Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p.

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.

p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint.

A recently identified ribonucleotide reductase (RR), p53R2, is directly regulated by p53 for supplying nucleotides to repair damaged DNA. We examined the role of this p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint, comparing it to R2-dependent DNA synthesis. The elevation of DNA synthesis activity through RR in response to gamma-irradiation was closely correlated with the level of expression of p53R2 but not of R2. The p53R2 product accumulated in nuclei, whereas R2 levels in cytoplasm decreased. We found a point mutation of p53R2 in cancer cell line HCT116, which resulted in loss of RR activity. In those cells, DNA damage-inducible apoptotic cell death was enhanced through transcriptional activation of p53AIP1. The results suggest that p53R2-dependent DNA synthesis plays a pivotal role in cell survival by repairing damaged DNA in the nucleus and that dysfunction of this pathway might result in activation of p53-dependent apoptosis to eliminate dangerous cells.

Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3.

We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3. In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert. The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB). The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G. The other gene F56 revealed no significant homology to any known genes. These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1.

The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin-dependent protein kinase Pho85p.

Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.

Quantification of sphingosine derivatives in human platelets: inducible formation of free sphingosine.

To elucidate the physiologic role of sphingolipid-derived products as signaling molecules, we analyzed the levels of endogenous sphingosine (Sph) derivatives in human platelets. When the platelets were stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate, neither ceramide formation nor sphingomyelin hydrolysis was observed, which suggests that the sphingomyelin cycle may not be an essential part of the signaling pathway under these conditions. In contrast, Sph was found to increase in platelets upon stimulation. The level of Sph 1-phosphate, which is formed from Sph by the action of Sph kinase, was not affected under our conditions. Although it has been established that Sph inhibits protein kinase C, which regulates the functional responses of the platelets, Sph levels which exert an inhibitory effect on protein kinase C cannot be attained under physiological conditions (without exogenous Sph). Considering the stimulation of the synthesis of Sph by the physiological agonist thrombin, we speculate that Sph is a signaling molecule of physiological importance in platelets, but protein kinase C may not be its target.

Topographical difference of cytoskeletal organization in smooth muscle cells of rat duodenum revealed by quick-freezing and deep-etching method.

The sarcolemmal domain of rat duodenal smooth muscle cells includes caveolae and associated cytoskeletal or filamentous elements. We have used the quick-freezing, deep-etching method to examine the three dimensional relationships between these components. Replica membranes for separated strips of rat duodenal muscle layers were routinely prepared after extraction soluble proteins from cytoplasm and extracellular matrix. As results, 1) cytoskeletal elements in smooth muscle cells consisted mainly of striated thin filaments; 2) thin filaments were connected with some plasma membranes through filaments associated with the sarcolemma, which formed fine network structures beneath the sarcolemma; 3) many bridging structures between the filaments associated with the sarcolemma and the extracellular matrix were frequently detected in the plasma membrane; and 4) compact filaments associated with the sarcolemma almost disappeared near the caveolae, and only thin filaments were anchored to their neck parts. The special arrangement of the cytoskeletal components, which is probably necessary for the intestinal motility, characterizes the topographical difference of the smooth muscle sarcolemma.

X-ray microanalysis of rat mast cells stimulated with compound 48/80 in combination with quick-freezing method.

X-ray microanalysis was performed on rat mast cells prepared by quick-freezing, cryosectioning and freeze-drying (QF-FD) method, or quick-freezing and freeze-substitution (QF-FS) method. Peritoneal cells including mast cells were stimulated with compound 48/80 for 0, 10 or 30 s at 17 degrees C, and the mast cells stimulated for 30 s started exocytosis. In X-ray spectra of the QF-FD specimen, mast cells stimulated for 10 s increased their levels of phosphorus, sodium and chlorine in the intergranular cytoplasm prior to exocytosis, and kept this increase until 30 s after stimulation. In the QF-FS specimen, where soluble elements were removed, peaks of phosphorus, sulphur and potassium could be detected as elements in X-ray spectra. Phosphorus increased and potassium decreased in intergranular cytoplasm of mast cells stimulated for 10 s, and these changes became more obvious after 30 s. However, supplemental increase of other cations such as sodium could not be detected in the QF-FS specimens.

Ultrastructural study of mast cells stimulated with compound 48/80 as revealed by quick-freezing method.

The ultrastructure of mast cells stimulated with compound 48/80 was examined by quick-freezing and deep-etching (QF-DE) or freeze-substitution (QF-FS) methods. Peritoneal cells including mast cells of adult male rats were stimulated in vitro with compound 48/80 at 17 degrees C for 0, 10, 30, 60 or 180 s. The QF-DE replicas revealed that the mast cells stimulated with compound 48/80 for 30 s decreased filamentous actin around secretory granules. In the QF-FS specimens, perigranular membranes in mast cells stimulated for 60 s formed pentalaminar structures between adjacent granules in their cytoplasm prior to degranulation. These findings suggest that preparatory states for degranulation occur in the whole cytoplasm of stimulated mast cells at early stages. Moreover, both QF-FS specimens and QF-DE replicas revealed a compact morphological appearance of discharged granules in the extracellular space, indicating the existence of considerable content within the granules. Skeletal structures in the granules were also demonstrated on QF-DE replicas prepared after extracting soluble elements from the cytoplasm. It is suggested that the granular contents associated with the skeletal structures are gradually detached from the discharged granules to ensure local concentration in the tissues.

Freeze-fracture immunocytochemistry for intracellular localization of serotonin in mast cells stimulated with compound 48/80.

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17 degrees C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.

Endomorphins have orexigenic and anxiolytic activities in mice.

This study was designed to investigate the effects of endomorphin 1 and 2, recently identified mu-opioid receptor selective peptides, on food intake and anxiety in non-food-deprived mice. The intracerebroventricular (i.c.v.) injection of either endomorphin 1 or 2 (0.03-30 nmol) increased food intake in a dose-related manner. A significant increase was observed 20 min after i.c.v. injection of endomorphin 1 or 2 and continued for 4 h. In the elevated plus maze test, the i.c.v. injection of endomorphin 1 (30 nmol) significantly decreased the normal preference for the closed arms. These results suggest that endomorphin produces orexigenic and anxiolytic effects, and that the mu-opioid receptor contributes to the regulation of feeding and anxiety in mice.

Motilin increases food intake in mice.

The effect of motilin on food intake was investigated in nonfood-deprived mice. A significant increase in food intake was observed 1 h after ICV administration of motilin (3 nmol/mouse) and continued for 2 h. This effect was attenuated markedly by the motilin receptor antagonist GM-109 (0.3-3 nmol/mouse) in a dose-related manner. GM-109 alone had no effect on food intake. These results indicate that motilin receptors are present in the brain and may have a role in the regulation of food intake.

Mouse pancreatic polypeptide modulates food intake, while not influencing anxiety in mice.

This study was designed to investigate the effects of synthetic mouse pancreatic polypeptide (mPP) on feeding and anxiety in mice. The intracerebroventricular (i.c.v.) injection of mPP (0.003-3 nmol) dose-dependently increased food intake. A significant increase was observed 20 min after i.c.v. injection and continued for 4 h. The intraperitoneal (i.p.) injection of mPP (0.03-30 nmol) dose-dependently decreased food intake. A significant decrease was observed 20 min after i.p. injection and continued for 4 h. In the elevated plus maze test, the i.c.v. injection of mPP (0.003-3 nmol) did not affect anxiety behavior. These results suggest that mPP modulates food intake and the Y4 receptor in the brain may contribute to the regulation of feeding, whereas appearing not to influence anxiety in mice.

Effect of peptide YY on gastric motor and secretory activity in vagally innervated and denervated corpus pouch dogs.

In this study, we examined the mechanism by which constant intravenous infusion of physiological doses of PYY affects gastric secretion and motility in the vagally innervated (Pavlov) and denervated (Heidenhain) corpus pouch. As a result, only in the Heidenhain pouch, PYY at a dose of 100 pmol/kg-h significantly inhibited gastric secretion in the interdigestive and postprandial states. A dose of 300 pmol/kg-h inhibited the gastric secretion in both types of pouch, but inhibition in the Pavlov pouch was less than in the Heidenhain pouch. The inhibitory effect of PYY on phase III contractile activity was dose-dependent and significant, except in the Heidenhain pouch, and no dose of PYY had any effect on postprandial gastric motility. After all, vagal denervation enhanced the inhibitory effect of PYY on gastric secretion, but abolished the inhibitory effect on phase III contractile activity. Our findings strongly suggest that the inhibitory effect of PYY on gastric secretion is in part mediated by a non-vagal pathway and the inhibitory effect of PYY on gastric motor activities is completely dependent on vagal innervation, but the vagus nerve acts as an inhibitory modulator of the inhibitory effect of PYY on gastric secretion.

Differential gene expression in the small intestines of wildtype and W/W(V) mice.

UNLABELLED: Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/W(V) mice. These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein. We examined gene expression in the small intestines of wildtype and W/W(V) mice. RNA expression in the jejunums of wildtype and W/W(V) mutants was studied using a differential gene expression METHOD: Seven known genes were differentially expressed in wildtype and W/W(V) mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug-resistance complex, class 4) were suppressed in both fed and fasted W/W(V) mice. Expression of another five genes was increased in W/W(V) mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6-5 (encoding phosphorylcholine [PC] T-cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/W(V) mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.

Morphologic changes in the pancreas detected by screening ultrasonography in a mass survey, with special reference to main duct dilatation, cyst formation, and calcification.

Subclinical morphologic changes in the pancreas detected by screening ultrasonography of 130,951 subjects were analyzed in relation to their incidence and background factors. Main pancreatic duct (MPD) dilatation, cystic lesion, and calcification were found in 644 (0.49%), 271 (0.21%) and 65 (0.05%) patients, respectively. The incidence of MPD dilatation and calcification was significantly higher in men (p < 0.0001), whereas cystic lesion was significantly more frequent in women (p < 0.01). Age-dependent increase in the incidence of MPD dilatation and cystic lesion was observed in both sexes whereas that of calcification was observed only in men. Further detailed examinations for 312 randomly selected patients with these findings revealed that 97% of MPD dilatation, 95% of cystic lesion, and 86% of calcification were correctly identified by ultrasonography. Finally, 18 (5.8%) patients with chronic pancreatitis, 16 (5.1%) with neoplastic cysts, 3 with mucin-producing tumors, and 3 with carcinomas (0.96%, respectively) were detected. On the other hand, in 84.0% of MPD dilatation, 87.4% of cystic lesion, and 50.0% of calcification, we could not attribute their etiology to any known pancreatic disease. It is indicated that aging and gender are major clinically related factors of these changes.

Selective uptake of intraluminal dextran sulfate sodium and senna by macrophages in the cecal mucosa of the guinea pig.

The involvement of macrophages in the passage of intraluminal substances into the lamina propria was examined in the large intestine of the guinea pig. Dextran sulfate sodium (DSS) and senna, which, experimentally, induce ulcerative colitis and melanosis coli, respectively, were chosen for examination, since these substances are visible under the microscope without any special treatment. DSS (MW 50,000) and senna were orally administered to guinea pigs. In tissue sections of the intestine, the presence of DSS was demonstrated by toluidine blue staining, while senna was visible under the light microscope as brown pigment. In the large intestine of guinea pigs, macrophages were most numerous in the cecum, decreasing in number towards the rectum. Metachromatic reaction due to DSS was first recognized in the epithelium of the cecum, and was subsequently incorporated by macrophages. The presence of DSS, either in the epithelium or in macrophages, was not recognized in the small intestine or the distal colon. Senna pigmentation was also limited to the cecum and proximal colon, in which pigmented macrophages aggregated in the lamina propria. The two different substances administered orally were taken up in the cecum, and partly also in the proximal colon; the substances passed through the epithelium and were incorporated by macrophages. This finding suggests the existence of a weak point in the intestinal barrier in this particular portion of the intestine.

Ultrastructure of rat duodenal myenteric plexus revealed by quick-freezing and deep-etching method.

A quick-freezing and deep-etching (QF-DE) method was employed with whole-mount strips of rat duodenal muscle walls to exhibit the cytoskeletons of the myenteric plexus. Nerve fibers in the myenteric plexus, which contained fewer neurofilaments than other types of neurons examined, had many varicosed contours, and were bundled by enteroglial cells. Cytoskeleton arrays were rarely observed in the varicosed regions, where synaptic vesicles were often seen, although other nerve regions contained many neurofilaments running almost in parallel with the nerve fiber bundle. Enteroglial cells had short cytoskeletons predominantly across the cytoplasm, becoming thinner the around varicosed regions of the nerve bundles. Such enteroglial extruded areas were often in close association with neighboring nerve fibers, indicating intercommunications between the nerve fibers. In distal parts of enteric nerve processes, there were numerous synaptic vesicles, but few neurofilaments. Smooth muscle cells were closely associated with the enteric nerve processes. Fine network structures, responsible for the extracellular matrix, were present between the smooth muscle cells and the enteric nerve processes. These specific structures of the myenteric plexus could be important for signalling or for the transportation of neurotransmitters involved in gut motility.

Electronic endoscopy in perspective.

Electronic endoscopy, developed in 1983, enables the endoscopic image to be transmitted through electric signals that can be easily processed by computer. An electronic endoscope is composed of three vital parts, i.e., a charge-coupled device (CCD) that converts the image to electric signals, a video processor that converts analog electric signals to the digital and processes them to become video signals, and a television monitor. The introduction of electronic endoscopy has enabled computer management of endoscopic images, on one hand, and provided an opportunity for image processing and analysis, on the other. A digital image management system will further develop into a database of endoscopic images. Combined with the technologies of remote control and remote sensing, the introduction of CCD to endoscopy has made it possible to transform the endoscope, so that it could become a capsule. Future advances in the research of image processing and analysis will lead to the development of a system of automated endoscopic diagnosis.

Urocortin reduces oxygen consumption in lean and ob/ob mice.

A vast number of intensive studies have been undertaken to clarify the mechanisms of energy balance. This study was undertaken to investigate the effect of urocortin, an endogenous ligand for corticotropin-releasing factor (CRF) type 2 receptor, on oxygen consumption in lean and genetically obese (ob/ob) mice. Oxygen consumption was measured after intraperitoneal injection in unrestrained mice at an environmental temperature of 22 degrees C of one of the following: urocortin, deamidated form of urocortin (urocortin OH) or CRF. The intraperitoneal injection of urocortin (0.3-3 nmol) dose-dependently decreased oxygen consumption in lean mice. The inhibitory effect induced by urocortin was more potent than that induced by CRF or urocortin OH. The ranking potency was urocortin > urocortin OH > CRF. Urocortin significantly reduced oxygen consumption in ob/ob mice as well as in lean mice. These results suggest that urocortin decreases oxygen consumption, and that the CRF type 2 receptor may influence energy balance in lean and ob/ob mice.

Case of ulcerative colitis associated with oesophageal ulcer.

A case of ulcerative colitis complicated by oesophageal ulcers is reported. A woman was admitted to our hospital because of exacerbations of ulcerative colitis both in 1992 (aged 15 years) and 1995 (aged 18 years). When she was admitted in 1995 she complained of bloody diarrhoea, sore throat and pain on swallowing. Oesophagogastro-duodenoscopy revealed oesophageal ulcers. Oesophageal pH monitoring (24-h) showed no evidence of gastro-oesophageal reflux disease. After the patient was treated she with oral prednisolone showed considerable improvement clinically and endoscopically. Initial dosage was 60 mg/day, and 1 week later, the dosage was gradually dropped since the patient responded favourably. The improvement of the oesophageal lesions coincided with the remission of ulcerative colitis. The oesophageal ulcers are, therefore, thought to be an extracolonic manifestation of ulcerative colitis.