Study on prediction of early adverse events by CapeOX therapy in patients with colorectal cancer

The Journal of Medical Investigation : JMI


Y chromosome haplogroups are associated with birth size in Japanese men


An update and frequency distribution of Y chromosome haplogroups in modern Japanese males


Analysis of the correlation between gene copy deletion in the AZFc region and male infertility in Japanese men

Deletion of the azoospermia factor c (AZFc), located on the long arm of the Y chromosome, is a cause of male infertility. The structure of the Y chromosome is diversified by the copy number of various genes, such as deleted in azoospermia (DAZ), basic protein Y2, chromodomain Y1, testis-specific transcript Y-linked 4, and Golgi autoantigen golgin subfamily a2 like Y, located in the AZF region. In this study, we investigated the deletion of each gene copy and analyzed its relationship with Japanese male infertility. Deletions of single nucleotide variants of each gene copy in 721 proven fertile men as controls, 139 patients with non-obstructive azoospermia (NOA), and 56 patients with oligozoospermia (OS) were analyzed via polymerase chain reaction-restriction fragment length polymorphism analysis. Their association with infertility was analyzed using logistic regression analysis adjusted for the Y-chromosome haplogroup, D1a2a. Deletions of DAZ/II in the r1 region and DAZ/V in the r1 and r2 regions showed significant associations with NOA (odds ratio [OR] = 4.15, 95 % confidence interval [CI] = 1.18-14.6, P = 0.026; OR = 4.19, 95 % CI = 1.19-14.7, P = 0.025, respectively). They did not show any association with OS. Partial deletion of the AZFc region affects spermatogenesis in Japanese male.


Lrriq1 is an essential factor for fertility by suppressing apoptosis.

Leucine-rich repeats and IQ motif containing 1 (LRRIQ1) gene is reportedly associated with plasma inhibin B levels. However, the function of LRRIQ1 remains unknown. In this study, we generated Lrriq1 knockout mice (Lrriq1 mice) and examined the effects of LRRIQ1 on inhibin B and fertility. Lrriq1 mice were generated using CRISPR/Cas9 genome editing technology. The expression of Inhibin B was examined by Western blotting using a protein extracted from the testis of a 3-month-old male mouse. Mating experiments were conducted using 7-week-old Lrriq1 mice and wild-type (WT) mice to examine fertility. Sperm concentration and sperm motility were measured using 3-month-old male mice. Expression analysis of inhibin B revealed that Lrriq1 mice exhibited reduced mRNA and protein levels of inhibin alpha (Inha), which constitutes the α subunit. In the mating experiment, the litter size of Lrriq1 male mice was 4.3 ± 2.9, which was significantly lower than that of WT male mice (8.3 ± 1.3) (p < 0.001). No difference in sperm count was observed between Lrriq1 and WT male mice; however, sperm motility (%) was significantly reduced in Lrriq1 mice (48.4 ± 4.9) when compared with WT mice (70.2 ± 4.7) (p < 0.001). Based on TUNEL staining, the testes and epididymal sperm of Lrriq1 mice showed high numbers of apoptosis-positive cells. Lrriq1 knockout reduced sperm motility and litter size by inducing apoptosis of testicular germ cells and epididymal sperm.

Journal of Assisted Reproduction and Genetics


A machine learning model using SNPs obtained from a genome-wide association study predicts the onset of vincristine-induced peripheral neuropathy

Vincristine treatment may cause peripheral neuropathy. In this study, we identified the genes associated with the development of peripheral neuropathy due to vincristine therapy using a genome-wide association study (GWAS) and constructed a predictive model for the development of peripheral neuropathy using genetic information-based machine learning. The study included 72 patients admitted to the Department of Hematology, Tokushima University Hospital, who received vincristine. Of these, 56 were genotyped using the Illumina Asian Screening Array-24 Kit, and a GWAS for the onset of peripheral neuropathy caused by vincristine was conducted. Using Sanger sequencing for 16 validation samples, the top three single nucleotide polymorphisms (SNPs) associated with the onset of peripheral neuropathy were determined. Machine learning was performed using the statistical software R package "caret". The 56 GWAS and 16 validation samples were used as the training and test sets, respectively. Predictive models were constructed using random forest, support vector machine, naive Bayes, and neural network algorithms. According to the GWAS, rs2110179, rs7126100, and rs2076549 were associated with the development of peripheral neuropathy on vincristine administration. Machine learning was performed using these three SNPs to construct a prediction model. A high accuracy of 93.8% was obtained with the support vector machine and neural network using rs2110179 and rs2076549. Thus, peripheral neuropathy development due to vincristine therapy can be effectively predicted by a machine learning prediction model using SNPs associated with it.


A genome-wide association study predicts the onset of dysgeusia due to anti-cancer drug treatment

Dysgeusia is a major side effect of anti-cancer drug treatment. Since dysgeusia significantly lowers the patient's QOL, predicting and avoiding its onset in advance is desirable. Accordingly, aims of the present study were to use a genome-wide association study (GWAS) to identify genes associated with the development of dysgeusia in patients taking anti-cancer drugs and to predict the development of dysgeusia using associated single nucleotide polymorphisms (SNPs). GWAS was conducted on 76 patients admitted to the Department of Hematology, Tokushima University Hospital. Using Sanger sequencing for 23 separately collected validation samples, the top two SNPs associated with the development of dysgeusia were determined. GWAS identified rs73049478 and rs41396146 SNPs on the retinoic acid receptor beta (RARB) gene associated with dysgeusia development due to the administration of anti-cancer drugs. Evaluation of the two SNPs using 23 validation samples indicated that the accuracy rate of rs73049478 was relatively high (87.0%). Thus, the findings of the present study suggest that the rs73049478 SNP of RARB can be used to predict the onset of dysgeusia caused by the administration of anti-cancer drugs.


Association analysis between adverse drug reactions to cytarabine therapy and single nucleotide polymorphisms in cytarabine metabolic genes in patients with hematopoietic tumor


Genome-wide association study of semen volume, sperm concentration, testis size, and plasma inhibin B levels

Semen quality is affected by environmental factors, endocrine function abnormalities, and genetic factors. A GWAS recently identified ERBB4 at 2q34 as a genetic locus associated with sperm motility. However, GWASs for human semen volume and sperm concentration have not been conducted. In addition, testis size also reportedly correlates with semen quality, and it is important to identify genes that affect testis size. Reproductive hormones also play an important role in spermatogenesis. To date, genetic loci associated with plasma testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels have been identified using GWASs. However, GWASs have not identified any relevant loci for plasma inhibin B levels. We conducted a two-stage GWAS using 811 Japanese men in a discovery stage followed by a replication stage using an additional 721 Japanese men. The results of the discovery and replication stages were combined into a meta-analysis. After setting a suggestive significance threshold for P values < 5 × 10 in the discovery stage, we identified ten regions with SNPs (semen volume: one, sperm concentration: three, testes size: two, and inhibin B: four). We selected only the most significant SNP in each region for replication genotyping. Combined discovery and replication results in the meta-analysis showed that the locus 12q21.31 associated with plasma inhibin B levels (rs11116724) had the most significant association (P = 5.7 × 10). The LRRIQ1 and TSPAN19 genes are located in the 12q21.31 region. This study provides new susceptibility variants that contribute to plasma inhibin B levels.


Novel formula using triceps skinfold thickness to revise the Cockcroft-Gault equation for estimating renal function in Japanese bedridden elderly patients

In recumbent elderly patients, creatinine clearance (eCCr) estimated by the Cockcroft-Gault (CG) equation may not necessarily reflect renal function. We aimed to develop a novel formula to revise the CG equation using anthropometric measurements in bedridden elderly patients and evaluate its clinical utility. The subjects included 77 bedridden Japanese patients aged ≦ 65, hospitalized at Naruto Yamakami Hospital. The actual CCr (mCCr) value was measured using the 24-hour urine collection method. Anthropometric data, such as skeletal muscle mass, body fat mass (BFM), and triceps skinfold thickness (TSF), were collected. We established a novel formula to estimate CCr or CCr by correcting the eCCr value with BFM or TSF. The stage of classification of renal dysfunctions in patients with eGFR or eGFR was equivalent to the GFR based on the mCCr. Notably, the novel equation for eCCr based on TSF (eCCr), dubbed the "Naruto" formula, can be useful to evaluate renal function in bedridden elderly patients without expensive equipment or additional costs. In this study, mCCr was considered to be the true renal function of the patient, but whether and to what extent mCCr correlates with inulin clearance is unknown. J. Med. Invest. 65:195-202, August, 2018.

The Journal of Medical Investigation : JMI


What is the correct adjustment protocol for serum creatinine value to reflect renal function in bedridden elderly patients?

The Japanese Journal of Nephrology and Pharmacotherapy


Genome-wide association study identifies ERBB4 on 2q34 as a novel locus associated with sperm motility in Japanese men.

The decrease in sperm motility has a potent influence on fertilisation. Sperm motility, represented as the percentage of motile sperm in ejaculated sperms, is influenced by lifestyle habits or environmental factors and by inherited factors. However, genetic factors contributing to individual differences in sperm motility remain unclear. To identify genetic factors that influence human sperm motility, we performed a genome-wide association study (GWAS) of sperm motility. A two-stage GWAS was conducted using 811 Japanese men in a discovery stage, followed by a replication study using an additional 779 Japanese men. In the two-staged GWAS, a single nucleotide polymorphism rs3791686 in the intron of gene for erb-b2 receptor tyrosine kinase 4 () on chromosome 2q34 was identified as a novel locus for sperm motility, as evident from the discovery and replication results using meta-analysis (β=-4.01, combined P=5.40×10). Together with the previous evidence that Sertoli cell-specific -knockout mice display an impaired ability to produce motile sperm, this finding provides the first genetic evidence for further investigation of the genome-wide significant association at the locus in larger studies across diverse human populations.


Association of TUSC1 and DPF3 gene polymorphisms with male infertility

Recently, genome-wide association studies of a Hutterite population in the USA revealed that five single nucleotide polymorphisms (SNPs) with a significant association with sperm quality and/or function in ethnically diverse men from Chicago were significantly correlated with family size. Of these, three SNPs (rs7867029, rs7174015, and rs12870438) were found to be significantly associated with the risk of azoospermia and/or oligozoospermia in a Japanese population. In this study, we investigated whether the rs10966811 (located in an intergenic region between the TUSC1 and IZUMO3 genes) and rs10129954 (located in the DPF3 gene) SNPs, previously related to family size, are associated with male infertility. In addition, we performed association analysis between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility. We genotyped 145 patients with infertility (including 83 patients with azoospermia and 62 with oligozoospermia) and 713 fertile controls by PCR-RFLP technique for polymorphism. Because rs10966811 has no restriction sites, the SNP rs12376894 with strong linkage disequilibrium was selected as an alternative to rs10966811. There was a statistically significant association between rs12376894 proxy SNP of rs10966811 and oligozoospermia. Also, a statistically significant association between rs10129954 and azoospermia, and oligozoospermia was observed. When we assessed the relationship between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility traits, we found that rs12348 in TUSC1 was significantly associated with azoospermia and oligozoospermia, but rs2772579 in IZUMO3 was not associated with male infertility. We found that the polymorphisms in TUSC1 and DPF3 displayed strong associations with male infertility.

Journal of Assisted Reproduction and Genetics


An independent validation study of three single nucleotide polymorphisms at the sex hormone-binding globulin locus for testosterone levels identified by genome-wide association studies


Protein kinase C-δ signaling regulates glucagon secretion from pancreatic islets

Accumulating evidence supports the "glucagonocentric hypothesis", in which antecedent -cell failure and inhibition of glucagon secretion are responsible for diabetes progression. Protein kinase C (PKC) is involved in glucagon secretion from -cells, although which PKC isozyme is involved and the mechanism underlying this PKC-regulated glucagon secretion remains unknown. Here, the involvement of PKC in the onset and progression of diabetes was elucidated. Immunofluorescence studies revealed that PKC was expressed and activated in -cells of STZ-induced diabetic model mice. Phorbol 12-myristate 13-acetate (PMA) stimulation significantly augmented glucagon secretion from isolated islets. Pre-treatment with quercetin and rottlerin, PKC signaling inhibitors, significantly suppressed the PMA-induced elevation of glucagon secretion. While Go6976, a Ca(2+)-dependent PKC selective inhibitor did not suppress glucagon secretion. Quercetin suppressed PMA-induced phosphorylation of Tyr(311) of PKC in isolated islets. However, quercetin itself had no effect on either glucagon secretion or glucagon mRNA expression. Our data suggest that PKC signaling inhibitors suppressed glucagon secretion. Elucidation of detailed signaling pathways causing PKC activation in the onset and progression of diabetes followed by the augmentation of glucagon secretion could lead to the identification of novel therapeutic target molecules and the development of novel therapeutic drugs for diabetes. J. Med. Invest. 64: 122-128, February, 2017.


スーパーサイエンスハイスクール(SSH)コンソーシアムによる日本人のY染色体とミトコンドリアDNAハプログループ地域分布調査

The Super Science High School Consortium

Anthropological Science (Japanese Series)


A replication study of a candidate locus for follicle-stimulating hormone levels and association analysis for semen quality traits in Japanese men

In men, follicle-stimulating hormone (FSH) acts on the seminiferous tubules and enhances spermatogenesis. Recently, a candidate locus (rs2414095) for FSH levels was identified by a genome-wide association study (GWAS) in Chinese men. The rs2414095 single-nucleotide polymorphism (SNP) is found on the third intron of the cytochrome P450, family 19, subfamily A, peptide 1 (CYP19A1) gene encoding an aromatase. In the present study, we performed a replication study in 1687 Japanese men (901 from cohort 1 and 786 from cohort 2) to assess whether this SNP is associated with circulating FSH levels. Furthermore, we investigated whether the rs2414095 SNP is correlated with semen quality traits in 2015 Japanese men (1224 from cohort 1 and 791 from cohort 2). The rs2414095 SNP was significantly associated with circulating FSH levels (STD=0.15, P=9.7 × 10(-5)), sperm concentration (STD=0.073, P=0.032) and total sperm number (TSN) (STD=0.074, P=0.027) in a combined analysis of the two Japanese male cohorts. We successfully replicated, in Japanese men, the results of the previous GWAS for the rs2414095 SNP in Chinese men, and found that the rs2414095 SNP was related with sperm production.


Labelling of endogenous target protein via N-S acyl transfer-mediated activation of N-sulfanylethylanilide


Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.


Lack of replication of four candidate SNPs implicated in human male fertility traits: a large-scale population-based study

Are the four candidate loci (rs7867029, rs12870438, rs7174015 and rs724078) for human male fertility traits, identified in a genome-wide association study (GWAS) of a Hutterite population in the USA, associated with semen quality traits in a Japanese population? The four single nucleotide polymorphisms (SNPs) rs7867029, rs12870438, rs7174015 and rs724078 have no association with semen parameters in a meta-analysis of two Japanese male cohorts. Four (rs7867029, rs12870438, rs7174015 and rs724078) of the SNPs associated with family size or birth rate in the GWAS of a Hutterite population in the USA were associated with semen parameters in ethnically diverse men from Chicago, USA. This is a replication study in a total of 2015 Japanese subjects, including 791 fertile men and 1224 young men from the general population. We performed a replication study in two cohorts to assess whether the SNPs rs7867029, rs12870438, rs7174015 and rs724078 are associated with sperm concentration, semen volume, total sperm numbers, total motile sperm numbers or sperm motility. The rs12870438 SNP was detected by restriction fragment length polymorphism PCR while rs7174015, rs724078 and rs7867029 SNPs were genotyped using TaqMan probes. This study indicated that none of the four SNPs rs7867029, rs12870438, rs7174015 and rs724078 displayed a significant association with semen parameters in the meta-analysis of two Japanese male cohorts. Only four SNPs identified in the Hutterite GWAS were examined for associations with semen quality traits in a Japanese population. In addition, the linkage disequilibrium structures around the testing markers were different between ethnic groups. Locus mapping studies using a set of tagging SNPs across the loci will be necessary in populations with larger sample sizes in order to understand the contribution of specific genes to semen quality. This study was supported in part by the Ministry of Health and Welfare of Japan (1013201) (to T.I.), Grant-in-Aids for Scientific Research (C) (23510242) (to A.Ta.) from the Japan Society for the Promotion of Science, the European Union (BMH4-CT96-0314) (to T.I.), and the Takeda Science Foundation (to A.Ta.). None of the authors has any competing interests to declare.


An association study of four candidate loci for human male fertility traits with male infertility

Are the four candidate loci (rs7867029, rs7174015, rs12870438 and rs724078) for human male fertility traits, identified in a genome-wide association study (GWAS) of a Hutterite population in the USA, associated with male infertility in a Japanese population? rs7867029, rs7174015 and rs12870438 are significantly associated with the risk of male infertility in a Japanese population. Recently, a GWAS of a Hutterite population in the USA revealed that 41 single-nucleotide polymorphisms (SNPs) were significantly correlated with family size or birth rate. Of these, four SNPs (rs7867029, rs7174015, rs12870438 and rs724078) were found to be associated with semen parameters in ethnically diverse men from Chicago. This is a case-control association study in a total of 917 Japanese subjects, including 791 fertile men, 76 patients with azoospermia and 50 patients with oligozoospermia. Azoospermia was diagnosed on the basis of semen analysis (the absence of sperm in ejaculate), serum hormone levels and physical examinations. Oligozoospermia was defined as a sperm concentration of <20 × 10(6)/ml. We excluded patients with any known cause of infertility (i.e. obstructive azoospermia, varicocele, cryptorchidism, hypogonadotropic hypogonadism, karyotype abnormalities or complete deletion of AZF a, b or c). The SNPs rs7867029, rs7174015, rs12870438 and rs724078 were genotyped using DNA from peripheral blood samples and either restriction fragment length polymorphism PCR or TaqMan probes. Genetic associations between the four SNPs and male infertility were assessed using a logistic regression analysis under three different comparative models (additive, recessive or dominant). The genotypes of all four SNPs were in Hardy-Weinberg equilibrium in the fertile controls. The SNPs rs7867029 and rs7174015 are associated with oligozoospermia [rs7867029: odds ratio (OR) = 1.70, 95% confidence interval (CI) = 1.07-2.68, P = 0.024 (log-additive); rs7174015: OR = 6.52, 95% CI = 1.57-27.10, P = 0.0099 (dominant)] and rs12870438 is associated with azoospermia (OR = 10.90, 95% CI = 2.67-44.60, P = 0.00087 (recessive)] and oligozoospermia [OR = 8.54, 95% CI = 1.52-47.90, P = 0.015 (recessive)]. The association between rs7174015 and oligozoospermia under a dominant model and between rs12870438 and azoospermia under additive and recessive models remained after correction for multiple testing. There were no associations between rs724078 and azoospermia or oligozoospermia. Even though the sample size of case subjects was not very large, we found that three SNPs were associated with the risk of male infertility in a Japanese population. The three infertility-associated SNPs may be contributing to a quantitative reduction in spermatogenesis. This study was supported in part by the Ministry of Health and Welfare of Japan (1013201) (to T.I.), Grant-in-Aids for Scientific Research (C) (23510242) (to A.Ta.) from the Japan Society for the Promotion of Science, the European Union (BMH4-CT96-0314) (to T. I.) and the Takeda Science Foundation (to A.Ta.). None of the authors has any competing interests to declare.


Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (β = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.


Overview of genetic variation in the Y chromosome of modern Japanese males


The role of dipeptidyl peptidase 4 (DPP4) in the preservation of renal function: DPP4 involvement in hemoglobin expression

In a previous study, we demonstrated that dipeptidyl peptidase 4 (DPP4)-deficient rats were susceptible to reduced glomerular filtration rate as a result of streptozotocin (STZ)-induced diabetes. Therefore, we proposed that DPP4 might be responsible for the preservation of renal function. In this study, to verify the role of DPP4 in the preservation of renal function, we performed a microarray analysis of the kidneys of WT and DPP4-deficient rats after STZ treatment, and gene expression analysis using rat kidneys, human embryonic kidney 293 (HEK293) cells, and human renal cancer cells (CakI-1). The microarray analysis indicated that the expression levels of the transporter activity, heme-binding, and pheromone binding-related genes changed significantly. The results of gene expression analysis indicated that there were no significant differences in the expression levels of hemoglobin mRNA between the DPP4-deficient and WT rats; however, the expression levels of hemoglobin mRNA in the kidneys of DPP4-deficient rats tended to decrease when compared with those of both the non-STZ-treated and STZ-treated WT rats. The expression levels of hemoglobin in HEK293 and Caki-1 cells were significantly decreased when DPP4 was knocked down by siRNA, were significantly increased by the addition of soluble human DPP4, and were also significantly increased by the addition of the DPP4 inhibitor, sitagliptin. The expression level of DPP4 was also significantly increased by the addition of sitagliptin in both cell types. Our findings indicate that DPP4 regulates the expression of the hemoglobin genes, and might play a role in the preservation of renal function; however, the underlying mechanism of this preservation remains to be elucidated.


Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker


Y chromosome gr/gr subdeletion is associated with lower semen quality in young men from the general Japanese population but not in fertile Japanese men

Several case-control studies have investigated whether Y chromosome haplogroups or deletions are associated with spermatogenic failure. However, the relationships between Y chromosome haplogroups or deletions and semen quality in general population have not been elucidated. In this study, we assessed relationships between Y chromosome haplogroups or deletions and semen parameters in 791 fertile Japanese men and 1221 young men from the general Japanese population. We found that the haplogroup D2 (M55 lineage) was significantly associated with lower semen parameters, especially total motile sperm count (P = 0.00051, beta = -0.097), in men from the general population but not in fertile men. In addition, we found that the gr/gr subdeletion was associated with semen quality and in particular, strongly associated with decreased sperm motility (P = 0.00041, beta = -3.14) and total motile sperm count (P = 0.00031, beta = -0.099) in men from the general population but not in fertile men. The combined analysis of fertile Japanese men and men from the general Japanese population showed that the haplogroup D2 (M55 lineage) and the gr/gr subdeletion were strongly associated with reduced sperm motility (P = 0.00056, beta = -2.71, and P = 7.7 × 10(-5), beta = -3.05, respectively) and that haplogroup O2b1 was strongly associated with elevated sperm motility (P = 0.00089, beta = 2.94). These observations add further support for the view that the gr/gr subdeletion diminishes sperm motility that consequently may result in male infertility.


Meta-Analysis of Interleukin Polymorphisms and NSAID Usage Indicates Correlations to the Risk of Developing Cancer

International Journal of Genomic Medicine


A meta-analysis of the association of PPARγ rs1801282 polymorphism and NSAID usage with the risk of developing cancer

Use of nonsteroidal anti-inflammatory drugs (NSAIDs) is correlated with a reduced risk of cancer through the reduction of inflammation, which is an important risk factor. Several studies have investigated polymorphisms in the peroxisome proliferator-activated receptor gamma (PPARγ) gene and NSAID use in association with cancer risk. However, these studies yielded mixed results. Therefore, we performed a meta-analysis to evaluate the association of PPARγ polymorphisms and NSAID usage with cancer risk. We conducted a comprehensive search of PubMed through May 2013. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated using the fixed-effect or random-effect model. A comprehensive search of the database revealed 6 studies that fulfilled the inclusion criteria. NSAID use was significantly associated with decreased cancer risk regardless of PPARγ rs1801282 genotypes. In a stratified analysis by cancer type, NSAID users who were minor allele carriers had significantly decreased colon cancer risk compared to non-NSAID users (OR=0.73, 95% CI=0.57-0.93), whereas NSAID users homozygous for the major allele had significantly decreased risk for cancers other than colon cancer compared to non-NSAID users (OR=0.79, 95% CI=0.69-0.91). Our results suggest that the association of PPARγ rs1801282 polymorphism and NSAID use with the risk of cancer may differ according to cancer type.


Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification.


A Meta-Analysis of PTGS1 and PTGS2 Polymorphisms and NSAID Intake on the Risk of Developing Cancer

Several studies have investigated whether the polymorphisms in the prostaglandin endoperoxide synthase 1 (PTGS1) and PTGS2 genes and nonsteroidal anti-inflammatory drug (NSAID) use are associated with cancer risk; however, those studies have produced mixed results. Therefore, we performed a meta-analysis to evaluate the association between the PTGS1 and PTGS2 polymorphisms and the effect of NSAID use on the risk of developing cancer. We conducted a comprehensive search in PubMed through March 2012. The odds ratios (ORs) with the corresponding 95% confidence intervals (CIs) were calculated using the fixed-effect model or the random-effect model. The database search generated 13 studies that met the inclusion criteria. For PTGS1 rs3842787, NSAID users homozygous for the major allele (CC) had a significantly decreased cancer risk compared with non-NSAID users (OR = 0.73, 95% CI = 0.59-0.89). For PTGS2 rs5275 and rs20417, there were no significant differences between the gene polymorphism and NSAID use on cancer risk among the 8 and 7 studies, respectively. However, in the stratified analysis by the type of cancer or ethnicity population, NSAID users homozygous for the major allele (TT) in rs5275 demonstrated significantly decreased cancer risk compared with non-NSAID users in cancer type not involving colorectal adenoma (OR = 0.70, 95% CI = 0.59-0.83) and among the USA population (OR = 0.67, 95% CI = 0.56-0.82). NSAID users homozygous for the major allele (GG) in rs20417 displayed a significantly decreased cancer risk than non-NSAID users among the US population (OR = 0.72, 95% CI = 0.58-0.88). For the PTGS2 rs689466 and rs2745557 SNPs, there were no significant differences. This meta-analysis suggests that the associations between PTGS polymorphisms and NSAID use on cancer risk may differ with regard to the type of cancer and nationality.


Y chromosome haplogroup D2* lineage is associated with azoospermia in Japanese males

Several studies have investigated whether particular Y chromosome haplogroups are associated with spermatogenic failure in Japanese males; however, they produced differing results. In this study, to investigate the association of Y chromosome haplogroup with spermatogenic failure, we recruited 451 infertile patients and 730 fertile men from a Japanese population and typed their Y chromosome haplogroups. The infertile patients were suffering from varicocele, azoospermia, oligozoospermia, asthenozoospermia, obstructive azoospermia, karyotype abnormalities, microdeletions of the long arm of the Y chromosome, or other conditions that affect fertility. The frequency of haplogroup D2* was significantly higher (odds ratio = 2.28, 95% confidence interval = 1.44-3.61, P = 0.00034 using chi-square test) among the men with azoospermia than among the fertile men. None of the other Y haplogroups displayed associations with particular types of infertility. In conclusion, Y chromosome haplogroup D2* is associated with spermatogenic failure in Japanese males, suggesting that the Y chromosome lineage can have significant effects on spermatogenesis.


Replication study and meta-analysis of human nonobstructive azoospermia in Japanese populations

Recently, a Chinese genomewide association study (GWAS) identified four autosomal single-nucleotide polymorphism (SNP) loci as being significantly associated with risk factors for nonobstructive azoospermia (NOA; P < 5 × 10(-8)). In the present study, we performed a replication study on two Japanese cohorts from different institutions in order to evaluate whether SNP loci are associated with NOA. The four SNPs (rs12097821, rs2477686, rs10842262, and rs6080550) reported in the Chinese GWAS were genotyped in 490 NOA patients and 1167 controls. To assess the significance of the associations between each of the four SNPs and NOA in the Japanese population, the association results for the two cohorts were combined by meta-analysis. In the meta-analysis, the combined per-allele odds ratios (ORs) for the four SNPs and their respective 95% confidence intervals (CIs) were as follows: rs12097821, OR = 1.10 (CI = 0.89-1.37); rs2477686, OR = 1.11 (CI = 0.87-1.43); rs10842262, OR = 1.11 (CI = 0.94-1.32); and rs6080550, OR = 0.96 (CI = 0.76-1.21). None of the SNPs was significantly associated with NOA (P > 0.05). However, three of four SNPs (rs12097821, rs2477686, and rs10842262) showed associations in the same direction in Japanese men as those reported in the Chinese GWAS. To determine whether the four SNPs are genetic risk factors for NOA, the effect sizes of NOA risk factors require further investigation using larger independent sets of case-control samples of populations, including Japanese and Chinese populations.


Plasma dipeptidyl peptidase 4 activity correlates with body mass index and the plasma adiponectin concentration in healthy young people

We previously found that plasma dipeptidyl peptidase 4 (DPP4) activity was associated with the development of obesity, type 2 diabetes, and type 1 diabetes using animal models. In this study, we investigated whether DPP4 activity is correlated with the clinical parameters of obesity and/or diabetes in healthy young subjects. Body mass index (BMI), plasma DPP4 activity, total cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, fasting blood glucose, adiponectin concentration, and body fat were measured in 165 subjects (110 males and 55 females, age 23.2 ± 2.4 years). In correlation analyses, DPP4 activity displayed strong positive correlations with BMI (p = 5.5 × 10(-5)) and total cholesterol (p = 0.0014), and a negative correlation with the plasma adiponectin concentration (p = 0.013), but not fasting blood glucose. Our findings suggest that plasma DPP4 activity is correlated with the clinical parameters of obesity rather than diabetes in young people.


Climatic influence on the reproductive characteristics of Japanese males

We previously performed a survey of the sperm characteristics of the partners of pregnant women in four cities in Japan. In the present study, we analyzed the sperm characteristics of these subjects and the correlations between these sperm characteristics and climatic changes or Y chromosome haplogroups. Our results showed that more haplogroup D2a1 males than O2b1 males were born in the first half of the year (January to June), whereas more O2b1 males were born in the last half of the year (July to December) (P<0.05). This was agreed and correlated with the seasonal variations in their mean sperm concentrations. The haplogroup C, D* and D2a1 males displayed lower sperm concentrations from March to May, followed by an increase in their sperm concentrations starting in June or July, while the O2b1 males displayed higher sperm concentrations in the first half of the year followed by a sudden decrease from July to August (P<0.05). We hypothesize that the Japanese climate has different effects on the sperm characteristics and reproductive seasonality of males from different lineages; and therefore, has influenced the modern population of Japan.


Anti-obesity effects of Schisandra chinensis fruit water extract in rats fed a high-fat diet

Obesity has become a major public health problem worldwide. To date, many studies on obesity prevention have been performed. In this study, we examined the anti-obesity effects of <I>Schisandra chinensis</I> (Turcz.) Baill. fruit (SC) water extract in rats fed a high-fat diet. We gave the rats high-fat chow including SC water extract for 10 weeks. Although these rats ate the same amount of chow as the high-fat diet group, their weight gain was significantly inhibited compared to that in the high-fat diet group. Moreover, the SC group showed significantly decreased epididymal fat weight and levels of plasma triglycerides, total cholesterol, and leptin compared to the high-fat diet group. In the oral glucose tolerance test (OGTT), the SC group showed lower blood glucose levels than the high-fat diet group 90 min after eating. This is the first study to show that SC water extract has anti-obesity effects. It may be beneficial to drink SC water extract or tea after eating to prevent obesity.

肥満は社会的問題となっており， 予防対策に関する研究が進められている． 本研究では， 五味子 (チョウセンゴミシ果実) の肥満予防に対する有効性について検討した． 五味子水抽出エキスを高脂肪食に混合してラットに投与し， 肥満に関わるパラメーターに対する影響を検討した． 10週間自由摂食させた結果， 摂食量は変わらないにもかかわらず五味子水抽出エキス混合食投与群では高脂肪食のみ投与されていた群より有意な体重減少， 内臓脂肪量の減少， 血漿中のトリグリセリド減少が見られた． さらに， 五味子水抽出液混合食を投与した群では， 高脂肪食のみ投与した群と比較して脂肪組織は普通食摂食群と同程度に小さく， ブドウ糖負荷試験において90分後の血糖値は高脂肪食投与群より有意に低かった． 以上のことより五味子水抽出エキスは肥満の予防に有効である可能性が示唆された．


SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.


Altered dipeptidyl peptidase 4 activity during the progression of hyperinsulinemic obesity and islet atrophy in spontaneously late-stage type 2 diabetic rats.

Altered dipeptidyl peptidase-4 (DPP4) activity during the progression of late-stage type 2 diabetes was measured in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Compared with OLETF rats subjected to 30% food restriction, food-satiated OLETF rats exhibited spontaneous hyperphagic obesity, insulin resistance, hyperglycemia, hyperinsulinemia, and increased plasma DPP4 activity during the early phase of the experiment (up to ∼30 wk). Subsequently, their plasma DPP4 activity as well as their body weight, body fat, and plasma insulin concentration declined to control levels during the late phase, resulting in excessive polyuria, proteinuria, dyslipidemia, pancreatic islet atrophy, hypoinsulinemia, and diabetes, which changed from insulin-resistant diabetes to hypoinsulinemic diabetes secondary to progressive islet insufficiency, and their fasting blood glucose level remained high. Since plasma DPP4 activity demonstrated significant positive correlations with body weight and the fasting plasma insulin level but not with the fasting blood glucose level during the late stage of diabetes, body fat and fasting plasma insulin levels may be useful factors for predicting the control of plasma DPP4 activity. In contrast, pancreatic DPP4 activity was significantly increased, and the expression of pancreatic insulin was significantly reduced in late-stage diabetic OLETF rats, suggesting that a relationship exists between the activation of pancreatic DPP4 and insulin depletion in pancreatic islet atrophy. In conclusion, it is suggested that plasma DPP4 activity changes in accordance with the progression of hyperinsulinemic obesity and pancreatic islet atrophy. DPP4 activity may play an important role in insulin homeostasis.

American Journal of Physiology, Endocrinology and Metabolism


Role of dipeptidyl peptidase IV (DPP4) in the development of dyslipidemia: DPP4 contributes to the steroid metabolism pathway.

We previously reported that dipeptidyl peptidase IV (DPP4)-deficient rats were susceptible to dyslipidemia induced by streptozotocin (STZ). Hence, it is suggested that DPP4 is important for lipid metabolism. In this study, to verify the role of DPP4 in the development of dyslipidemia, we carried out a microarray analysis of the livers of STZ-treated wild-type and DPP4-deficient rats and showed that the expression levels of genes involved in metabolic processes (steroid metabolic processes and cellular lipid metabolic processes) were significantly altered by STZ treatment. In the wild-type rats, the expression of hydroxysteroid (17-beta) dehydrogenase 2 (Hsd7b2), which catalyzes sex steroid synthesis from cholesterol, was significantly increased by about 15-fold after STZ treatment; however, it did not change in the DPP4-deficient rats. In the STZ untreated group of DPP4-deficient rats, the expression levels of cytochrome P450, subfamily 51 (Cyp51) and sterol-C4-methyl oxidase-like (Sc4mol), which catalyze intermediate steps in cholesterol synthesis, were significantly elevated compared to those of other groups. Similar results were demonstrated in HuH7-cells after DPP4 overexpression or the addition of human sera containing DPP4. DPP4 is crucial for regulating the expression of factors related to steroid metabolism such as Cyp51, Sc4mol, and Hsd17b2, and DPP4 deficiency or inhibition may cause dyslipidemia.


The male-determining gene SRY is a hybrid of DGCR8 and SOX3, and is regulated by the transcription factor CP2.

In mammals, sex is determined by the presence or absence of the Y chromosome that bears a male-dominant sex-determining gene SRY, which switches the differentiation of gonads into male testes. The molecular signaling mechanism turning on the switch, however, has remained unclear for 18 years since the identification of the gene. Here, we describe how this gene emerged and started to work. From amino acid homology, we realized that SRY is a hybrid gene between a portion of the first exon of DiGeorge syndrome critical region gene 8 (DGCR8) and the high-mobility group (HMG) box of SRY box-3 (SOX3) gene. We identified the regulatory sequence in the SRY promotor region by searching for a common motif shared with DGCR8 mRNA. From the motif search between DGCR8 mRNA and the SRY upstream sequence, we found that the transcription factor CP2 (TFCP2) binding motif is present in both. TFCP2 overexpression did not show a significant increase of SRY mRNA expression, and TFCP2 suppression by RNA interference (RNAi) significantly reduced SRY mRNA expression. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that TFCP2 acts as a regulator by directly binding to the SRY promoter. We conclude that SRY is a hybrid gene composed of two genes, DGCR8 and SOX3; and TFCP2 is an essential transcription factor for SRY expression regulation.


Prediction of Japanese children at risk for complications of childhood obesity: gender differences for intervention approaches.

Childhood obesity is one of the most serious public health problems in Japan, especially in Tokushima compared with other prefectures. This study was designed to clarify the life habits which predispose to development of obesity and can be modified through an appropriate intervention program to combat childhood obesity and its lifestyle-related diseases. A total of 216 school children from Itano Town, a municipality of Tokushima Prefecture, Japan, who are attending the fourth grade (9-10 years) of elementary schools, participated in the study from 2004 to 2007. The study included child's life habits questionnaire, investigating physical activity by recording the daily steps using a pedometer, anthropometric measurements, hematological examination and hemodynamometry in a cross-sectional survey during a two-month period from June to July every year. We conclude that there are considerable gender-related differences for developing obesity and other lifestyle-related diseases; and all intervention strategies against obesity must consider such gender differences. For example, restriction of television watching hours must be intervened for controlling obesity in boys, however for girls, promotion of exercise practice or making more steps per day with adequate sleeping periods should be intervened as the proper approaches for preventing and controlling obesity and other lifestyle-related diseases.

The Journal of Medical Investigation : JMI


Proteasome activity correlates with male BMI and contributes to the differentiation of adipocyte in hADSC

We have previously reported that 26S proteasome subunit mRNA expressions correlate with male body mass index (BMI). In this study, to investigate whether proteasome activities are correlated with BMI, we recruited 61 healthy young Japanese male subjects, measured proteasome activities in their plasma, and correlated them with their BMI and various metabolic factors. We found that among three different proteasome activities, chymotrypsin-like activity in plasma was positively correlated with BMI in healthy Japanese male subjects. Furthermore, we analyzed proteasome activity in vitro during the differentiation of human adipose-derived stem cell (hADSC) into mature adipocytes. In the early stage of differentiation, proteasome activity was at its highest level, and proteasome inhibitor could inhibit hADSC adipocyte differentiation. Our findings suggest that proteasome is an important controlling factor for the development of obesity and adipogenesis.


日本の子どもの肥満は増加しているのか(平成1∼20年度)

日本の子どもの肥満は増加しているのか(平成1∼20年度)

Journal of Shikoku Public Health Society


Proteomics and transcriptome approaches to investigate the mechanism of human sex determination

The SRY gene (sex-determining region on the Y chromosome) was isolated in 1990 and is known as the testis-determining factor on the Y chromosome. The SRY has been considered as a transcription factor since it contains an HMG box, which functions as a DNA-binding domain. However, a direct target for SRY remains to be identified. We have investigated the function of SRY through proteomics and transcriptome approaches, and by using two stable SRY-overexpressing cell lines (SRY1 and SRY2) in NT2/D1 cells derived from human testicular embryonal cell carcinoma. The results of 2-dimensional gel electrophoresis show that SRY overexpression causes a considerable downregulation of many chaperone proteins. SRY also upregulates laminin, which is important for Sertoli cell differentiation. Additionally, transcriptome analysis shows that SRY overexpression upregulates many zinc finger proteins and downregulates cellular growth factors with S or G(2)/M arrest of the cell cycle and inhibition of cellular proliferation.


Proteasome subunits mRNA expressions correlate with male BMI; implications for a role in obesity

Obesity as well as its associated chronic diseases and adverse health consequences such as type 2 diabetes mellitus, dyslipidemia, hypertension, and coronary artery disease are afflicting middle-aged adults and an ever greater number of children globally. We planned to investigate new obesity-related factors using proteomics approaches in a randomly selected three high and three low BMI samples of Epstein-Barr-transformed B (EBV-B) lymphoblastoid cell lines prepared from two groups of young Japanese men with different BMI. To search novel obesity-related factors, comparisons of protein expressions between high and low BMI groups were carried out by two-dimensional gel electrophoresis (2-DE). Gene transcripts of proteasome subunits found out from 2-DE were further determined by quantitative real-time PCR. Results from proteomics approach showed that the expression of proteasome alpha subunit type 5 (PSMA5) was significantly lower in the high BMI male group than in those with low BMI (P < 0.05). To validate these results, we expanded the study to include 20 more men and used real-time PCR to quantify the mRNA expression level in their EBV-B cells. Both PSMA5 and PSMA2 of EBV-B cells showed negative correlation with BMI. Furthermore, the mRNA levels measured in the peripheral blood B lymphocytes for many proteasome subunits in 75 healthy men and women showed significant negative correlation with BMI in healthy men. Our findings suggest that proteasome expression may play a key role in obesity.


Increased Plasma Dipeptidyl Peptidase IV (DPP IV) Activity and Decreased DPP IV Activity of Visceral But Not Subcutaneous Adipose Tissue in Impaired Glucose Tolerance Rats Induced by High-Fat or High-Sucrose Diet

Several studies have investigated whether dipeptidyl peptidase IV (DPP IV) activity is correlated to the severity of diabetes; however, it remains unclear. To investigate the roles of DPP IV activity in metabolic abnormalities, impaired glucose tolerance rats were produced using a high-fat (HF) or high-sucrose (HS) diet. HF diet-fed rats obviously exhibited impaired glucose tolerance, with increases in subcutaneous and epididymal fat mass, insulin resistance and dyslipidaemia. In rats fed a HS diet rather than a normal diet, lower body weight and fasting blood glucose were observed temporarily in the early period after HS diet feeding; however, impaired glucose tolerance was evoked to some extent with an increase in epididymal fat mass. Both HF and HS diet-fed rats showed significantly higher plasma DPP IV activity than normal diet-fed rats, in the order of HF diet>HS diet>normal diet. HF and HS diets did not significantly affect DPP IV activity and mRNA expression in the kidney. On the other hand, HF, but not HS, diet caused a significant decrease in DPP IV activity in the liver as compared to the control. Of note, both HF and HS diets caused a significant decrease in DPP IV activity in epididymal fat, even though they did not change DPP IV activity in subcutaneous fat. In conclusion, HF or HS diet-induced impaired glucose tolerance with visceral fat accumulation may be interrelated with increased plasma DPP IV activity and decreased DPP IV activity of visceral but not subcutaneous adipose tissue.


Proteasome subunits are regulated and expressed in comparable concentrations as a functional cluster

The proteasome is the main proteolytic enzyme that functions in the ubiquitin-proteasome system. The 26S proteasome has multi-subunit protease complexes consisting of 20S subunits composed of four seven-numbered rings with two outer rings containing alpha subunits and two central rings composed of beta subunits, and 19S caps of 6 ATPase and 11 non-ATPase subunits; however, it is unclear how these subunits are regulated and the 26S proteasomes assembled. To verify whether each subunit's mRNA expression is associated with the mRNA expression of other proteasome subunits, we carried out expression analysis of 34 proteasome subunits mRNA on peripheral blood from 75 subjects. The expression of proteasome subunits mRNA was comparable in each individual of the studied population and the mRNA expression has been investigated in each 20S or 19S proteasome. Our results suggest that each type of subunit is regulated by respectively common factors, and that the 20S and 19S proteasomes are regulated by different systems.

Biochemical and Biophysical Research Communications


Interrelationship of dipeptidyl peptidase IV (DPP IV) with the development of diabetes, dyslipidaemia and nephropathy: A streptozotocin-induced model using wild-type and DPP IV-deficient rats

We examined the role of dipeptidyl peptidase IV (DPP4) in the development of diabetes, dyslipidaemia and renal dysfunction induced by streptozotocin (STZ). F344/DuCrlCrlj rats, which lack DPP4 activity, and wild-type rats were treated with STZ. Plasma DPP4 activity and biochemical parameters were measured until 42 days after STZ treatment. At the end of the experiment, renal function and DPP4 expressions of the kidney, liver, pancreas and adipose tissues were determined. Increases in blood glucose, cholesterol and triglycerides were evoked by STZ in both rat strains; however, the onset of hyperglycaemia was delayed in DPP4-deficient rats as compared with wild-type rats. By contrast, more severe dyslipidaemia was observed in DPP4-deficient rats than in wild-type rats after STZ treatment. Plasma DPP4 activity increased progressively with time after STZ treatment in wild-type rats. The kidney of wild-type rats showed decreased DPP4 activity with increased Dpp4 mRNA after STZ treatment. In addition, kidney weight, serum creatinine and excreted amounts of urinary protein, glucose and DPP4 enzyme were enhanced by STZ. DPP4-deficient rats showed increased serum creatinine in accordance with decreased creatinine clearance as compared with wild-type rats after STZ treatment. In conclusion, plasma DPP4 activity increased after STZ treatment, positively correlating to blood glucose. DPP4-deficient rats were resistant to developing diabetes, while susceptible to dyslipidaemia and reduction of glomerular filtration rate by STZ. DPP4 activation may be responsible for hyperglycaemia, lipid metabolism and preservation of renal function.


Overexpression of SOX15 Inhibits Proliferation of NT2/D1 Cells Derived from a Testicular Embryonal Cell Carcinoma

SOX (Sry-related HMG box) family proteins, which have an evolutionarily conserved DNA binding domain, have crucial roles in cell differentiation. However, their target genes remain enigmatic. Some members of the SOX family may have roles in regulation of cell proliferation. We established stable NT2/D1 cell lines overexpressing SOX15 (SOX15-NT2/D1), and a modified 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the SOX15-NT2/D1 cells exhibited significantly slower growth than the controls. Flow cytometry analysis revealed that an increased fraction of the SOX15-NT2/D1 cells were in G1-G0. In addition, a microarray analysis identified 26 genes that were up-regulated in the SOX15-NT2/D1 cells, but none that were down-regulated genes. Among the up-regulated genes, IGFBP5, S100A4, ID2, FABP5, MTSS1, PDCD4 have been shown to be related to cell proliferation and/or the cell cycle.


A/G heterozygote of the A-3826G polymorphism in the UCP-1 gene has higher BMI than A/A and G/G homozygote in young Japanese males

UCP-1 is suggested to have important roles for thermogenesis and energy expenditure. To elucidate whether the A-3826G polymorphism that is located in the 5' flanking region of the UCP-1 gene has roles in healthy young people, the polymorphism was genotyped among 251 young Japanese men whose mean age is 22.7 years old. We analyzed relationship between the A-3826G polymorphism and body mass index (BMI) or six biochemical parameters, serum concentration of total cholesterol (TC), high density lipoprotein (HDL) cholesterol, triglyceride (TG), asparatate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose. The genotype frequencies were observed at the frequencies of 24.3% for AA, 48.2% for AG and 27.5% for GG, respectively. When BMI and the biochemical parameters were compared by ANOVA among individuals with each genotype, the statistical difference was observed only for BMI (P=0.016). Bonferroni's test demonstrated that the men with the AG genotype have higher BMI than those with the AA genotype (22.4+/-2.8 vs. 21.4+/-2.2) (P=0.04). The individuals with the AG genotype also showed trend to have higher BMI than those with the GG, although the difference was not statistically apparent (22.4+/-2.8 vs. 21.5+/-2.3) (P=0.07). Our results indicated that the young healthy Japanese men with the AG heterozygote showed higher BMI than those with other genotypes.

The Journal of Medical Investigation : JMI


Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.


A rapid and simple system of detecting deletions on the Y chromosome related with male infertility using multiplex PCR

Around 10% of males with idiopathic azoospermia or oligozoospermia, which are important causes of male infertility, have partial deletions on the long arm of the Y chromosome. To develop a rapid and accurate detection system for screening major Y deletions found in infertile men, we developed a multiplex PCR system that can simultaneously amplify five loci on the Y chromosome, SRY, AMELY, DBY, RBMY, DAZ and one locus on the X chromosome, AMELX. The size of the PCR products was designed to increase gradually from the distal Yp to the distal Yq. Our system could detect deletions of three major candidate regions for the azoospermic factor, AZFa, AZFb and AZFc on the Y chromosome together with sex. The gradual increase in the size of the PCR products was convenient for imaging the location of deletions on the Y chromosome. Moreover, the multiplex PCR system was combined with microchip-based electrophoresis, and the PCR products derived from each locus were separated within 4 min. Our system is useful for screening Y chromosomes bearing the structural anomalies including three major AZF deletions found among azoospermic or oligozoospermic males.


Survey of the two polymorphisms in DAZL, an autosomal candidate for the azoospermic factor, in Japanese infertile men and implications for male infertility

The DAZL (DAZ-like) gene is suggested to be an ancestral gene of the DAZ (deleted in azoospermia) gene on the Y chromosome, which is a strong candidate for the azoospermic factor. Recently, it has been reported that the T54A (Thr54-->Ala) polymorphism in exon 3 of the DAZL gene is associated with spermatogenic failure in the Taiwanese population. In this study, to investigate whether this polymorphism is associated with spermatogenic failure in Japanese males, we analysed genomic DNA derived from 234 patients with azoospermia or oligozoospermia and 131 fertile controls. The T54A polymorphism was completely absent in both the patients and the controls. The T12A (Thr12-->Ala) polymorphism in exon 2 of the DAZL gene was found at a similar frequency in the patients and controls, 15.4% and 13.7%, respectively (P = 0.67). However, the frequency of T12A was higher for the azoospermic (20.5%) than oligozoospermic (9.6%) individuals in infertile men without DAZ deletions, although statistical difference was not so apparent (chi2 test: P = 0.037, OR = 2.413, 95% CI = 1.035-5.629; Yate's chi2 test: P = 0.058, OR = 2.319, 95% CI = 0.973-6.166). Our results show that the T54A polymorphism in DAZL has no major role in Japanese males with azoospermia or oligozoospermia. The distribution of the T54A polymorphism may be restricted to the narrow area including Taiwan.


Molecular analysis of TBL1Y, a Y-linked homologue of TBL1X related with X-linked late-onset sensorineural deafness

Recent progress in sequencing the human Y chromosome has unveiled a series of X-Y homologous genes. In the present study, we focused on Transducin beta-like 1Y (TBL1Y), which is a Y-linked homologue of TBL1X that is related with X-linked late-onset sensorineural deafness. Recently, it has been shown that TBLR1, another homologue whose gene resides on chromosome 3, and TBL1X act as a corepressor/coactivator exchanger for several nuclear receptors and transcription factors. However, the expression pattern and function of TBL1Y remain unknown. The RT-PCR analysis of the TBL1 family revealed that TBL1Y was expressed in all 13 tissues examined but not in leukocytes. Among the cell lines tested, however, it was only expressed in NT2/D1 cells and in lymphoblasts transformed with Epstein Barr (EB) virus. To compare the functions of the TBL1 family, we generated a series of expression plasmids for GAL4DBD-fused proteins of the TBL1 family. We carried out dual luciferase assays using these plasmids in combination with a plasmid having a luciferase reporter gene harboring 5xGAL4 binding sites. Unlike the other constructs, GAL4DBD-fused TBL1Y did not repress the promoter activity. Moreover, we found three novel polymorphisms in the TBL1Y gene, IVS7+9G>A, G268C, and IVS7+1G>C, which is presumed to cause splicing error. These polymorphisms are found in males within Y-haplogroup O3 (XO3e), which is defined as the Y-haplogroup O3 excluding O3e, a branch of O3. The results show that TBL1Y differs from other members of the TBL1 family in expression and function, suggesting other roles in maleness.


Alkyl Gallates, Intensifiers of ß-Lactam Susceptibility in Methicillin-Resistant Staphylococcus aureus

We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified beta-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and beta-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 microg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to beta-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for beta-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to beta-lactam antibiotics.


Proteomics approach for the mechanism of sex determination

性分化と精子形成機構に対するプロテオミクスからのアプローチ

SRY(sex determining region on the Y chromosome)was a gene isolated as the testis determiningfactor on the Y chromosome in1990．The gene consisted of single exon and encodes a204aminoacid protein. Since SRY protein has a HMG domain, which functions as DNA binding domain, it hasbeen considered to be a transcription factor. Direct target of SRY, however, have not yet identifiedfor14years.In this study, we have performed a proteomics approach to analyze the function of SRY. Twodimensionalgel electrophoresis after over expression of SRY showed considerable down regulationin many proteins. Among the proteins, mitochondrial 60‐kDa heat shock protein(HSP60)andprobable protein disulfide isomerase(ER60)remarkably decreased. Since they were reported tobe associated with cell proliferation and differentiation, we investigated the effects of SRY on cellcycle profiles. It was shown that the over‐expression of SRY negatively regulated the cell growthwith significant S or G2/M arrest of cell cycle.


Variation in synergistic activity by flavone and its related compounds on the increased susceptibility of various strains of methicillin-resistant Staphylococcus aureus to β-lactam antibiotics

We found that some flavonoids had a weak antibacterial effect on methicillin-resistant Staphylococcus aureus (MRSA), but that at sub-MIC concentrations they greatly increased the susceptibility of these strains to beta-lactam antibiotics. Flavone showed diverse synergistic effects on the susceptibility of MRSA to beta-lactam antibiotics. The variation of the synergistic effects of the flavones to increase the susceptibility of strains of MRSA to beta-lactam antibiotics coincided with their varying effects on growth-inhibition of these strains. Based on these findings, we have proposed a model for the mechanisms of high resistance of MRSA to beta-lactams and the massive reduction in the beta-lactams MIC caused by flavones.

International Journal of Antimicrobial Agents


6,7-dihydroxyflavone dramatically intensifies the susceptibility of methicillin-resistant or -sensitive Staphylococcus aureus to beta-lactams.

We have demonstrated that 6,7-dihydroxyflavone by itself has only a weak antibacterial effect on methicillin-resistant Staphylococcus aureus (MRSA) but that at concentrations less than MIC it synergistically elevates the susceptibility of clinically isolated MRSA and methicillin-sensitive S. aureus strains to beta-lactam antibiotics from 8- to 32,800-fold.


Flavone markedly affects phenotypic expression of beta-lactam resistance in methicillin-resistant Staphylococcus aureus strains isolated clinically.

Flavone and its derivatives had very weak antibacterial effects on niethicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus, but dramatically intensified MRSA's susceptibility to β-lactams. We named these compounds "ILSMR (intensifier of β-lactam-susceptibility in MRSA)." We also found discrepancies among MRSA strains in their responses to flavone; some strains showed phenotypic susceptibility to methicillin while others showed phenotypic resistance to it. To understand the mechanism underlying this discrepancy, we characterized 20 MRSA strains in detail, analyzed their conventional and molecular typings, and examined each strain's resistance to β-lactams, with COL serving as a reference. Neither SCCmec typing nor coagulase typing explained the diverse effects of flavone on the β-lactam MICs of these strains. Likewise, changes in pulsed-field gel electrophoresis (PFGE) type were not associated with the profiles of ILSMR effects. However, the present observations suggest that the ILSMR effects on MRSA is strain-specific, and that this effect depends on an as-yet unknown mechanism that is essential for the expression of the phenotype conferring β-lactam resistance to MRSA strains, independently of an interaction with the mecA-encoded penicillin-binding protein 2a or with the β-lactamase.


Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus.

A crude extract prepared from Scutellaria barbata D. Don (Lamiaceae) was analyzed in the effort to discover antibacterial compounds against high-level strains of methicillin-resistant Staphylococcus aureus (MRSA). Apigenin and luteolin were isolated from the plant as active constituents against the bacteria. These flavonoid congeners were selectively toxic to S. aureus, including the MRSA and methicillin-sensitive S. aureus strains.


A Xanthanolide with Potent Antibacterial Activity against Methicillin-resistant Staphylococcus aureus.

The Journal of Pharmacy and Pharmacology


Extraction and Purification of Effective Antimicrobial Constituents of Terminalia chebula RETS. against Methicillin-Resistant Staphylococcus aureus.


男性不妊症関連遺伝子の精子形成メカニズム

男性不妊症関連遺伝子の精子形成メカニズム


Spermatogenesis mechanism of male infertility related genes

男性不妊症関連遺伝子の精子形成メカニズム


Development of thiol-responsive traceable linker for efficient enrichment and selective labeling of target proteins


日本人類学会遺伝分科会平成22年度公開シンポジウム「日本人の成り立ちについての新たな考察―遺伝と形態から―Y染色体からみた日本人の成り立ち

Anthropological Science (Japanese Series)