Generation of dyspeptic symptoms by direct acid and water infusion into the stomachs of functional dyspepsia patients and healthy subjects.

BACKGROUND: The mechanisms of the development of symptoms in functional dyspepsia (FD) patients have not been fully elucidated. We previously reported that acid directly infused into the stomach causes dyspeptic symptoms in asymptomatic healthy controls (HCs); however, the response to acid infusion of FD patients was not determined. AIM: To investigate the severity of dyspeptic symptoms induced by direct acid infusion in FD subjects and HCs. METHODS: This was a multi-centre, cross-over, randomised, double-blind study in 23 FD subjects and 32 HCs. FD was defined using the Rome III criteria. All subjects were Helicobacter pylori negative. Each subject received two tests; 0.1 mol/L hydrochloric acid and water infused into the stomach. The presence and severity of 12 dyspeptic symptoms were assessed using a visual analogue scale. RESULTS: The proportion of subjects developing symptoms by acid or water infusion was significantly greater in FD subjects than HCs. All of the FD subjects experienced at least one symptom by water or acid infusion. In the FD subjects, the severity of symptoms was significantly greater with acid infusion than water infusion. The severity of symptoms in total and the scores for eight of the 12 symptoms induced by acid infusion was significantly greater in FD subjects than in HCs. CONCLUSIONS: The severity of dyspeptic symptom generation induced by direct acid infusion into the stomach was significantly greater in functional dyspepsia subjects than in healthy controls, suggesting that hypersensitivity to acid is one of the important mechanisms of the development of symptoms in functional dyspepsia patients.

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.

Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like proteases.

IL-1beta converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

A novel SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses.

Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. Here we have cloned, identified and characterized a novel member of the mammalian MAPKKs, designated MKK7. MKK7 is most similar to the mediator of morphogenesis, hemipterous (hep), in Drosophila. Immunochemical studies have identified MKK7 as one of the major SAPK/JNK-activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor alpha (TNFalpha) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFalpha. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFalpha-stimulated pathway. Moreover, we have found that overexpression of MKK7 enhances transcription from an AP-1-dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform which is specific for SAPK/JNK, is involved in AP-1-dependent transcription and may be a crucial mediator of TNFalpha signalling.

Purification and identification of a major activator for p38 from osmotically shocked cells. Activation of mitogen-activated protein kinase kinase 6 by osmotic shock, tumor necrosis factor-alpha, and H2O2.

A stress-activated, serine/threonine kinase, p38 (also known as HOG1 or MPK2) belongs to a subgroup of mitogen-activated protein kinase (MAPK) superfamily molecules. An activity to activate p38 (p38 activator activity) as well as p38 activity itself were greatly stimulated by hyperosmolar media in mouse lymphoma L5178Y cells. The activator activity has been purified by sequential chromatography. A 36-kDa polypeptide that was coeluted with the activity in the final chromatography step was identified as MAPK kinase 6 (MAPKK6) by protein microsequencing analysis. Monoclonal and polyclonal antibodies raised against recombinant MAPKK6 recognized specifically the 36-kDa MAPKK6 protein but did not cross-react with MKK3 proteins. The use of these anti-MAPKK6 antibodies revealed that two major peaks of the p38 activator activity in the first chromatography step reside in the activated MAPKK6. Using a genetic screen in yeast, we isolated MKK3b, an alternatively spliced form of MKK3. Like MKK3 and MAPKK6, MKK3b was shown to be a specific activator for p38 and was activated by osmotic shock when expressed in COS7 cells. Immunoblotting analysis revealed that MAPKK6 is expressed highly in HeLa and KB cells and scarcely in PC12 cells, whereas MKK3 and MKK3b are expressed in all cells examined. Immunodepletion of MAPKK6 from the extracts obtained from L5178Y cells and KB cells exposed to hyperosmolar media depleted them of almost all of the p38 activator activity, indicating that MAPKK6 is a major activator for p38 in an osmosensing pathway in these cells. In addition, MAPKK6 was activated strongly by tumor necrosis factor-alpha, H2O2, and okadaic acid and moderately by cycloheximide in KB cells. Thus, there are at least three members of p38 activator, MKK3, MKK3b, and MAPKK6, and MAPKK6 may function as a major activator for p38 when expressed.