A case of acute gastric mucosal lesions associated with Helicobacter heilmannii infection

A 69-year-old-woman presented with acute epigastric pain, nausea, vomiting and heartburn. Endoscopy disclosed acute gastric mucosal lesions including mucosal edema, erosions, and ulcers with blood crusts in the antrum. Touch cytology and histological assessment obtained from the affected mucosa revealed acute neutrophilic gastritis and single longer and more coiled organisms than Helicobacter pylori, suggesting Helicobacter heilmannii. Electron micropragh confirmed the characteristic morphology. Despite a positive rapid urease test, H. pylori was not isolated by culture or detected by histology and Gram smears. Based on these findings, a diagnosis of acute gastric mucosal lesions associated with H. heilmannii infection was established. This was successfully treated with a 2-week triple therapy consisting of lansoprazole, clarithromycin and metronidazole with persistent endoscopic and histological remission. This is a rare case of H. heilmannii-associated acute gastric mucosal lesions, diagnosed by morphology using touch cytology and histology. The patient might benefit from antimicrobial treatment employing the regimen effective for H. pylori.     

Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway

We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.     

Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis

The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated. The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk. Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls. The results show the effectiveness of oral vaccination against BCWD in ayu.     

Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases

The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

Synthesis of chemotactic peptide analogs with a photoaffinity cross-linker as new probes for analysis of receptor-ligand interaction

Formylpeptide receptors are well-characterized receptors which participate in host defense responses of neutrophils. We designed and synthesized chemotactic peptide analog with p-benzoylphenylalanine (Bpa) and biotin to probe structural and mechanistic aspects of peptide-receptor interaction. These peptides possess biological activities which were dependent upon spacer residue length of and Bpa position. The covalent photoaffinity label was detected by Streptavidine-blot, which was inhibited by the parent peptide.