Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

Factors predicting successful discontinuation of continuous renal replacement therapy.

This multicentre, retrospective observational study was conducted from January 2010 to December 2010 to determine the optimal time for discontinuing continuous renal replacement therapy (CRRT) by evaluating factors predictive of successful discontinuation in patients with acute kidney injury. Analysis was performed for patients after CRRT was discontinued because of renal function recovery. Patients were divided into two groups according to the success or failure of CRRT discontinuation. In multivariate logistic regression analysis, urine output at discontinuation, creatinine level and CRRT duration were found to be significant variables (area under the receiver operating characteristic curve for urine output, 0.814). In conclusion, we found that higher urine output, lower creatinine and shorter CRRT duration were significant factors to predict successful discontinuation of CRRT.

Height, weight, and alcohol consumption in relation to the risk of colorectal cancer in Japan: a prospective study.

Colorectal cancer incidence in relation to body size, smoking, and alcohol consumption was studied in a cohort of 29 051 city residents of Japan. In 1992, each participant completed a self-administered questionnaire on sociodemographic characteristics, drinking, cigarette smoking, diet, exercise, and reproductive and medical histories. The response rate was 92%. From 1993 to 2000, 161 men and 134 women were diagnosed with colorectal cancer at two major hospitals in the city. Relative risks and 95% confidence intervals were calculated by using Cox proportional hazard models. A positive relation between height and colorectal cancer was seen in both sexes, controlling for age, body mass index (BMI), smoking and drinking habits, and years of education. The findings were statistically significant only for men (relative risk 2.13 for the tallest compared with the shortest height tertile; 95% confidence interval=1.26-3.58). Body mass index was also associated positively with colon cancer risk for men, whereas the pattern for women was not clear. There was a positive association between pack-years of cigarette smoking and the risk of rectal cancer in men. A positive dose-response relation between alcohol consumption and colon cancer risk was observed for men and women.

Overexpression of deletion-mutant epidermal growth factor receptor is associated with altered genotoxic stress-provoked p53 mRNA induction in a human glioblastoma cell line.

A distinct 801-bp deletion mutation of the epidermal growth factor receptor (EGFR) gene is frequently present in primary glioblastoma multiforme (GBM), confers enhanced tumorigenicity in vivo and is prognostic of a shorter interval to clinical relapse. This study sought to investigate whether overexpression of deletion-mutant (delta) EGFR affects genotoxic stress-provoked mRNA inductions of p53 and murine double minute 2 (MDM2), two other genes strongly involved in the pathogenesis of GBM. In a set of human wild-type (wt) p53 GBM cell lines (U-87MG and U-87MG.delta EGFR) that exclusively differ in EGFR expression (endogenous wt EGFR expression and exogenous delta EGFR overexpression, respectively), ultraviolet (UV) light irradiation-mediated EGFR, p53 and MDM2 genotoxic stress-provoked mRNA inductions were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and densitometry of electrophoretically separated and stained RT-PCR products. Although baseline (at 0 J/m2) p53 mRNA expression in U-87MG.delta EGFR was 42-fold reduced, maximum p53 induction (at 8 J/m2) amounted to 130% compared to U-87MG. Thus, ultimate UV light-mediated p53 mRNA induction was 131.5-fold in U-87MG.delta EGFR and 2.8-fold in U-87MG. In contrast, neither wt/delta EGFR nor MDM2 mRNA expressions were significantly inducible, and MDM2 mRNA profiles were essentially the same among U-87MG and U-87MG.delta EGFR. These data suggest that in human GBM overexpression of delta EGFR is associated with differential genotoxic stress-provoked p53 mRNA induction whereas MDM2 mRNA expression is apparently not directly affected by EGFR status.

Diet and colorectal adenoma in Japanese males and females.

PURPOSE: The purpose of this study was to assess the relationship between risk of colorectal adenoma and dietary intake of nutrients and foods. METHODS: In 1992, diet was assessed by a semiquantitative food-frequency questionnaire in a cohort of the Takayama Study in Japan. Patients were 181 male and 98 female cohort members who were newly histologically proved to have colorectal adenoma at colonoscopic examination between January 1, 1993, and December 31, 1995. Controls were 12,607 males and 15,754 females who had no history of colorectal polyp, adenoma, and cancer at baseline (1992) and were not diagnosed to have these diseases during the follow-up period. RESULTS: In males, the risk of adenoma was significantly associated with intake of animal protein and vitamin A (relative risk, 1.42; 95 percent confidence interval, 1.00-2.04; and relative risk, 1.51; 95 percent confidence interval, 1.04-2.20, for the highest vs. lowest tertiles, respectively; P for trend = 0.048 and 0.03, respectively) after controlling for age, years of smoking, and alcohol intake. A significantly inverse association was observed for carbohydrate intake after controlling for the covariates (relative risk, 0.52; 95 percent confidence interval, 0.32-0.82, for the highest vs. lowest tertiles; P for trend = 0.02). Intakes of animal fat and cholesterol were marginally associated with risk of adenoma. CONCLUSION: Some dietary components such as animal protein and carbohydrate, which have been associated with risk of colorectal adenoma or cancer in western populations, were also associated with risk of colorectal adenoma in the Japanese population.

Gene expression of PSD95 in prefrontal cortex and hippocampus in schizophrenia.

A number of studies have suggested that disturbance in glutamatergic transmission in the cerebral cortex may underlie, or contribute to the pathophysiology of schizophrenia. In this study we examined expression of the postsynaptic density protein 95 (PSD95) mRNA in the prefrontal cortex and hippocampus in postmortem material from neuroleptic-treated schizophrenics and normal controls. PSD95 is known to bind to NMDA receptor subunits and is known to be involved in synaptic plasticity. In situ hybridization analysis showed that the expression of PSD95 was significantly decreased in Brodmann area 9 of the prefrontal cortex but not in the hippocampus. These results further implicate the prefrontal cortex in the pathophysiology of schizophrenia and suggest dysfunction of NMDA receptors in the schizophrenic cortex.

No genetic association between alpha-2 macroglobulin I1000V polymorphism and Japanese sporadic Alzheimer's disease.

Alpha-2 macroglobulin (A2M) is a serum pan-protease inhibitor that is related with the pathogenesis of Alzheimer's disease (AD) through its ability to mediate amyloid beta degradation. Recently, it has been reported that the I1000V polymorphism in A2M gene might increase the risk of AD. In the present study, we investigated this mutation in 95 healthy controls and in 111 sporadic AD cases by polymerase chain reaction-restriction fragment length polymorphism method in order to study this hypothesis in the Japanese AD population. Allelic frequencies with the I1000V polymorphism in the gene were 7.4 and 6.8% in the control and AD groups, respectively. Our results failed to demonstrate an association between this polymorphism and Japanese sporadic AD, and the A2M I1000V mutation does not seem to be a risk factor per se for sporadic AD.

Lack of an association of estrogen receptor alpha gene polymorphisms and transcriptional activity with Alzheimer disease.

BACKGROUND: Long-term cognitive decline in postmenopausal women is associated with aging and Alzheimer disease (AD). Estrogen replacement therapy has been reported to reduce the risk of developing AD. The distribution of estrogen receptors (ERs) in neurons overlaps that of the brain neurons known to develop AD. Estrogen increases the secretion and metabolism of amyloid precursor protein, may help synapse formation, and is reported to protect neurons from toxins. Restriction fragment length polymorphisms (RFLPs) of the ERalpha gene at intron 1 and exon 2 were associated with a low bone mineral density in postmenopausal women and also with AD in a Japanese population. OBJECTIVE: To determine whether ERalpha gene polymorphisms are associated with transcriptional activity and AD. METHODS: A luciferase reporter assay analyzed enhancer activity of the ERalpha gene at intron 1 and exon 2. This activity was evaluated according to the RFLPs. The RFLPs of the ERalpha gene were determined in Japanese patients clinically diagnosed as having AD, white patients diagnosed as having AD at autopsy, and corresponding healthy control subjects. The RFLPs were also evaluated for the contribution of the ERalpha gene RFLPs to AD. RESULTS: We found weak (about 2-fold) enhancer activity of the ERalpha gene, which differed among RFLPs. Although there were racial differences in these polymorphisms, we could not confirm the previously reported association between ERalpha gene polymorphisms and AD. CONCLUSION: Regulatory element of the ERalpha gene was found in intron 1, but we found no association between ERalpha gene polymorphisms and AD.

Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA.

OBJECT: The goal of this study was to evaluate the activity of certain hairpin ribozymes against deletion-mutant epidermal growth factor receptor (deltaEGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 801-bp deletion mutation associated with amplification of the EGFR gene is present in a large subgroup of primary GBMs and confers enhanced tumorigenicity in vivo. As a result of the deletion mutation, the fusion junction of the gene is created directly upstream of a GTA triplet, which is subsequently transcribed into a ribozyme target codon (GUA). METHODS: In attempts to intercept deltaEGFR gene expression at the mRNA level, the authors designed three different hairpin ribozymes derived from the negative strands of satellite RNAs in tobacco ringspot virus, chicory yellow mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV1, identified as the most active anti-deltaEGFR hairpin ribozyme motif, was cloned into the retroviral plasmid N2A+tRNAi(met). High-titer recombinant retrovirus-containing supernatants (> 10(5) colony-forming units/ml) derived from an amphotropic GP+envAM 12 packaging cell line transfected with the N2A+tRNAi(met)-anti-deltaEGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.deltaEGFR glioblastoma cells, which overexpress exogenous deltaEGFR. Using a virus/target cell ratio of 40:1 in the absence of drug selection, the ribozyme transfer resulted in a greater than 90% reduction of deltaEGFR mRNA levels, a 69% inhibition of deltaEGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions in vitro; transfer of a control mutant ribozyme that was rendered incapable of cleaving its target yielded none of these effects. CONCLUSIONS: These findings indicate that the anti-deltaEGFR-sCYMV1 hairpin ribozyme is capable of specifically inhibiting the expression of deltaEGFR and reversing the deltaEGFR-associated malignant phenotype of GBM cells. This strategy may constitute a promising gene therapy approach for a molecularly defined subgroup of GBMs.

Gene expression of metabotropic glutamate receptor 5 and excitatory amino acid transporter 2 in the schizophrenic hippocampus.

A disturbance in glutamatergic transmission has been suggested to contribute to the pathophysiology of schizophrenia and recent studies on ionotropic glutamate receptors are consistent with altered glutamatergic function in the hippocampus of schizophrenics. In order to investigate this hypothesis further, the expression of two 'glutamatergic' markers, the mRNAs of metabotropic glutamate receptor 5 (mGluR5) and human excitatory amino acid transporter (EAAT2) were compared in the hippocampus of control subjects and schizophrenics. We examined the regional/cellular mRNA expression of mGluR5 and EAAT2 in postmortem hippocampal sections from schizophrenics and control subjects, using in situ hybridization. Regions of interests were dentate gyrus, cornu ammonis 4, 3, 1 and parahippocampal gyrus. The regional/cellular mGluR5 mRNA content was not different between the two groups. The cellular EAAT2 mRNA content was significantly decreased in schizophrenic parahippocampal gyrus, but not in other hippocampal regions. Furthermore, only in the parahippocampal gyrus, schizophrenics had a significantly increased mGluR5/EAAT2 ratio at both the regional and cellular mRNA level. The results suggest that a disturbance of glutamatergic neurotransmission in schizophrenia was not apparent using these indices in the hippocampus, but 'hypo-glutamatergic' neurotransmission may be present in the schizophrenic parahippocampal gyrus.

[Electromyographic study of the striated urethral sphincter by using the bulbocavernosus reflex: study on change of sacral reflex activity caused by bladder filling].

PURPOSE: The change of sacral reflex activity of the striated urethral sphincter in the urine storage phase is investigated using evoked potential reaction of the bulbocavernosus reflex (BCR). METHODS: Eleven normal male subjects and 13 male patients with neurogenic bladder due to suprasacral (C3-C7) spinal cord injury (SCI patients) were investigated. Within the SCI patients, five were complete SCI and 8 were incomplete SCI. BCR was elicited by electrical stimulation of dorsal nerve of the penis, and the evoked potential of the BCR was recorded with a concentric needle electrode from the periurethral striated muscle. BCR was performed both at empty and at filled bladder respectively, and changes of the amplitudes (AMP) were examined. Moreover, the changes of AMP affected by bladder filling were compared between the normal subjects and the SCI patients. RESULTS: In both the normal subjects and the SCI patients, AMP increased at the filled bladder as compared with that of the empty bladder. In addition, the change of AMP was statistically bigger in the SCI patients (a ratio of amplitude at filled bladder/amplitude at empty bladder: 4.73 +/- 3.90) than in the normal subjects (the ratio: 1.32 +/- 0.44). CONCLUSION: Sacral reflex activity was accelerated by bladder filling in both the normal subjects and SCI patients. And the acceleration in the SCI patients was more remarkable than that in the normal subjects. In addition to the conventional evaluation of the integrity of sacral reflex arc by BCR examination, the observation of changes of BCR affected by bladder filling may provide the information for the continuity of sacral segment and supraspinal micturition center.

Anti-MDR1 Ribozyme Gene Therapy.

Multidrug resistance (MDR) in human cancer seriously limits the efficacy of anticancer agents. Circumvention of MDR is, thus, one of the urgent goals for successful cancer chemotherapy.

Genetic association between cytochrome P-450 2D6 gene polymorphism and plasma concentration of haloperidol in Japanese schizophrenics.

Haloperidol is one of the major neuroleptics partly metabolized by cytochrome P-450 2D6 (CYP2D6), which has over 10 genetic polymorphisms. The purpose of this study was to investigate whether individual haloperidol plasma concentration was affected by CYP 2D6 gene polymorphisms. The genomic DNA of 56 subjects who have been taking haloperidol were analysed by restriction fragment length polymorphism (RFLP) method to detect the major Caucasian mutations, CYP 2D6A, 2D6B, and the major oriental population mutation, 2D6J. We found 10 cases of the CYP 2D6J mutation, only one case of the CYP 2D6B mutation, and no cases of the CYP 2D6A mutation in the present subjects. Contrary to expectations, haloperidol plasma concentration of the CYP 2D6B mutation case showed a lower haloperidol plasma concentration than the mean value of that of wild type homozygous cases. Haloperidol plasma concentration of 10 subjects who had the CYP 2D6J mutation cases showed no significant difference from that of the wild type homozygous group. These results suggest that the major genetic polymorphisms of CYP 2D6 do not seem to be one of the factors which affect individual haloperidol plasma concentration in Japanese schizophrenics.

Inhibitory effect of telomere-mimic phosphorothioate oligodeoxy nucleotides (S-ODNS) on human tumor cell lines.

To clarify the inhibitory effect of telomere-mimic oligonucleotides on human cancer cell lines, we synthesized 18-mers (18T; n = 3), 24-mers (24T; n = 4) and 30-mers (30T; n = 5) of telomere-mimic phosphorothioate oligodeoxy nucleotides [5'-d(TTA GGG)n-3'] and examined their effects on the proliferation of human tumor cells by XTT assay. After 7 days of continuous exposure to 24T and 30T at concentrations ranging from 0.1 to 10 microM, concentration-dependent cell growth inhibition was observed in MCF-7 clone E3, ZR-75-1, MDA-MB 231, Colo 201 and WiDr. All of these cell lines highly expressed telomerase using the telomeric repeat amplification protocol. None of these tumor cell lines were affected by 18T. In MCF-7, ZR-75-1 and Colo 201 cell lines, a more than 50% growth inhibition was obtained by 3 microM of 24T and 30T whereas, in MDA-MB 231 and WiDr cell lines, cell growth inhibition was less than 50%. 30T was more effective than 24T. Estrogen-dependent growth of both MCF-7 and ZR-75-1 was inhibited by 3 microM of 24T and 30T, however, in the absence of estrogen, no growth inhibition was seen. The MCF-10A cell line, which was developed from normal human breast tissue and expressed telomerase only weakly, was inhibited by 10 microM of 18T. In conclusion, these observations indicate that S-ODNs inhibit tumor growth in cell lines expressing telomerase in a concentration-dependent manner and that cell growth inhibition is dependent on the length of S-ODNs. In addition, the short-length S-ODNs may inhibit growth of cells weakly expressing telomerase, but not of cells with high telomerase expression.

Genetic association between alpha-2 macroglobulin and Japanese sporadic Alzheimer's disease.

Alpha-2 macroglobulin (encoded by the gene A2M) is a serum pan-protease inhibitor that may be related with the pathogenesis of Alzheimer's disease (AD) because of its ability to mediate amyloid beta degradation. Recently, several groups have reported that the five-nucleotides deletion in A2M gene at the 5' splice site of exon 18 might increase risk for AD. In the present study, therefore, this mutation was studied in 69 healthy controls and 55 sporadic AD cases by polymerase chain reaction- restriction fragment length polymorphism method. The allelic frequencies with the deletion (A2M-2) are 9.4 and 6.4% in the control and AD groups, respectively. There is no significant difference in the A2M-2 frequencies between the controls and sporadic AD cases. This is the first report to study the frequencies of A2M-2 in Japanese AD cases, suggesting its no genetic association with sporadic AD.

Measurement of GABAergic parameters in the prefrontal cortex in schizophrenia: focus on GABA content, GABA(A) receptor alpha-1 subunit messenger RNA and human GABA transporter-1 (HGAT-1) messenger RNA expression.

The hypothesis that the pathophysiology of schizophrenia may be associated with a dysfunction in GABA transmission in the human prefrontal cortex was investigated. Human post mortem brain tissue from 10 control cases and six cases of schizophrenia were processed for amino acid analysis and for radioactive in situ hybridization. Laminae III and V of three prefrontal cortical areas were examined in detail, namely Brodmann areas 9, 10 and 11. Of these three areas significant changes in GABAergic markers were found only in areas 9 and 10. Of note, a significant decrease in the tissue content of GABA was observed and this was accompanied by a marked increase in the cellular expression of the GABA(A) receptor alpha-1 subunit messenger RNA and a marked decrease in the expression of human GABA transporter-1, the messenger RNA encoding the neuronal GABA transporter protein. The amino acid analysis data provided in this study coupled with the detailed cellular study of several GABAergic markers in the human prefrontal cortex provide direct evidence in support of a disturbance in GABA transmission in the prefrontal cortex, which may be loosely termed "hypofrontality".

[Screening of prostate cancer with PSA and transperineal six sextant biopsy].

OBJECT: The objectives of this study are to examine how many cancer patients we can detect among the outpatients whose PSA values are above 4.0 ng/ml, and to compare the usefulness of transperineal six sextant biopsy (ss-biopsy) with that of transrectal one. METHODS: All the male outpatients (above 50 years old) were inspected Tandem-R PSA levels and digital rectal examination (DRE). Among them, 129 patients showed more than 4.0 ng/ml of PSA values and/or positive finding of DRE, and underwent subsequent transperineal ss-biopsy. RESULTS: Cancers were detected in 52 patients (40.3%) without major complications. Among 64 gray zone (PSA 4.1-10.0 ng/ml) patients, 17 (26.6%) were found to be cancer by ss-biopsy, meanwhile only 2 cancer patients (8.9%) were detected from 23 gray zone ones by traditional directed biopsy. Application of PSA density could not be found practicable to eliminate unnecessary biopsies in the gray zone group. CONCLUSION: Prostate cancer could be found nearly a fourth in the gray zone group of the outpatients. To enhance the detection rate, obtaining at least 6 core samples are recommended from either perineal or rectal root.

Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.