A role for chromogranin A (4-16), a vasostatin-derived peptide, on human colonic motility. An in vitro study

The hypothesis that CgA-derived peptides may be involved in mechanisms modulating motility was tested. Human colonic smooth muscles were studied using an organ bath technique. Acetic acid (AA) effects were characterized on spontaneous mechanical activities (SMA) and on responses to transmural nerve stimulation (NS). AA induced a significant decrease in tone and abolished SMA; this effect was insensitive to either TTX or L-NAME/apamin. The AA-induced inhibitory effects were significantly reduced in the presence of CgA4-16. This effect was insensitive to TTX or L-NAME/apamin. Furthermore, AA-induced effects were blocked in the presence of BAYK8644 and CgA4-16 together. The inhibitory effect of nifedipine was delayed in the presence of CgA4-16. NS induced a triphasic response. Only the excitatory components were reduced in the presence of AA. This effect was dose-related and remained unchanged in the presence of CgA4-16 alone, but was blocked in the presence of simultaneous administration of CgA4-16 and L-NAME/apamin. AA application induced inhibition of human colon motility in vitro. This effect may be mediated through an action on L-type calcium channels. CgA4-16 may display a protective role, which prevents the inhibition of motility due to AA to occur, by acting on both smooth muscle and afferent terminals.     

Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Effects of acetic acid-alcohol,trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes

Summary In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irrversibly changed the conformation of HI and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73–212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.     

Effect of acetic acid on astaxanthin production by Phaffia rhodozyma

Summary Low concentrations of acetic acid decreased the growth rate of and astaxanthin production by Phaffia rhodozyma on glucose, with growth completely inhibited by 2 g acetic acid/l. Using H₂SO₄ for pH control after sugar depletion caused a decline in the biomass concentration, whereas using acetic acid as titrant resulted in an increase in the biomass with a high astaxanthin content of 1430 g/g cells. An extended culture with a continuous glucose feed failed to maintain a high astaxanthin content.     

Effect of DOPA and dopamine on the motility of isolated rat duodenum in vitro]

The motor effects of DOPA and Dopamine on the isolated rat duodenum in vitro have been studied by establishing successive dose- response curves. These effects are either excitatory or inhibitory according to the concentrations used. In every case they are of small amplitude. The inhibitory effects do not exist in the presence of alpha, and beta blocking agents. The excitatory effects are suppressed by using a serotoninergic blocking agent.     

Actions of galanin fragments on rat, guinea-pig, and canine intestinal motility

The 1-20 fragment of synthetic porcine galanin, prepared by tryptic digestion of the intact molecule, was equipotent to synthetic porcine galanin 1-29 in the smooth muscle actions of exciting the rat jejunal longitudinal muscle in vitro and inhibiting circular muscle contractions of the canine small intestine in vitro and in vivo, but was less potent in inhibiting nerve-stimulated contractions of the guinea-pig taenia coli. Fragment 21-29 was effective at high doses only in the canine ileum. Activity of galanin 1-11 was greatly reduced in the dog in vivo. These results may reflect species or cell type differences.     

The effects of extracellular sodium on acid release and motility initiation in rat caudal epididymal spermatozoa in vitro

When rat caudal epididymal spermatozoa were incubated in a sodium-free solution, they suffered a progressive fall in motility, and by 40 min the motility was completely suppressed. However, upon resuspending the spermatozoa in a sodium containing solution, motility was completely restored within 15 min. During this reinitiation period, H⁺ ions were found to be released from spermatozoa. Both motility reinitiation and acid release were found to be closely dependent on the extracellular sodium. Of the other monovalent cations studied, only NH₄⁺ could replace Na⁺ in these events. Both processes were partially inhibited by amiloride (10^?4-10^?3 M) and ouabain (10^?4-10^?3 M) but was unaffected by acetazolamide (10^?4 M). The motility activation and acid release were studied under various conditions. It was found that there was a close correlation between the two processes. The H⁺ efflux during motility activation was accompanied by a rise in the intracellular pH of the sperm. It is proposed that the requirement of Na⁺ for the motility initiation is attributed to an increase in the intracellular pH via the Na⁺-H⁺ exchange. An intracellular pH shift might be involved in motility activation in mammalian sperm.     

The effect of genetically expressed cardiac titin fragments on in vitro actin motility.

Titin is a striated muscle-specific giant protein (M(r) approximately 3,000,000) that consists predominantly of two classes of approximately 100 amino acid motifs, class I and class II, that repeat along the molecule. Titin is found inside the sarcomere, in close proximity to both actin and myosin filaments. Several biochemical studies have found that titin interacts with myosin and actin. In the present work we investigated whether this biochemical interaction is functionally significant by studying the effect of titin on actomyosin interaction in an in vitro motility assay where fluorescently labeled actin filaments are sliding on top of a lawn of myosin molecules. We used genetically expressed titin fragments containing either a single class I motif (Ti I), a single class II motif (Ti II), or the two motifs linked together (Ti I-II). Neither Ti I nor Ti II alone affected actin-filament sliding on either myosin, heavy meromyosin, or myosin subfragment-1. In contrast, the linked fragment (Ti I-II) strongly inhibited actin sliding. Ti I-II-induced inhibition was observed with full-length myosin, heavy meromyosin, and myosin subfragment-1. The degree of inhibition was largest with myosin subfragment-1, intermediate with heavy meromyosin, and smallest with myosin. In vitro binding assays and electrophoretic analyses revealed that the inhibition is most likely caused by interaction between the actin filament and the titin I-II fragment. The physiological relevance of the novel finding of motility inhibition by titin fragments is discussed.     

Effect of hypothalamic extracts and synthetic LH-RH on proteic synthesis by rat hypothalamus in vitro (author's transl)]

The incorporation of 14C-Leucine in pituitary proteins in rats, in vitro, has been studied. In absence of stimulation, the pituitaries of adult female rats have shown approximately twice the capacity of protein biosynthesis in vitro than the pituitaries of prepuberal female rats (21 days old). For the stimulation in vitro of the pituitaries, synthetic LH-RH or hypothalamic extracts from adult or prepuberal female rats were used. The pituitaries of adult female rats did not respond to any of the stimulation tests employed. The pituitaries of prepuberal female rats increased their biosynthetic activity significantly, when synthetic LH-RH or adult female rat hypothalamic extract was added to the culture medium. The addition of prepuberal female rat hypothalamic extract did not alter the basic response. The female prepuberal rats injected during 5 consecutive days with FSH and LH, have shown a greater sensibility to LH-RH in vitro than the ones injected with estradiol and progesterone, or with synthetic LH-RH.     

乙酸对土壤胶体矿物吸附酸性磷酸酶的影响

研究了不同pH值、不同浓度乙酸对酸性磷酸酶在土壤胶体和矿物表面吸附的影响,结果表明,在pH2～8的乙酸体系中,酶在胶体矿物表面的最大吸附pH一般出现在蛋白的等电点和矿物的零电荷点(PZC)之间,各土壤胶体和粘粒矿物对酶的吸附量大小顺序为针铁矿》黄棕壤>砖红壤>高岭石>二氧化锰,乙酸浓度对酶在胶体矿物表面的吸附量和吸附结合能具有较显著影响,在0～200mmol·L^-1范围内,随着乙酸浓度的增加,酶吸附量呈现先升高、后降低、再稳定的趋势,而吸附结合能的变化与此相反,并就乙酸对酶在胶体矿物表面吸附影响的可能机理进行了初步探讨。     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.