L-NAME pre-treatment partially inhibits the agmatine-evoked depression of the electrically induced twitch contraction of isolated rat vas deferens

The effect of the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors agmatine was studied in sympathetic neurotransmission in the rat epididymal vas deferens. Tissues were obtained from N(varpi)-nitro-l-arginine methyl ester (l-NAME)-treated or normal animals and were contracted by electrical stimulation or by exogenous adenosine 5'-triphosphate (ATP). In the electrically stimulated epididymal end, agmatine produced an inhibitory effect on twitch contraction that was partially reversed in l-NAME-treated animals, whereas the inhibition produced by clonidine was not affected by l-NAME treatment. The nitric oxide (NO)-donor S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited twitch contraction. Neither agmatine nor SNAP interfered with the responses induced by exogenous ATP in the epididymal end. Removal of the epithelium of the preparation did not modify the agmatine response. We conclude that a nitrergic pathway activated by agmatine plays a role in its inhibitory effect in rat vas deferens, but it remains to be investigated whether it results from a direct action on the enzyme NO-synthase or a receptor-mediated mechanism.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

A combination of ischemic preconditioning and allopurinol protects against ischemic injury through a nitric oxide-dependent mechanism

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50 mg/kg) was intraperitoneally administered 18 and 1 h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5 h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Diazepam effects on carrageenan-induced inflammatory paw edema in rats: role of nitric oxide

High doses of diazepam (10.0-20.0 mg/kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenan administration. This effect was attributed to an action of diazepam on the peripheral-type benzodiazepine receptor (PBR) present in the adrenal and/or immune/inflammatory cells. The present study was undertaken to analyze the involvement of nitric oxide (NO) on the effects of diazepam on carrageenan-induced paw edema in rats (CIPE) and to look for the presence of PBR and inducible/constitutive NO synthases (NOS) on slices taken from the inflamed paws of diazepam-treated rats. For that, an acute inhibition of NO biosynthesis was achieved using 50.0 mg/kg No mega-nitro-L-arginine (L-NAME), L-arginine (300.0 mg/kg), the true precursor of NO, and D-arginine (300.0 mg/kg), its false substrate, were also used. The following results were obtained: (1) diazepam (10.0 and 20.0 mg/kg) decreased CIPE values in a dose- and time-dependent way; (2) diazepam effects on CIPE were increased by L-NAME pretreatment; (3) treatment with L-arginine but not with D-arginine reverted at least in part the decrements of CIPE values observed after diazepam administration; (4) PBR were found in endothelial and inflammatory cells that migrated to the inflammatory site at the rat paw; (5) confocal microscopy showed the presence of both PBR and NOS in endothelial and inflammatory cells taken from inflamed paw tissues of rats treated with diazepam a finding not observed in tissues provided from rats treated with diazepam's control solution. These results suggest an important role for NO on the effects of diazepam on CIPE. Most probably, these effects reflect a direct action of diazepam on PBR present in the endothelium of the microvascular ambient and/or on immune/inflammatory cells. An action like that would lead, among other factors, to a decrease in NO, generated by NO synthase, and thus in the mechanisms responsible for CIPE.     

Influence of orexin A on the mechanical activity of mouse gastric strips

The presence of orexins and orexin receptors has been revealed not only in the central nervous system but also in the gastrointestinal tract. The present study was aimed to investigate the influence of orexin A (OXA) on the mechanical activity of fundal and antral strips of the mouse stomach. In the fundus, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive, frequency-dependent contractile responses whose amplitude was markedly reduced by OXA and enhanced by the orexin-1 type receptor antagonist SB-334867. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), OXA was no longer effective. Methacholine caused a sustained contracture whose amplitude was not influenced by OXA, TTX or L-NNA. In carbachol-precontracted strips, the neurally-induced relaxant responses elicited during EFS were increased in amplitude by OXA. Antral strips showed a spontaneous contractile activity that was unaffected by TTX or L-NNA and transiently depressed by EFS. OXA did not influence either the spontaneous motility or the EFS-induced effects. The results indicate that OXA exerts region-specific effects and that, in the fundus, depresses EFS-induced contractile responses by acting at the nervous level. It is likely that NO is involved in the effects of the peptide.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

V1 receptor in ENS mediates the excitatory effect of vasopressin on circular muscle strips of gastric body in vitro in rats

The purpose of this study was to localize vasopressin (VP) V_1a receptor in stomach and to characterize the role of VP in the regulation of gastric motility in rats. Double staining was used to locate the V_1a receptor in the gastric body of the rat. The contraction of the circular muscle strips of gastric body was monitored by a polygraph. V_1a receptor was expressed on the neurons of myenteric plexus of the gastric body. VP (10^− 10–10^− 6 M) caused a concentration-dependent contractile effect on the circular muscle strips of gastric body in vitro. V-1880 ([deamino-Pen¹, O-Me-Tyr², Arg⁸]-Vasopressin, 10^− 7 M), a V₁ receptor antagonist, inhibited the spontaneous contraction of the strips. Tetradotoxin (TTX, 10^− 6 M) and V-1880 (10^− 7 M) abolished the excitatory effect of VP. Atropine (10^− 6 M) partially inhibited VP-induced excitatory effect on the muscle strips but hexamethonium (10^− 4 M) did not influence it. These results suggest that V_1a receptor was expressed on the neurons of myenteric nerves. The cholinergic nerve was involved in the excitatory effect of VP on the contraction of gastric body.     

Protein L-isoaspartyl methyltransferase repairs abnormal aspartyl residues accumulated in vivo in type-I collagen and restores cell migration

Abnormal aspartyl residue formation such as L-isoaspartates occurs frequently during aging in long-lived proteins, resulting in the alteration of their structures and biological functions. In this study, we investigated the alteration of aspartyl residues in extracellular matrix (ECM) proteins, type-I collagen and fibronectin, and in integrin- and ECM-binding motifs during aging, as well as the resulting effects on cell biological functions such as migration and attachment. Using protein L-isoaspartyl methyltransferase (PIMT) to monitor the presence of L-isoaspartyl residues, we showed their accumulation during in vivo aging in type-I collagen from rats. In vitro aging of fibronectin as well as of peptides containing an integrin- or ECM-binding motif such as RGDSR, KDGEA and KDDL also resulted in the formation of L-isoaspartyl residues. While aged fibronectin does not alter cell adhesion and migration, type-I collagen aged 20 months reduced by 65% cell motility, but not adhesion, when compared to 3-month-aged type-I collagen. Finally, by repairing 20-month-old type-I collagen with recombinant PIMT (rPIMT), cell migration was recovered by 72%. These results strongly suggest that L-isoaspartyl residue formation in ECM proteins such as type-I collagen could play an important role in reducing cell migration and that PIMT could be a therapeutic tool to restore normal cell migration in pathological conditions where cell motility is crucial.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

mGluR1 in the fundic glands of rat stomach

l-glutamate not only confers cognitive discrimination for umami taste in the oral cavity, but also conveys sensory information to vagal afferent fibers in the gastric mucosa. We used RT-PCR, western blotting, and immunohistochemistry to demonstrate that mGluR1 is located in glandular stomach. Double staining revealed that mGluR1 is found at the apical membrane of chief cells and possibly in parietal cells. Moreover, a diet with 1% l-glutamate induced changes in the expression of pepsinogen C mRNA in stomach mucosa. These data suggest that mGluR1 is involved in the gastric phase regulation of protein digestion.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

缩胆囊素和促胰液素对豚鼠离体胃平滑肌运动的影响

用８个肌槽同时记录豚鼠胃不同部位肌条的收缩活动,以观察八肽缩胆囊素（ＣＣＫ－８）和促胰液素的影响。结果表明：ＣＣＫ－８能（１）增高各部位纵行肌和环行肌的张力;（２）加快胃体纵行肌,胃窦纵行肌、环行肌和幽门环行肌的收缩频率;（３）增大胃窦环行肌收缩波平均振幅和（４）增加幽门环行肌收缩波运动指数,但减少胃体和胃窦纵行肌收缩波平均振幅。上述作用均不能被阿托品和消炎病所阻断。而促胰液素对各部位肌条的收缩活动没有明显的影响。     

Low levels of mammalian TGF-beta1 are protective against malaria parasite infection, a paradox clarified in the mosquito host

Nitric oxide (NO), derived from catalysis of inducible NO synthase (iNOS), limits malaria parasite growth in mammals. Transforming growth factor (TGF)-beta1 suppresses iNOS in cells in vitro as well as in vivo in mice, but paradoxically severe malaria in humans is associated with low levels of TGF-beta1. We hypothesized that this paradox is a universal feature of infection and occurs in the mosquito Anopheles stephensi, an invertebrate host for Plasmodium that also regulates parasite development with inducible NO synthase (AsNOS). We show that exogenous human TGF-beta1 dose-dependently regulates mosquito AsNOS expression and that parasite killing by low dose TGF-beta1 depends on AsNOS catalysis. Furthermore, induction of AsNOS expression by TGF-beta1 is regulated by NO synthesis. These results suggest that TGF-beta1 plays similar roles during parasite infection in mammals and mosquitoes and that this role is linked to the effects of TGF-beta1 on inducible NO synthesis.     

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.