The genetic basis of interspecific differences in genital morphology of closely related carabid beetles.

Marked diversification of genital morphology is common in internally fertilizing animals. Although sexual selection may be the primary process controlling genital evolution, factors promoting genital evolution are controversial, and the genetic background of genital morphology is poorly understood. We analyzed the genetic basis of species-specific genital morphologies in carabid beetles of the subgenus Ohomopterus (genus Carabus, Carabidae) using two parapatric species with hybrid zones. Biometric analyses on experimental F(1) and backcross populations revealed that inheritance of genital morphology is polygenic. Applying Lande's modification of the Castle-Wright estimator to population means and variances to estimate the minimum number of genes involved, we found that a relatively small number of loci is responsible for species differences in genital morphology. In addition, joint-scaling tests indicated that the additive genetic effect accounts for most interspecific differences in genital traits, but dominance and epistatic genetic effects also play roles. Overall, the genetic basis of male and female genitalia is fairly simple, enabling these traits to respond quickly to selection pressures and to diverge rapidly. Our results provide insight into the diversification of genital morphology in carabid beetles, and will hopefully stimulate further studies on the genetic basis of genitalia, such as mapping of quantitative trait loci affecting species-specific genital morphology.

Unrelated donor bone marrow transplantation for 100 pediatric patients: a single institute's experience.

In all, 100 unrelated donor bone marrow transplantations (UD-BMT) were performed in our institute between October 1993 and January 2003. Of 93 evaluable patients, 73 patients had hematological malignancy, 13 had nonmalignancy and seven had lymphoproliferative disease. The estimated 9-year event-free survival (EFS) rate was 57.1+/-5.5% in all patients. In the following analyses of the patients with hematological malignancy, the standard group had significantly better EFS than the high-risk group (61.5+/-7.0 vs 35.6+/-9.7%, P=0.02), and the EFS rate of the tacrolimus (FK-506)+methotrexate (MTX)+/-methylprednisolone prophylactic group for graft-versus-host disease was superior to that of the FK-506 without MTX group (75.7+/-8.0 vs 55.8+/-7.6%, P=0.02). When we compared the EFS rates of the FK506+MTX+/-methylprednisolone (mPSL) group and the HLA-matched related donor BMT group in our institute, these were almost similar (75.7+/-8.1 vs 68.4+/-9.3%). Therefore, UD-BMT using FK-506+MTX+/-mPSL is a safe and useful method for children with hematological malignancy who require allogeneic BMT.

Effect of SMP-300, a new Na+/H+ exchange inhibitor, on myocardial ischemia and experimental angina models in rats.

We evaluated the effects of SMP-300 (N-(aminoiminomethyl)-11-chloro-5,6,7,8-tetrahydro-8-oxo-4H-pyrrolo[3,2,1-kl][1]benzazocine-2-carboxamide monomethanesulfonate monohydrate), a newly synthesized compound, on Na+/H+ exchange activity in rat cardiomyocytes and on other ion transporters, channels and receptors. We also investigated the protective effects of SMP-300 in isolated ischemic rat hearts and rat isoproterenol- or vasopressin-induced experimental angina models. SMP-300 concentration-dependently inhibited recovery from acidosis in rat myocytes, and its IC50 for Na+/H+ exchange was 6 nM. In comparison, its IC50s for Na+/Ca2+ exchange and for the Na+ channel were >1000 nM, and those for other channels or receptors tested were >10,000 nM. In rat isolated perfused hearts, SMP-300 (10(-8)-10(-7) M), administered only at preischemia and not during reperfusion, significantly improved the postischemic recovery of cardiac function. SMP-300 (0.03-0.3 mg/kg, i.v.) or 5-(N-ethyl-N-isopropyl)-amiloride (1 mg/kg, i.v.) prevented the isoproterenol-induced ST-segment depression in the ECG of anesthetized rats, in a dose-dependent manner. SMP-300 (0.1 mg/kg, i.v.) and 5-(N-ethyl-N-isopropyl)-amiloride (1 mg/kg, i.v.) also inhibited the vasopressin-induced ST-segment depression in the ECG of anesthetized rats. This is the first report presenting the protective effect of Na+/H+ exchange inhibitors on isoproterenol- or vasopressin-induced ECG changes in rats, providing the future perspective of SMP-300, a potent Na+/H+ exchange inhibitor, as an anti-anginal drug.

Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture.

We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.

Association of Helicobacter pylori-dependent gastritis with gastric carcinomas in young Japanese patients: histopathological comparison of diffuse and intestinal type cancer cases.

AIMS: The causal relationship of H. pylori gastric colonization with gastric cancer development has not as yet been fully elucidated. The prevalence of H. pylori infection increases with age in the asymptomatic population in Japan, and reaches a high plateau in those older than 40 years. The objective of this study was to assess the link between H. pylori and gastric carcinomas in patients younger than 40 years. METHODS AND RESULTS: Detection of H. pylori and assessment of background mucosa based on the Sydney system was performed histopathologically for 40 Japanese gastric cancer cases younger than 40 years and compared with 40 age- and sex-matched controls. H. pylori infection in gastric mucosa was detected significantly more frequently (P < 0.001) in patients with cancer (29/40; 72.5%) than in controls (11/40; 27.5%). Additionally, by histopathological comparison between intestinal (18 cases) and diffuse (70 cases) types of young gastric cancer patients, mucosal atrophy and intestinal metaplasia were found to coexist with acute and chronic inflammation in the background mucosa of both intestinal and diffuse types, being significantly more prevalent than in young controls. CONCLUSIONS: As well as the high prevalence of H. pylori in young subjects with gastric cancer, it is clear that persistent infection induces mucosal damage, resulting in atrophy and intestinal metaplasia. Thus, acute/chronic gastritis could play an essential role in the early development of neoplasia in the stomach.

[Prophylaxis with FK-506 for graft-versus-host disease after transplantation of bone marrow from unrelated donors].

Forty-eight patients who underwent bone marrow transplantation (BMT) from serologically HLA-matched unrelated donors received tacrolimus (FK506) alone or with methotrexate (MTX) and/or methylprednisolone (mPSL) to prevent graft-versus-host disease (GVHD). We analyzed retrospectively the efficacy of FK506 for GVHD prophylaxis, and its toxicity, by comparing three groups of patients: those given FK506 alone, those given FK506 + mPSL, and those given FK506 + MTX + mPSL. Grade III and IV acute GVHD occurred in five of 10 patients given FK506 alone and in 11 of 30 patients given FK506 + mPSL. In these groups, severe acute GVHD was commonly seen in the patients who discontinued FK506 administration early after BMT and in those who received bone marrow from genotypically HLA-mismatched donors. Early withdrawal of FK506 was due mainly to severe nephrotoxicity. The incidence of nephrotoxicity was very high in patients who received high-dose FK506 as well as melphalan-containing preconditioning (80% and 50%). None of eight patients who received FK506 + mPSL + MTX developed grade III-IV acute GVHD even though five of them received bone marrow from genotypically HLA-mismatched donors. In patients receiving bone marrow from unrelated donors, adjustment of the initial dose of FK506 seems essential in order to avoid severe nephrotoxicity, and combination of MTX and FK506 is useful for preventing severe acute GVHD.

Relations of vascular calcium channels with blood pressure and endothelium in hypertension and with aging.

To investigate the relationships between the activity in potential operated Ca2+ channels (POC), blood pressure, and endothelium in hypertension, we tested the contractile responses to a Ca2+ channel agonist Bay K 8644 (BAY K) in aorta from deoxycorticosterone-acetate-saline (DOCA-S) and reduced renal mass-saline (RRM-S) hypertensive rats. The effects of mechanical rubbing, N omega-Nitro-L-Arginine Methyl Ester (l-NAME) and indomethacin were also examined. Sensitivity to BAY K increased in experimental rats before they became hypertensive and contractile responses were enhanced as hypertension developed. Force development to BAY K was correlated with blood pressure levels. Endothelium removal enhanced the contractile response to BAY K. L-NAME, but not indomethacin, potentiated the response to BAY K. Contractile response to BAY K was negatively correlated with relaxation to acetylcholine. An enhanced contractile response to BAY K was observed also in aged rats. Enhanced activation of vascular POC in hypertension results from elevated blood pressure and partly from diminished inhibitory action of endothelium. Senescence also enhances vascular POC activity.

Lack of active lung anaphylaxis in congenitally mast cell-deficient Ws/Ws rats sensitized with the nematode Nippostrongylus brasiliensis.

Ws/Ws rats are deficient in both mucosal- and connective tissue-type mast cells. To study the role of mast cells in active anaphylaxis, changes in vascular permeability in the trachea upon intravenous antigen challenge with Evans blue dye were examined in Ws/Ws, heterogenic Ws/+, and normal +/ + rats sensitized with the nematode Nippostrongylus brasiliensis. Antigen challenge resulted in fatal anaphylactic shock in some +/+ and Ws/+ rats, but not in Ws/Ws rats. Marked dye leakage developed within 30 min in the trachea of +/+ and Ws/+ rats, while Ws/Ws rats showed no substantial increases in the levels of vascular permeability. Ex vivo stimulation of sensitized lung fragments from +/+ animals with specific antigen induced significant releases of histamine and leukotriene (LT) C4, while sensitized Ws/Ws rat-lung fragments did not. In Ws/Ws rats, levels of nematode-specific IgE, IgG1 and IgG2a antibodies as well as levels of lung eosinophilia were not significantly different from those in +/+ rats. These results show that mast cell-deficient Ws/Ws rats fail to develop active anaphylaxis, and this is mediated probably by the lack of mast cell-derived mediators required for initiation of the reaction.

dad-1, A putative programmed cell death suppressor gene in rice.

The human dad-1 cDNA homolog was isolated from rice plants. The amino acid sequence of the predicted protein product is well conserved in both animals and plants. This rice dad-1 homolog can rescue the temperature-sensitive dad-1 mutants of hamster cells from apoptotic death, suggesting that the rice dad-1 homolog also functions as a suppressor for programmed cell death.

Conformational change of DNA on the formation of DNA-anti-DNA antibody immune complex.

Monoclonal antibodies were purified from the culture medium of hybridoma from systemic lupus erythematosus (SLE)-prone MRL/Mp-Ipr-Ipr mice with affinity chromatography. Binding activity of the antibodies to double-stranded DNA from salmon milt was actually shown in enzyme-linked immunosorbent assay (ELISA). Circular dichroism (CD) spectrum of DNA solution (150 mM NaCl, 20 mM Na2HPO4, pH 7.4) which showed B-type conformation, changed significantly by addition of the anti-DNA antibodies. These results indicate that conformational change of DNA occurred by binding of anti-DNA antibodies, forming DNA-anti-DNA antibody immune complex.

Lung granulomatous response induced by infection with the intestinal nematode Nippostrongylus brasiliensis is suppressed in mast cell-deficient Ws/Ws rats.

Certain nematode infections induce eosinophil infiltration and granulomatous responses in the lungs. To examine the role of mast cells in the development of lung lesions, normal +/+ and genetically mast cell-deficient Ws/Ws rats were infected with the nematode Nippostrongylus brasiliensis. In +/+ rats, numbers of eosinophils in bronchoalveolar lavage fluid (BALF) increased significantly 3-7 days after infection, and granulomatous responses composed of histiocytes/ macrophages and multinucleate giant cells were triggered in the lungs 3-14 days after infection. Challenge infection, which was carried out on day 28 after primary infection, induced much higher levels of granulomatous response than after primary infection, suggesting that the response is mediated at least in part by an immunological mechanism. In Ws/Ws rats, both the eosinophil percentage in BALF and the size of the granulomas in the lungs were significantly smaller than in +/+ rats after primary as well as after challenge infection. The amount of rat mast cell protease (RMCP) II in +/+ rat BALF was increased 1 day after primary infection and more significantly after challenge infection, suggesting that lung mucosal mast cells were activated more markedly after the challenge infection. In Ws/Ws rats, RMCP II was undetectable throughout the observation period. The time course of nematode migration in the lungs did not differ in +/+ and Ws/Ws rats. These results suggest that mast cell activation might be relevant to eosinophil infiltration and granulomatous response in the lungs, although the responses do not affect lung migration of the nematode.

Modulation of vascular calcium channel activity in response to acute volume expansion in rats.

The mechanisms of the increased resistance in hypertension are still unclear. Several studies have indicated that the potential-sensitive Ca2+ channels (PSC) are altered in arteries isolated from hypertensive patients or animals. An expansion of body fluid volume may trigger local autoregulatory responses or may induce the release of humoral factors, either of which could increase systemic vascular resistance and cause volume-dependent forms of hypertension. We tested the hypothesis that volume expansion per se may cause the alterations of PSC similar to those seen in hypertension. For this, we examined the alterations of PSC in aortas from volume-expanded rats with the use of dihydropyridine-type Ca2+ channel activator, BayK 8644, in parallel with the changes in endothelium-dependent relaxation. Volume expansion was produced by a rapid intravenous infusion of saline (10% of body weight) over 30 min in rats. At the end of infusion, rats were killed and aorta removed for in vitro measurement of isometric tension. Relaxation to acetylcholine (10(-7)-10(-5) mol/L, % relaxation to 10(-7) mmol/L norepinephrine contraction) was not significantly changed. In contrast, contractile response to BayK 8644 (10(-9)-10(-6) mol/L, % response to 50 mmol/L KCl) was significantly enhanced in rats with volume expansion (12 control rats: 11.6 +/- 4.9%; 18 volume-expanded rats: 40.9 +/- 10.4% at 10(-6) mol/L, p < 0.05). These findings suggest that acute volume expansion could induce a similar enhanced vascular Ca2+ channel activity to that seen in hypertension in rats.

Role of ouabainlike compound in rats with reduced renal mass-saline hypertension.

Ouabainlike compound (OLC) has recently been identified as a likely mammalian endogenous digitalis-like factor (EDLF) from human plasma. In this study, plasma levels of OLC were determined to assess the role of OLC in a model known as volume-expanded, reduced renal mass (RRM)-saline (S) hypertension in rats with use of a newly developed radioimmunoassay for ouabain. In the first experiment, at 3 wk after subtotal nephrectomy and drinking 1% saline solution, sysolic blood pressure (SBP) of 18 rats with reduced renal mass (RRM-S rats) was significantly higher than in 17 sham-operated saline-drinking control (C-S) rats [154 +/- 4 (SE) vs. 132 +/- 2 mmHg; P < 0.01]. Plasma OLC levels were 355 +/- 68 pmol/l in RRM-S rats, sevenfold higher than in C-S rats (54 +/- 4 pmol/l; P < 0.01). In the second experiment, we measured plasma OLC levels of 10 RRM-S, 12 sham-operated control (C), and 10 subtotally nephrectomized rats drinking distilled water (RRM rats). Concomitant with a marked increase in blood pressure (203 +/- 5 mmHg), RRM-S rats showed significantly higher plasma OLC levels compared with C and RRM rats (RRM-S 114 +/- 24, C 47 +/- 11, and RRM 52 +/- 9 pmol/l; P < 0.05). In both experiments, plasma OLC levels correlated significantly with SBP (P < 0.05). These findings suggest that plasma OLC shows a similar behavior to that of EDLFs or Na(+)-K(+)-adenosinetriphosphatase inhibitors reported in previous publications and may play a role in hypertensive mechanisms in rats with RRM and excess Na intake.

Participation of ouabainlike compound in reduced renal mass-saline hypertension.

We examined the role of ouabainlike compound in reduced renal mass-saline hypertension using a population of rats immunized with ouabain. To develop ouabain-immunized rats, ouabain-bovine serum albumin conjugates were injected subcutaneously three times at 4-week intervals. Titer determinations were made 2 weeks after the third immunization, and rats with high titers were used in the study. Immunoglobulin G fractions from ouabain-immunized rats effectively inhibited the contractile response of guinea pig aorta to exogenous ouabain (150 nmol). Fourteen ouabain-immunized and seven nonimmunized control rats underwent subtotal nephrectomy. An additional eight ouabain-immunized and six nonimmunized rats served as sham-operated rats. Four groups of rats drank 1% NaCl solution for 3 weeks, and systolic blood pressure was measured weekly by the tail-cuff method. Two groups of sham-operated rats remained normotensive. In contrast, two groups of subtotally nephrectomized rats developed hypertension. However, among these rats, systolic blood pressure was significantly lower in ouabain-immunized rats than in nonimmunized rats (161 +/- 5 versus 180 +/- 3 [+/- SEM) mm Hg, P < .01). The decrease in blood pressure was accompanied by a significant inhibition of aortic hypertrophy (P < .05). These results indicate that chronic blockade of circulating ouabainlike compound partly ameliorates reduced renal mass-saline hypertension and suggest that circulating ouabainlike compound may be involved in the pathophysiology in this model of hypertension.

Electron microscopic study of intercellular junctions in human gastric mucosa with special reference to their relationship to gastric ulcer.

The development of gap junctions in the human gastric mucosa has been examined to see if there is any relation to gastric ulcer. Freeze fracture replicas were prepared from the endoscopic biopsy specimens of 20 patients with gastric ulcer (15 men and five women, aged 49 (13) years) and seven healthy volunteers (four men and three women, aged 41 (19) years). Large fractured areas of lateral cell membranes of surface mucous cells were observed randomly at a direct magnification of 15,000 using electron microscopy. Small gap junctions were observed between gastric surface mucous cells in all healthy volunteers. Gap junctions in the patients with gastric ulcer were significantly fewer than in the healthy volunteers. In addition, gap junctions in patients with recurrent ulcer were significantly fewer than in those with first onset ulcer. There was no obvious relationship between age and the development of gap junctions in patients with gastric ulcer or in healthy volunteers. In areas of intestinal metaplasia, gap junctions were occasionally seen between absorptive cells of the villi, but not in the lateral membranes of goblet cells. These findings suggest that loss of intercellular communication via gap junctions is associated with gastric ulcer formation.

Digitalis-like factors from human urine.

We isolated two candidates for endogenous digitalis-like factors from human urine based on the inhibition of [3H]ouabain binding to intact human erythrocytes. The more-polar ouabain-displacing compound-1 (ODC-1) closely resembled ouabain in biological, physicochemical, and chromatographic properties. Moreover, anti-ouabain IgG dose-dependently neutralized the action of ODC-1. The less-polar ODC-2 behaved identically to digoxin in three analytical high-performance liquid chromatography and thin layer chromatography systems. Fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum supported the notion that ODC-2 may be indistinguishable from digoxin. The possibility that substances quite similar to cardenolides are synthesized in the mammalian body must be seriously considered.

A high frequency of detection of Helicobacter pylori in whitish exudate of gastric ulcer.

Helicobacter pylori has been implicated in the pathogenesis of chronic gastritis and peptic ulcer. However, no report to date has been made on the presence of H. pylori in the whitish exudate of peptic ulcers. We examined the biopsy specimens of 52 patients (42 men and 10 women, aged 29-78, not taking steroids or nonsteroid antiinflammatory drugs), and evaluated the frequency of detecting the organism in the whitish exudate of gastric ulcers in comparison with that in the ulcer border, gastric fundic gland, and pyloric gland using microaerobic culture and acridine-orange stain. H. pylori was detected in high rates: 38 (73%) of 52 cases by culture, and 50 cases (96%) by staining of the whitish exudate, 42 (81%) of 52 cases by culture, and 46 cases (88%) by staining of the ulcer border. The frequency for the other regions were 81% by culture and 90% by staining of the fundic gland, and 81 and 90% of the pyloric gland, respectively. Of all sites studied by the staining method, H. pylori was detected at the highest frequency in the whitish exudate. The fact that many H. pylori live in the whitish exudate of gastric ulcer, suggests a causal relationship between H. pylori and gastric ulcer.