The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

Oxidative bioconversion of toluene to 1,3-butadiene-1,4-dicarboxylic acid (cis,cis-muconic acid)

Pseudomonas sp. strains, isolated from soil, utilized toluene as their sole carbon source through ameta cleavage pathway. Strains metabolizing toluene through anortho cleavage pathway were selected from the wild typemeta strain. Theortho pathway strains were subjected to chemostat selection to obtain a fast-growing strain with doubling time reduced from 14 to 1.2 h. Benzoale and antibiotics enrichment selection procedures were utilized to select a blocked mutant. The blocked mutant grew on acetate as its sole carbon source and oxidatively converted toluene tocis, cis-muconic acid. Double-blocked and muconate-permeable mutants were also selected to reduce reversion frequency and to enhance muconic acid production. In shake-flask experiments, muconic acid at 3.5 g/l was obtained after 2 days of fermentation. In a 14 l fermenter, muconic acid was produced at 45 g/l in 4 days of controlled fed-batch fermentation. The oxidative bioconversion process was also demonstrated in a 1500 l fermenter.

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Résumé Des souches dePseudomonas sp., isolées du sol, ont utilisé le toluène comme seule source de carbone par la vole de la rupture de cycle enmeta. On a sélectionné des souches métabolisant le toluène par la voie de la rupture de cycle enortho, à partir de la souche sauvage de typemeta. Les souches de la voieortho ont été soumises à la sélection en chémostat pour obtenir des souches à croissance rapide dont le temps de doublement est rédult de 14 à 1.2 h. Les procédures de sélection par enrichissement sur benzoale et antibiotiques ont été utilisées pour sélectionner un mutant bloqué. Le mutant bloqué croît sur acétate comme seule source de carbone et convertit le toluène par voie oxydative en acidecis,cis-muconique. On a également sélectionné des mutants doublement bloqués et perméables au muconate pour réduire la lréquence de réversion et pour augmenter la production d'acide muconique. En expérimentation en flacons agités, on a obtenu 3.5 g/l d'acide muconique après 2 jours de fermentation. En fermentuer de 14 l, on a produit 45 g/l d'acide muconique en 4 jours de fermentation contrôlée en milieu non renouvelé à allmentation étagée. Le processus de bioconversion oxydative a également été démontré en fermenteur de 1500 l.

     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase

Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS.     

Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description

The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.     

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig

Summary The peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.