Dual effects of acupuncture on gastric motility in conscious rats

The effects of manual acupuncture on gastric motility were investigated in 35 conscious rats implanted with a strain gauge transducer. Twenty (57.1%) rats showed no cyclic groupings of strong contractions (type A), whereas 15 (42.9%) rats showed the phase III-like contractions of the migrating motor complex (type B) in the fasting gastric motility. Acupuncture at the stomach (ST)-36 (Zusanli), but not on the back [Weishu, bladder (BL)-21], increased the peak amplitude of contractions to 172.4 +/- 25.6% of basal in the type A rats (n = 20, P < 0.05). On the other hand, the motility index for 60 min after the acupuncture was not affected by the acupuncture in this group. On the contrary, acupuncture decreased the peak amplitude and motility index to 72.9 +/- 14.0% and 73.6 +/- 16.2% in the type B rats (n = 15, P < 0.05), respectively. The stimulatory and inhibitory effects of acupuncture observed in each type were reproducible on the separate days. In 70% of type A rats, acupuncture induced strong phase III-like contractions lasting for over 3 h that were abolished by atropine, hexamethonium, atropine methyl bromide, and vagotomy. Naloxone significantly shortened the duration of the stimulatory effects from 3.52 +/- 0.21 to 1.02 +/- 0.15 h (n = 3, P < 0.05). These results suggest that acupuncture at ST-36 induces dual effects, either stimulatory or inhibitory, on gastric motility. The stimulatory effects are mediated in part via vagal efferent and opioid pathways.     

Restraint stress delays solid gastric emptying via a central CRF and peripheral sympathetic neuron in rats

Central corticotropin-releasing factor (CRF) delays gastric emptying through the autonomic nervous system. CRF plays an important role in mediating delayed gastric emptying induced by stress. However, it is not clear whether a sympathetic or parasympathetic pathway is involved in the mechanism of central CRF-induced inhibition of solid gastric emptying. The purpose of this study was to investigate whether 1) CRF inhibits solid gastric emptying via a peripheral sympathetic pathway and 2) stress-induced inhibition of solid gastric emptying is mediated via a central CRF and peripheral sympathetic pathways. Using male Sprague-Dawley rats, CRF was injected intracisternally with or without various adrenergic-blocking agents. To investigate whether central CRF-induced inhibition of solid gastric emptying is mediated via a peripheral sympathetic pathway, rats underwent celiac ganglionectomy 1 wk before the gastric emptying study. After solid meal ingestion (90 min), gastric emptying was calculated. To investigate the role of endogenous CRF in stress-induced delayed gastric emptying, a CRF type2 receptor antagonist, astressin2-B, was intracisternally administered. Rats were subjected to a restraint stress immediately after the feeding. Intracisternal injection of CRF (0.1-1.0 microg) dose-dependently inhibited solid gastric emptying. The inhibitory effect of CRF on solid gastric emptying was significantly blocked by guanethidine, propranolol, and celiac ganglionectomy but not by phentolamine. Restraint stress significantly delayed solid gastric emptying, which was improved by astressin2-B, guanethidine, and celiac ganglionectomy. Our research suggests that restraint stress inhibits solid gastric emptying via a central CRF type2 receptor and peripheral sympathetic neural pathway in rats.     

Restraint stress augments postprandial gastric contractions but impairs antropyloric coordination in conscious rats

Central corticotropin-releasing factor (CRF) plays an important role in mediating restraint stress-induced delayed gastric emptying. However, it is unclear how restraint stress modulates gastric motility to delay gastric emptying. Inasmuch as solid gastric emptying is regulated via antropyloric coordination, we hypothesized that restraint stress impairs antropyloric coordination, resulting in delayed solid gastric emptying in conscious rats. Two strain gauge transducers were sutured onto the serosal surface of the antrum and pylorus, and postprandial gastric motility was monitored before, during, and after restraint stress. Antropyloric coordination, defined as a propagated single contraction from the antrum to the pylorus within 10 s, was followed by > or = 20 s of quiescence. Restraint stress enhanced postprandial gastric motility in the antrum and pylorus to 140 +/- 9% and 134 +/- 9% of basal, respectively (n = 6). The number of episodes of antropyloric coordination before restraint stress, 2.4 +/- 0.4/10 min, was significantly reduced to 0.6 +/- 0.3/10 min by restraint stress. Intracisternal injection of the CRF type 2 receptor antagonist astressin 2B (60 microg) or guanethidine partially restored restraint stress-induced impairment of antropyloric coordination (1.6 +/- 0.3/10 min, n = 6). The restraint stress-induced augmentation of antral and pyloric contractions was increased by astressin 2B and guanethidine but abolished by atropine, hexamethonium, and vagotomy. Restraint stress enhanced postprandial gastric motility via a vagal cholinergic pathway. Restraint stress-induced delay of solid gastric emptying is due to impairment of antropyloric coordination. Restraint stress-induced impairment of antropyloric coordination might be mediated via a central CRF pathway.     

Serine/threonine protein kinase SpkA in Synechocystis sp. strain PCC 6803 is a regulator of expression of three putative pilA operons, formation of thick pili, and cell motility

Previous studies showed that a Ser/Thr protein kinase, SpkA, in Synechocystis sp. strain PCC 6803 is involved in cell motility. The present study, in which DNA microarray analysis and electron microscopy were used, demonstrated that SpkA regulates the expression of putative pilA9-pilA10-pilA11-slr2018, pilA5-pilA6, and pilA1-pilA2 operons and is essential for the formation of thick pili.     

Endogenous ghrelin and 5-HT regulate interdigestive gastrointestinal contractions in conscious rats

Endogenous ghrelin causes interdigestive contractions of the stomach in rats. In contrast, previous studies showed that 5-HT(3) and 5-HT(4) receptors were involved in regulating intestinal interdigestive contractions. We studied the possible role of endogenous ghrelin and 5-HT regulating interdigestive gastrointestinal (GI) contractions in rats. Four strain gauge transducers were implanted on the antrum, duodenum, and proximal and distal jejunum. After an overnight fast, GI contractions were recorded in freely moving conscious rats and ghrelin receptor antagonists [(d-lys3)GHRP6; 1 micromol/kg], 5-HT(3) antagonists (Ondansetron; 0.5 mg/kg) and 5-HT(4) antagonists (GR 125,487; 1 mg/kg) were administered (bolus iv). To evaluate the relationship between the luminal concentrations of 5-HT and phase III-like contractions of the duodenum, duodenal juice was collected via the intraduodenal catheter. 5-HT content of the duodenal juice was measured by HPLC. (d-lys3)GHRP6 significantly attenuated the occurrence and amplitude of phase III-like contractions of the antrum, but not the duodenum and jejunum. 5-HT(4) antagonists significantly reduced spontaneous phase III-like contractions of the jejunum, without affecting those of the antrum and duodenum. In contrast, 5-HT(3) antagonists did not affect phase III-like contractions in GI tract. Luminal concentration of 5-HT at the phase III-like contraction (36.0 +/- 13.3 ng/ml, n = 9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 +/- 1.6 ng/ml, n = 9, P < 0.05). It is suggested that released ghrelin from the gastric mucosa mediates gastric phase III-like contractions, whereas 5-HT released from enterochromaffin cells of the duodenal mucosa mediates intestinal phase III-like contractions via 5-HT(4) receptors.     

Phosphorylation of ribosomal protein S19 at Ser59 by CaM Kinase Iα

In order to examine the possible involvements of Ca²⁺/calmodulin-dependent protein kinases (CaM kinases) in the regulation of ribosomal functions, we tested the phosphorylation of rat ribosomal protein S19 (RPS19) by various CaM kinases in vitro . We found that CaM kinase Iα, but not CaM kinase Iβ1, Iβ2, II, or IV, robustly phosphorylated RPS19. From the consensus phosphorylation site sequence, Ser59, Ser90, and Thr124 were likely to be phosphorylated; therefore, we mutated each amino acid to alanine and found that the mutation of Ser59 to alanine strongly attenuated phosphorylation by CaM kinase Iα, suggesting that Ser59 was a major phosphorylation site. Furthermore, we produced a specific antibody against RPS19 phosphorylated at Ser59, and found that Ser59 was phosphorylated both in GT1-7 cells and rat brain. Phosphorylation of RPS19 in GT1-7 cells was inhibited by KN93, an inhibitor of CaM kinases. Immunoblot analysis after subcellular fractionation of rat brain demonstrated that phosphorylated RPS19 was present in 80S ribosomes. Phosphorylation of RPS19 by CaM kinase Iα augmented the interaction of RPS19 with the previously identified S19 binding protein. These results suggest that CaM kinase Iα regulates the functions of RPS19 through phosphorylation of Ser59.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.     

Golgi membrane‐associated degradation pathway in yeast and mammals

Autophagy is a cellular process that degrades subcellular constituents, and is conserved from yeast to mammals. Although autophagy is believed to be essential for living cells, cells lacking Atg5 or Atg7 are healthy, suggesting that a non‐canonical degradation pathway exists to compensate for the lack of autophagy. In this study, we show that the budding yeast Saccharomyces cerevisiae, which lacks Atg5, undergoes bulk protein degradation using Golgi‐mediated structures to compensate for autophagy when treated with amphotericin B1, a polyene antifungal drug. We named this mechanism Golgi membrane‐associated degradation (GOMED) pathway. This process is driven by the disruption of PI(4)P‐dependent anterograde trafficking from the Golgi, and it also exists in Atg5‐deficient mammalian cells. Biologically, when an Atg5‐deficient β‐cell line and Atg7‐deficient β‐cells were cultured in glucose‐deprived medium, a disruption in the secretion of insulin granules from the Golgi occurred, and GOMED was induced to digest these (pro)insulin granules. In conclusion, GOMED is activated by the disruption of PI(4)P‐dependent anterograde trafficking in autophagy‐deficient yeast and mammalian cells.     

Effects of low dose actinomycin D treatment in vivo on the biosynthesis of ribosomal proteins in rat liver

The long-term effects (up to 12 h) of low dose in vivo actinomycin D treatment, which selectively inhibits rRNA synthesis, on the activity of rat liver for the synthesis of ribosomal proteins relative to that for the synthesis of total protein were investigated. The effects of actinomycin D treatment in vivo and in vitro on the template activity of poly(A)-containing mRNA of rat liver for ribosomal proteins were examined by using a wheat germ cell-free system. The following results were obtained. 1. The activity of rat liver for synthesizing total protein observed in vivo and in vitro was inhibited by actinomycin D treatment even at a small dose. 2. A double-labeling technique using [3H] and [14C]leucine in vivo showed that the rate of synthesis of the ribosomal protein fraction relative to that of total protein in actinomycin-treated rat liver (6 + 6 h) was 1.45 times higher than that in the control rat. 3. By using a wheat germ cell-free system, it was shown that the template activity of poly(A)-containing mRNA for the synthesis of total protein was increased slightly by actinomycin D treatment in vivo. Furthermore, the template activity for the ribosomal protein fraction relative to that for total protein was increased. This increase was observed in most of the ribosomal proteins separated on two-dimensional acrylamide gel electrophoresis, although the extents of increase were different among individual ribosomal proteins examined. On the other hand, the selective increase of the template activity for the ribosomal protein fraction was not observed when poly(A)-containing mRNA was incubated with actinomycin D in vitro, although the template activity for total protein was increased slightly.