Clinical characteristics and associated factors of Japanese patients with adenocarcinoma of the esophagogastric junction: a multicenter clinicoepidemiological study.

Gastroesophageal reflux disease-related diseases, such as Barrett's esophagus and adenocarcinoma of the esophagogastric junction (AEGJ), are believed to occur less frequently in Asia than in Western countries. However, the number of reported cases is increasing, yet little is known regarding the epidemiology of AEGJ in Japan. The primary study aim is to investigate the clinicoepidemiological characteristics of AEGJ. The secondary aim is to identify factors associated with it. In the 6.5 years between January 2008 and June 2014, we reviewed 88,199 esophagogastroduodenoscopy (EGD) reports and associated medical records (Study 1). We conducted a case-control study to identify factors associated with AEGJ (Study 2). Control subjects were randomly selected and age and sex matched from among subjects who underwent EGD during medical evaluations. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using an unconditional logistic regression method. During the study period, 83 patients with AEGJ (72 men and 11 women; mean age 68 years) were diagnosed. Six cases were Siewert type I and 77 were type II. The incidence rate of AEGJ was 0.6-1.7/100,000 person-years. Compared with the 101 control subjects, obesity (body mass index ≧ 25 kg/m2; [OR = 2.82; 95% CI: 1.13-7.01]) was associated with AEGJ. The incidence rate of AEGJ is lower in Japan than in Western countries, but associated factors similar to those in Western patients were detected, including obesity, a hiatal hernia, smoking, and the male sex.

Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy.

We determined expression and localization of the anti-apoptotic cellular FLICE inhibitory protein (cFLIP) in the porcine corpora lutea (CL) and corpus albicans (CA) during estrous and pregnancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein expression. The mRNA expression of the short cFLIP isoform (cFLIPS) was higher at the early and mid CL stages, and lower by the late CL stage (P < 0.01); mRNA expression of the long cFLIP isoform (cFLIPL) was higher at the mid CL stage, and lower at the early and late CL stages (P < 0.01). Levels of cFLIPS and cFLIPL were steady and high during the early and mid CL stages, and had significantly decreased (P < 0.01) by the late stage. The cFLIP protein was highly expressed in the early and mid CL stages of estrous, but weakly ex-pressed in the late stage. Expression of cFLIPS showed no significant difference between preovulatory corpus albicans (CA1) and corpus albicans (CA2) coexistent with the CL from the previous estrus, but cFLIPL mRNA expression was higher during CA1 than CA2. The expression of cFLIPS showed no significant difference between CA1 and CA2, but cFLIPL was not detected. The cFLIP protein was weak-ly expressed in the CA. Expression of cFLIPS and cFLIPL mRNA and proteins was observed in the CL, and the cFLIP protein was highly expressed during pregnancy. We propose that cFLIPS/L acts as a survival factor, and performs an anti-apoptotic function in the porcine CL.

Efficacy of adding sodium alginate to omeprazole in patients with nonerosive reflux disease: a randomized clinical trial.

Nonerosive reflux disease (NERD) is the most common form of gastroesophageal reflux disease. Patients with NERD have a lower response rate to proton pump inhibitors (PPIs) than patients with erosive esophagitis when gauged from relief of heartburn. Sodium alginate decreases the acidity of refluxate and protects the esophageal mucosa. However, whether the addition of sodium alginate to PPI therapy can improve NERD symptoms remains unknown. Accordingly, the aim of this study was to evaluate the efficacy of adding sodium alginate to basal PPI therapy for NERD. Patients who had experienced heartburn on at least 2 days per week during the 1-month period before entering the study and had no endoscopic mucosal breaks (grade M or N according to Hoshihara's modification of the Los Angeles classification) were randomized to one of two treatments for 4 weeks: omeprazole (20 mg once daily) plus sodium alginate (30 mL four times a day) (group A) or omeprazole (20 mg once daily) alone (group B). Eighty-seven patients were enrolled, and 76 patients were randomly assigned to group A (n = 36) or group B (n = 40). Complete resolution of heartburn for at least 7 consecutive days by the end of treatment was significantly more common in group A (56.7%) than in group B (25.7%). One patient from group A had mild drug-related diarrhea that was not clinically serious. In conclusion, omeprazole combined with sodium alginate was better than omeprazole alone in Japanese patients with NERD.

Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability.

Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.

Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression.

The proto-oncoproteins ETS1 and growth factor independent-1 (GFI1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant transformation. Here, we report that ETS1 and GFI1 interact and affect gene expression through their cross-talk. Co-immunoprecipitation analyses and glutathione-S-transferase pull-down assays revealed that ETS1 bound directly to GFI1 via its Ets domain, and GFI1 bound to ETS1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reporters showed that GFI1 repressed ETS1-mediated transcriptional activation and ETS1 repressed GFI1-mediated transcriptional activation, in a dose-dependent manner. However, in the Bax promoter where the Ets- and Gfi-binding sites (EBS and GBS) are adjacent, ETS1 and GFI1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the Bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses confirmed that an ETS1-GFI1 complex formed on the Bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against ETS1 and/or GFI1 enhanced endogenous Bax gene expression. Our results suggest that the interaction between ETS1 and GFI1 facilitates their binding to specific sites on the Bax promoter and represses Bax expression in vivo.

Pig cellular FLICE-like inhibitory protein (c-FLIP) overexpression in pig xenograft cells induces resistance to human CD8+ cytotoxic T lymphocyte-mediated xenocytotoxicity.

Although the use of organs from alpha1,3-galactosyltransferase gene knockout pigs may prolong xenograft survival, resulting in overcoming antibody-mediated hyperacute rejection, pig xenografts will be destroyed directly by cell-mediated immunity, such as NK cells, macrophages, and CD8+ cytotoxic T lymphocytes (CTLs). Therefore, conquering cell-mediated immunity, especially of human CD8+ CTLs, is of particular importance to the success of long-term xenograft survival. We have previously reported that the cytotoxicity of human CD8+ CTLs is strong against pig endothelial cells (PEC) and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as a potent inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP S) and a long, 55-kDa form (c-FLIP L). The present study demonstrated that overexpression of c-FLIP S/L genes in PEC markedly suppressed human CD8+ CTL-mediated xenocytotoxicity; moreover, the cytoprotective effects of c-FLIP L appeared to be significantly stronger than those of c-FLIP S. Furthermore, to prove the in vivo prolongation effects of xenograft survival, we transplanted PEC transfectants with c-FLIP(S/L) genes under the rat kidney capsule. Prolonged survival was displayed by xenografts of FLIP S/L PEC transfectants, whereas xenografts of parental PEC were completely rejected by day 5 posttransplantation. Thus, intracellular blocking of death receptor-mediated apoptotic signals by overexpression of c-FLIP S/L in xenograft cells may prevent innate cellular attacks against xenografts opening the window of opportunity for long-term xenograft survival.

Role of cell death ligand and receptor system on regulation of follicular atresia in pig ovaries.

Several hundred thousand primordial follicles are present in the mammalian ovary, however, only a limited number develop to the pre-ovulatory stage, and then finally ovulate. The others, more than 99%, will be eliminated through a degenerative process called 'atresia'. The endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, but the precise temporal and molecular mechanisms involved in the regulation of these events have remained unknown. From many recent studies, it is suggested that the apoptosis in ovarian granulosa cells plays a crucial role in follicular atresia. Notably, death ligand-receptor interaction and subsequent intracellular signalling have been demonstrated to be the key mechanisms regulating granulosa cell apoptosis. In this review, we provide an overview of granulosa cell apoptosis regulated by death ligand-receptor signalling. The roles of death ligands and receptors [Fas ligand (FasL)-Fas, tumour necrosis factor (TNF)alpha-TNF receptor (TNFR), and TNFalpha-related apoptosis-inducing ligand (TRAIL)-TRAIL receptor (TRAILR)] and intracellular death-signal mediators [Fas-associated death domain protein (FADD), TNF receptor 1-associated death domain protein (TRADD), caspases, apoptotic protease-activating factor 1 (Apaf1), TNFR-associated factor 2 (TRAF2), and cellular FLICE-like inhibitory protein (cFLIP), etc.] in granulosa cells will be discussed.

Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia.

We recently identified a new gene, klotho, which is involved in the suppression of multiple aging phenotypes. The mouse homozygous for a disruption of the klotho locus (kl/kl) exhibited multiple pathological conditions resembling human aging. Histomorphometric analysis revealed low-turnover osteopenia in kl/kl mice. The decrease in bone formation exceeded that of bone resorption, resulting in a net bone loss. The number of osteoblast progenitors determined by ex vivo bone marrow cultures was reduced in kl/kl mice. In addition, cultured osteoblastic cells derived from kl/kl mice showed lower alkaline phosphatase activity and matrix nodule formation than those from wild-type mice. Osteoclastogenesis in the coculture of marrow cells and osteoblastic cells was decreased only when marrow cells originated from kl/kl mice independently of the origin of osteoblastic cells. We also found that the expression of osteoprotegerin, an osteoclastogenesis inhibitor, was significantly upregulated in kl/kl mice. We conclude that a defect in the klotho gene expression causes the independent impairment of both osteoblast and osteoclast differentiation, leading to low-turnover osteopenia. Because this state represents a characteristic feature of senile osteoporosis in humans, kl/kl mice can be regarded as a useful model for investigating cellular and molecular mechanisms of age-related bone loss.

The effect of feeding piglets with the diet containing green tea extracts or coumarin on in vitro metabolism of aflatoxin B1 by their tissues.

To clarify whether enzymes involved in aflatoxin B1 (AFB1) metabolism in pigs respond to antioxidant agents, the effect of feeding piglets with diets containing green tea extracts (Sunphenon) and coumarin on in vitro AFB1 metabolism by their liver and intestinal tissues was studied. The results showed that coumarin reduced AFB1-DNA adduct formation by both liver and intestinal microsomes, while Sunphenon did not have any effects. Both coumarin and Sunphenon enhanced the glutathione S-transferase (GST) activity to conjugate AFB1 to glutathione GSH in the intestine, although no effects were noted in the liver. Changes of the expression of mRNA of GSTA2 and GSTO1 were not in parallel with the observed changes of GST activity, suggesting that other GST subtypes are involved in the GST activity toward AFB1. As for lipophilic-free AFB1 metabolites, coumarin reduced the liver microsomal conversion of AFB1 to aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), but Sunphenon exerted no effects. Both coumarin and Sunphenon enhanced the conversion of AFB1 to aflatoxicol in the liver. All the results suggest that feeding with a diet containing coumarin affects AFB1 metabolism to enhance AFB1 detoxification through the suppression of P450 enzyme activity in the liver and the enhancement of GST activity in the intestine. Feeding with a diet containing Sunphenon enhances AFB1 detoxification, but the effects are noted mainly in the intestine.

The usefulness of transabdominal ultrasound for the diagnosis of lower gastrointestinal bleeding.

BACKGROUND: Lower gastrointestinal bleeding is a frequent cause of hospitalization, but diagnostic methods for this condition are not fully established. Transabdominal ultrasound is a widely accepted diagnostic tool in bowel diseases. AIM: To evaluate the usefulness of transabdominal ultrasound for lower gastrointestinal bleeding. METHODS: We reviewed the medical records of consecutive patients who underwent transabdominal ultrasound as the first diagnostic procedure for acute haematochezia during the period June 1999 to June 2004. The study group comprised 111 patients and all underwent colonoscopy thereafter. Detection and diagnosis of lower gastrointestinal bleeding by ultrasonographic examination were evaluated by comparing the ultrasound diagnosis with the colonoscopic findings and final diagnosis. RESULTS: The bleeding site was localized by colonoscopy in 90 of the 111 patients (81%). The bleeding site was localized by ultrasound in 59 of the 90 patients (66%). When the bleeding site was in the rectum, ultrasonographic detectability was 30% (10/33); ultrasonographic detectability was 82-100% when the bleeding site was elsewhere. Rectal bleeding and diverticular bleeding were difficult to diagnose by ultrasound, but for the other diseases, diagnosis by ultrasonographic examination was possible in 91-100% of cases. CONCLUSIONS: Ultrasonographic examination may be an effective screening method for lower gastrointestinal bleeding.

A novel strategy for preventing human CD8+ cytotoxic T lymphocyte-mediated cytotoxicity against pig endothelial cells by overexpression of pig cellular FLICE-like inhibitory protein (c-FLIP) gene.

Human CD8+ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity in xenograft recipients is an important obstacle for successful xenotransplantation of pig organs into humans. In our previous study, we demonstrated that xenocytotoxicity of human CD8+ CTL detrimental to pig endothelial cells (PEC) is mediated mainly by the Fas/FasL apoptotic pathway. Furthermore, we developed new methods to prevent this CTL killing by extracellular remodeling using overexpression of human decoy Fas antigen and membrane-bound human FasL on pig xenograft cells. The cellular FLICE-inhibitory protein (c-FLIP), a caspase-8 inhibitor that lacks the cysteine domain, is a negative regulator of death receptor-mediated apoptosis. c-FLIP proteins exist as long (c-FLIP(L)) and short (c-FLIPs) splice variants, both capable of protecting cells from death receptor-mediated apoptosis. In this report, we have demonstrated that both pig c-FLIPs and pig c-FLIP(L) significantly inhibit human CD8+ CTL-mediated xenocytotoxicity toward stably transfected PEC, although the expression level of pig Fas antigen on cell surface was not changed. These data suggested that intracellular remodeling with overexpression of pig c-FLIP in xenograft cells may decrease the innate cellular responses against xenografts, facilitating long-term xenograft survival.

Famotidine prevents canine gastric blood flow reduction by NSAIDs.

AIM: To investigate the effect of famotidine on gastric blood flow reduction induced by diclofenac sodium, a common non-steroidal anti-inflammatory drug in Japan, using laser Doppler flowmetry in the canine stomach. METHODS: The gastric mucosal blood flow was measured by laser Doppler flowmetry in 15 healthy male beagles before and 60 min after the administration of diclofenac suppository (1.0 mg/kg) into the rectum. The examination was done in a crossover, single-blinded fashion. All dogs underwent both famotidine (0.5 mg/kg) and placebo (saline) injection simultaneously with the administration of diclofenac. In addition, the tissue concentration of prostaglandin E2 was measured. RESULTS: The blood flow decreased by 18.3 +/- 9.1% in the gastric body, by 26.3 +/- 8.1% in the antrum in the placebo group after the administration of diclofenac sodium, while the decreases seen were significantly smaller in the famotidine group: 3.2 +/- 12.6% in the gastric body and 7.9 +/- 16.5% in the antrum (P = 0.001 for the gastric body, P = 0.0034 for the antrum). Conversely, the percentage of mucosal prostaglandin E2 concentration decrease in each group did not show a significant difference. CONCLUSION: Famotidine alleviates the reduction of gastric blood flow induced by diclofenac sodium. Further, not only mucosal prostaglandins but also gastric acid may play an important role in non-steroidal anti-inflammatory drugs-induced gastric microcirculatory disturbance.

Pilot study of Biomarkers for predicting effectiveness of ramosetron in diarrhea-predominant irritable bowel syndrome: expression of S100A10 and polymorphisms of TPH1.

BACKGROUND: Serotonin type 3 receptor (5-HT3 R) antagonists are potentially useful therapeutic agents for diarrhea-predominant irritable bowel syndrome (IBS-D). To identify biomarkers predicting effectiveness of the 5-HT3 R antagonist (ramosetron) in IBS-D. METHODS: Irritable bowel syndrome-D Japanese subjects received 2.5 or 5 µg of ramosetron once daily for 4 weeks. Colonic mucosal S100A and tryptophan hydroxylase (TPH) mRNA expression levels were measured before treatment. Genomic DNA was extracted from blood and polymorphisms of TPH1 and TPH2 were analyzed. KEY RESULTS: Forty-two patients (27 men and 15 women, mean age 42 years) with IBS-D were included for analysis. Improvement of IBS symptoms was seen in 26 (61.9%). Baseline S100A10 (p = 0.02) and TPH1 (p = 0.02) expression were significantly higher in the ramosetron responders than in the non-responders. The frequencies of the TPH1 rs4537731G allele in linkage disequilibrium with the TPH1 rs7130929 T allele (11.5% vs 50%, p = 0.003; OR: 12; 95% CI: 2.1-69) along with TPH1 rs211105 C allele (3.8% vs 43.8%, p = 0.0003; OR: 19; 95% CI: 2.1-181) were significantly lower in the responders than in the non-responders. The mean scores of diarrhea at baseline were significantly higher (5.2 vs 3.7, p = 0.005) in patients with TPH1 rs211105 T/T than those with the G allele. CONCLUSIONS & INFERENCES: TPH1 gene polymorphisms and S100A10 expression, which correlate with 5-HT signaling were associated with ramosetron effectiveness in IBS-D, and may possibly lead to prospective identification of the resistance to treatment.

A novel synthetic triazolotriazepine derivative JTT-606 inhibits bone resorption by down-regulation of action and production of bone resorptive factors.

In the search for a new class of bone-sparing agents, we have conducted random screening of the domestic chemical library using 45Ca release assay from prelabeled cultured neonatal mouse calvariae and identified a novel synthetic triazolotriazepine JTT-606 as a candidate for a potent inhibitor of bone resorption. JTT-606 inhibited 45Ca release dose dependently not only in the control calvarial culture but also in the stimulated cultures by interleukin-1alpha (IL-1alpha), fibroblast growth factor 2 (FGF-2), and parathyroid hormone (PTH). JTT-606 also inhibited both basal and stimulated osteoclast-like (OCL) cell formation in the coculture of mouse osteoblastic cells and bone marrow cells dose dependently, indicating its inhibitory effect on osteoclast differentiation. Ex vivo OCL cell formation by cultured bone marrow cells collected from ovariectomized (OVX) mice also was decreased dose dependently by in vivo application of JTT-606 to a level similar to that from sham-operated mice. Furthermore, JTT-606 inhibited resorbed pit formation by isolated mature osteoclasts as well as by unfractionated bone cells derived from rabbit long bones in the control and FGF-2-stimulated cultures dose dependently, indicating both the direct and the indirect actions of JTT-606 on mature osteoclast function. In addition, JTT-606 reduced production of IL-1alpha, tumor necrosis factor alpha (TNF-alpha), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the human peripheral blood mononuclear cell culture. In vivo analyses of mature OVX rats revealed that the application of JTT-606 for 12 weeks increased the BMD of the lumbar spine and decreased the levels of serum osteocalcin and urine deoxypyridinoline to levels similar to those of 17beta-estradiol-treated OVX rats. We propose that JTT-606 may inhibit both osteoclast differentiation and function by down-regulating both the action and the production of bone resorptive factors. It is speculated that JTT-606 could be a potent agent for the treatment of osteopenic disorders with elevated osteoclastic bone resorption.

Additive effects of combined treatment with etidronate and alfacalcidol on bone mass and mechanical properties in ovariectomized rats.

The present study was undertaken to evaluate the effects of combined treatment with intermittent cyclical etidronate and daily alfacalcidol on the mass and the mechanical properties of bone in ovariectomized rats, and to compare the effects with those of single treatments. Seventy 14-week-old female rats underwent ovariectomy (ovx) or sham operation, and were assigned to seven groups (n = 10 each): sham-operated; ovx; ovx treated with etidronate; ovx treated with 0.1 microg/kg alfacalcidol; ovx treated with 0.2 microg/kg alfacalcidol; ovx treated with etidronate and 0.1 microg/kg alfacalcidol; and ovx treated with etidronate and 0.2 microg/kg alfacalcidol. One week after the operation, etidronate (4 mg/kg per day) was intermittently injected into rats for 2 weeks followed by a 10 week period of no treatment, and alfacalcidol was administered orally every day. After 24 weeks of treatment, all single and combined treatments increased the bone mineral densities (BMDs) of the proximal tibiae, midfemurs, and the fourth and fifth vertebral bodies, which had been decreased by ovx. Combined treatment groups showed higher BMDs than single treatment groups, and the effects were almost equal to the addition of those of respective single treatment groups. The combined treatment also showed additive effects on the mechanical properties of both midfemurs and L4 vertebral bodies. The increases in mechanical properties were proportional to those in BMDs. Analyses of microcomputed tomography images and histology confirmed the strong effects of combined treatments on both trabecular and cortical bone mass without impairment of mineralization or connectivity. We conclude that the combined treatment with etidronate and alfacalcidol additively increases the mass of bone with normal quality, resulting in bone strengthening in ovx rats.

Heterogeneity of presynaptic muscarinic receptors involved in modulation of transmitter release.

In order to extend the characterization of muscarinic receptors at presynaptic sites their inhibitory effect on the stimulation-evoked release of [3H]noradrenaline and [3H]acetylcholine from different axon terminals was studied and the dissociation constants and potencies of different antagonists were estimated, in guinea-pig and rat. While oxotremorine reduced the release of [3H]acetylcholine and [3H]-noradrenaline in a concentration-dependent manner from different release sites (Auerbach plexus, noradrenergic neurons in the right atrium, cerebral cortex), McN-A 343, an M1 receptor agonist, enhanced their release evoked by field stimulation. When the inhibitory effect of oxotremorine on transmitter release was studied, pancuronium, pirenzepine and atropine were competitive antagonists of presynaptic muscarinic receptors located on the noradrenergic axon terminals of the atrium. While atropine and pirenzepine inhibited the muscarinic receptors of cholinergic axon terminals in the Auerbach plexus, pancuronium and gallamine had a very low affinity. Significant differences were found in the affinity constants of antagonists for muscarinic receptors located in the cholinergic axon terminals of Auerbach plexus and cerebral cortex, and noradrenergic axon terminals of the atrium. While atropine and pirenzepine exerted similar effects on these presynaptic sites, pancuronium, gallamine and (11-(2-[diethylamino)-methyl)-1-piperidinyl)acetyl)-5, 11-dihydro-6(1-pyrido(2,3-b)(1,4)-benzodiazepin-6-on) were much more effective on muscarinic receptors controlling acetylcholine release from the cerebral cortex and noradrenaline release from the heart. There was more than 100-fold (2.0 pA2 units) difference in affinities of these antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel mutation in exon 18 of the cartilage oligomeric matrix protein gene causes a severe pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a common skeletal dysplasia characterized by disproportionate short stature, early-onset osteoarthrosis, and dysplasia of the spine, epiphysis, and metaphysis. Multiple epiphyseal dysplasia (MED) is a similar but less severe disorder characterized by dysplasia of the epiphysis. Both disorders are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. COMP mutations cluster in a region of the gene that encodes calmodulin-like repeats (CLRs) and correlate closely with disease severity. Typically, mutations in exon 13 that composes the seventh CLR produce severe PSACH phenotypes, whereas mutations found elsewhere in the gene produce mild PSACH or MED phenotypes. We have identified a PSACH patient carrying a novel mutation in exon 18 of COMP that composes the C-terminal globular domain. This mutation produced a severe PSACH phenotype with marked short stature and deformities of the spine and extremities. Our results extend the range of disease-causing mutations within the COMP gene and demonstrate the importance of the additional domain of COMP protein in its in vivo function.

Long-term observation of reflux oesophagitis developing after Helicobacter pylori eradication therapy.

BACKGROUND: Development of reflux oesophagitis after Helicobacter pylori eradication therapy has been reported, but the prognosis is not well known. AIM: To evaluate the prognosis of patients with reflux oesophagitis that developed after eradication therapy by long-term observation. METHODS: Forty-five patients who developed reflux oesophagitis after successful H. pylori eradication therapy were followed up prospectively. All 45 patients were followed up by endoscopy more than 3 years after onset of reflux oesophagitis (3-year follow-up group) and nine were followed up more than 5 years after onset (5-year follow-up group). Endoscopic observations were performed yearly or when upper gastrointestinal symptoms recurred. Reflux oesophagitis was graded according to the Los Angeles Classification System. Presence of gastro-oesophageal reflux symptoms and medication of proton pump inhibitors, H2-blockers or prokinetics were investigated at final endoscopy. RESULTS: All patients were classified as grade A or B at initial endoscopy. At final observation, the grade of reflux oesophagitis improved in 35/45 (78.8%) patients from the 3-year follow-up group and 7/9 (78.8%) patients from the 5-year follow-up group. Reflux oesophagitis progressed from grade A to B in only four (8.9%) patients from the 3-year follow-up group and in no patients in the 5-year follow-up group. No patient progressed to grade C or D. Gastro-oesophageal reflux symptoms were seen in 12 patients (26.7%) from the 3-year follow-up group and four patients (44.4%) from the 5-year follow-up group. Among them, medication was needed continuously in only six (13.3%) and two (22.2%) patients, respectively. CONCLUSIONS: Reflux oesophagitis, which develops after H. pylori eradication therapy, rarely becomes a long-term clinical problem among patients who complete therapy successfully.

Monoclonal antibodies against the mercaptoethanol-sensitive structure of a cell-cell adhesion protein of Polysphondylium pallidum.

Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64,000 (gp64), of the cellular slime mold, Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.