared with a control group (no operation, n = 8). The surgical procedures included: proximal gastric vagotomy (n = 7), truncal vagotomy and pyloroplasty (n = 7), truncal vagotomy and antrectomy (n = 8). Samples of gastric juice were aspirated half hourly or hourly over 24 hours for measurement of pH, counts of all identified bacteria, nitrite and total N-nitrosocompounds. Although the pH over 24 hours was significantly higher after proximal gastric vagotomy (p less than 0.05) and truncal vagotomy and antrectomy (p less than 0.001) than controls, there was no difference between truncal vagotomy and pyloroplasty and controls. Counts of nitrate reducing bacteria over 24 hours were also significantly higher after truncal vagotomy and antrectomy than controls (p less than 0.1) but no differences were observed between the other groups. Only after truncal vagotomy and antrectomy was nitrite over 24 hours significantly increased compared with controls (p less than 0.01). Despite these higher values after truncal vagotomy and antrectomy, there was no significant difference in total N-nitrosocompounds between any of the four groups. Whereas bacterial counts and nitrite increased with pH, no correlation was found between total N-nitrosocompounds and pH. These results provide no evidence that exposure to total N-nitrosocompounds is increased after operations for duodenal ulcer.

Intragastric acidity, bacteria, nitrite, and N-nitroso compounds before, during, and after cimetidine treatment.

Eight healthy subjects were studied half-hourly or hourly for 24 h periods before, during, and after cimetidine treatment. No significant differences in intragastric bacterial counts or bacterial species or in intragastric nitrite or N-nitroso-compound concentrations were found as a result of cimetidine treatment. Bacterial counts and nitrite concentrations tended to increase with pH, but N-nitroso-compound concentrations did not. This study provides no evidence that cimetidine treatment may increase the risk of gastric carcinoma by raising N-nitroso-compound concentrations.

Effects of certain muscarinic antagonists on the actions of anticholinesterases on cat skeletal muscle.

1. The effects of some muscarinic antagonists, namely, N-ethyl-2-pyrrolidylmethyl-cyclopentylphenyl glycollate (PMCG), N-methyl-4-piperidyl-phenylcyclohexyl glycollate (PPCG, racemate and R and S enantiomers) and 4'-N-methyl-piperidyl-1-phenyl-cyclopentane carboxylate (G3063) on organophosphate (sarin, soman)- and carbamate (neostigmine)-induced twitch augmentation have been studied in cat soleus muscle. 2. The results of a preliminary study comparing the potency of sarin and soman in inhibiting the acetylcholinesterase activity of muscle in relation to the effect on the maximal twitch response indicated that there is not a simple relationship between degree of enzyme inhibition by these drugs and alteration of muscle function. 3. The muscarinic antagonists studied were capable of preventing or reversing sarin-, soman- or neostigmine-induced twitch augmentation. Doses sufficient to give complete protection from the effects of the anticholinesterase agents had little or no effect on the twitch response of normal muscle. 4. The protective action of these muscarinic antagonists is dose-dependent but independent of known antagonist actions at muscarinic receptors. 5. The effects of some local anaesthetics (lignocaine, prilocaine, cinchocaine, procaine) and other membrane stabilizers (quinine, ketamine, chlorpromazine, triflupromazine) were compared with those of the muscarinic antagonists in an attempt to elucidate the mode of action of these acetylcholine antagonists. The evidence is insufficient to exclude the involvement of a membrane stabilizing action.

Characterization and development of cimetidine as a histamine H2-receptor antagonist.

The concept of two classes of histamine receptor, H1 and H2, is introduced and the chemical derivation of histamine H2-receptor antagonists is outlined briefly. Starting from the structure of histamine, chemical modification led eventually to burimamide, the first described histamine H2-receptor antagonist. Further stepwise modifications ultimately afforded metiamide and cimetidine. In vitro studies show that cimetidine is a specific competitive histamine H2-receptor antagonist. In vivo, it is a potent inhibitor of histamine-stimulated gastric acid secretion in rats and dogs after both intravenous and oral administration. It is equally potent as an inhibitor of pentagastrin-stimulated secretion. The evidence suggests that cimetidine inhibits gastric acid secretion through blockade of histamine H2-receptors in the gastric mucosa. Cimetidine has been shown to have low acute toxicity. Repeated dose studies of up to 24 months in rats and up to 12 months in dogs have been carried out and the results are presented and discussed. There is no known toxic effect which would limit the usefulness of cimetidine in man.

The pharmacology of burimamide and metiamide, two histamine H2-receptor antagonists.

Burimamide and metiamide are two histamine H2-receptor antagonists. Evidence is presented that indicates the competitive nature and the specificity of the antagonism. Metiamide is about ten times more potent than burimamide and is also more effective than burimamide when given orally. Both compounds inhibit gastric secretion and the evidence is consistent with this inhibition being due to competitive antagonism of H2 receptors in the gastric mucosa. Burimamide, unlike metiamide, causes release of catecholamines even at dose levels that are just sufficient to produce H2-receptor antagonism. Burimamide, but not metiamide, has alpha-adrenoceptor blocking activity. In certain models for inflammation, particularly rat paw edema induced by compound 48/80, burimamide in combination with the H1-receptor antagonist mepyramine shows anti-inflammatory activity. This may, in part, be associated with the catecholamine-releasing properties of the compound. Metiamide is less active in this respect.

Brain acetylcholine concentration and acetylcholinesterase activity in selectively-bred strains of rats.

Significant differences in whole brain and in brain area acetylcholine concentration have been observed between the two strains of rats selectively bred for differences in speed of acquisition of conditioned avoidance. Brain acetylcholinesterase activity was similar in the strains. The strain showing poor conditioned avoidance behaviour possessed the highest brain concentration of acetylcholine. The finding supports a theory which describes a central cholinergic inhibitory system which may be active in the control of certain types of learning behaviour.