Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua.

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria.

Detailed structural analyses revealed a family of periplasmic chaperones in Gram-negative prokaryotes which are structurally and possibly evolutionarily related to the immunoglobulin superfamily and assist in the assembly of adhesive pili. The members of this family have similar structures consistent with the overall topology of an immunoglobulin fold. Seven pilus chaperone sequences from Escherichia coli, Haemophilus influenzae and Klebsiella pneumoniae were aligned and their consensus sequence was superimposed onto the known three-dimensional structure of PapD, a representative member of the family. The molecular details of the conserved and variable structural motifs in this family of periplasmic chaperones give important insight into their structure, function, mechanism of action and evolutionary relationship with the immunoglobulin superfamily.     

Effect of a melatonin implant on the circadian variation of plasma prolactin and rectal temperature in newborn sheep.

Plasma prolactin and rectal temperature show a circadian rhythm in newborn sheep raised under continuous light. Melatonin lowers the concentration of plasma prolactin but it is not known if it affects its circadian rhythm. To detect whether melatonin acts on the circadian system we studied the effect of a subcutaneous melatonin implant in the circadian rhythms of prolactin and rectal temperature in newborn lambs raised under continuous light. We placed catheters in the pedal artery and vein in 9 newborn lambs (2-5 days of age). A subcutaneous melatonin implant was placed in 4 of the lambs at 9-12 days of age. Blood samples and rectal temperature measurements were obtained hourly for a period of 24 h, 11-15 days after the implant, at 20-27 days of age. To avoid interferences of heparin in our melatonin assay, serum melatonin concentration was measured before and during the implant in three additional newborns. Prolactin and melatonin were measured by RIA. Melatonin concentrations were 52.8 +/- 45.9 pg/ml (day) and 315.5 +/- 77.0 pg/ml (night) before treatment (SEM, P less than 0.001), and increased to 594.1 +/- 54.5 pg/ml after placing the implant (there was no difference in melatonin concentration between day and night during the time that the implant was in place). Melatonin had no effect on rectal temperature or its rhythm, but decreased basal plasma prolactin concentration (control: 97.5 +/- 11.3 ng/ml; treated: 25.1 +/- 2.4 ng/ml, P less than 0.001) and abolished the prolactin circadian rhythm, (Cosinor analysis): control: log prolactin (ng/ml) = 1.8 + 0.26 cos 15 (t - 11.16), p = 0.05; treated: log prolactin (ng/ml) = 1.2 + 0.14 cos 15 (t - 9.43), P = 0.36.     

Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1

The binding specificity of thirteen mouse monoclonal antibodies reacting with Fuc-GM1, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)-Gal beta 1-4Glc beta 1-1Cer, a ganglioside found to be associated with small cell lung carcinoma (O. Nilsson et al. (1984) Glycoconjugate J. 1, 43-49) was studied. The results are based upon radioimmunodetection of their binding to structurally related glycolipids adsorbed to microtiter plates or chromatographed on thin-layer plates. Four of thirteen antibodies reacted only with Fuc-GM1 and both the fucose and the sialic residues were necessary for binding. Optimal binding was obtained when the sialic acid was N-acetylneuraminic acid. When this sialic acid residue was substituted with N-glycoloylneuraminic acid the binding activity was reduced and up to 10-times more Fuc-GM1 was needed for detection. The ceramide composition did not influence the binding. The other nine monoclonal antibodies cross-reacted with glycolipids containing structures closely related to Fuc-GM1 and differed from the specific ones by recognizing a smaller portion of the carbohydrate moiety in Fuc-GM1. These results indicate that anticarbohydrate monoclonal antibodies, recognizing structures involving a large proportion of the sugar in the glycolipid, possess a high specificity and might be useful for detection of tumor-associated ganglioside antigen.     

Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with charcoal inactivates glucocorticoid-binding capacity and receptors can be reactivated to the steroid-binding state by an endogenous reducing system utilizing NADPH and a Mr = 12,000, heat-stable, endogenous, cytosolic protein (Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 13658-13664). In this paper we show that NADPH-dependent conversion of the rat liver glucocorticoid receptor from a nonbinding to a steroid-binding form is blocked in an immune-specific manner by antisera raised against purified rat liver thioredoxin reductase or thioredoxin. The inhibition produced by thioredoxin reductase antiserum may be circumvented by dithiothreitol or overcome by addition of purified thioredoxin reductase. These observations prove that the endogenous glucocorticoid receptor-activating factor is thioredoxin and that the enzyme required for generating the steroid-binding conformation of the glucocorticoid receptor by the endogenous receptor-activating system is thioredoxin reductase.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

D- and L-isoprenaline have different effects on adrenoceptors in the systemic vasculature of the cod, Gadus morhua

The effects of D- and L-isoprenaline on systemic vascular adrenoceptors of the cod was studied using blood pressure recording from conscious cod in vivo and isolated coeliac artery strip preparations in vitro. Experiments with isolated artery preparations demonstrated that the D-, but not the L-isomer of isoprenaline acts as an alpha-adrenoceptor antagonist. A dose-dependent reduction of ventral and dorsal aortic blood pressures together with a dose-dependent increase in the heart rate could be produced by injection of L-isoprenaline. D-Isoprenaline, on the other hand, had no effect on the blood pressures or heart rate except in the highest dose injected. The results show an alpha-adrenoceptor blocking capacity of D-isoprenaline in the cod. The effect of L-isoprenaline on the blood pressure supports the idea that the hypotensive effect of DL-isoprenaline seen in previous studies of the cod may be attributed to an effect of the L-isomer on beta-adrenoceptors mediating vasodilation of the cod systemic vasculature.     

T7 DNA polymerase is not a zinc-metalloenzyme and the polymerase and exonuclease activities are inhibited by zinc ions

Phage T7 DNA polymerase purified to homogeneity by an antithioredoxin immunoadsorbent technique was resolved into its active subunits the gene 5 protein and Escherichia coli thioredoxin by a novel technique involving chromatography on Sephadex G-50 at pH 11.5. Analysis of the metal content of the holoenzyme by atomic absorption spectroscopy showed that it did not contain stoichiometric amounts of zinc. Determination of polymerase and exonuclease activities of the holoenzyme and the gene 5 protein in assay mixtures containing enzyme concentrations in excess of the Zn2+ concentration showed full activity. Addition of Zn2+ resulted in no stimulation and the activities were completely inhibited by 0.1 mM Zn2+. These results demonstrate that the essential T7 DNA polymerase is not a zinc-metalloenzyme and suggest that DNA polymerases show no functional requirement for Zn2+.