Kenta Nagahori

Haplotype Analysis of GSK-3β Gene Polymorphisms in Bipolar Disorder Lithium Responders and Nonresponders

Abstract The GSK-3&bgr; gene, GSK3B, codes for an enzyme that is a target for the action of mood stabilizers, lithium and possibly valproic acid. In this study, the relationship between haplotypes consisting of single nucleotide polymorphisms (SNPs) of GSK3B −50T/C and −1727A/T and the effect of lithium was studied among Japanese bipolar disorder lithium nonresponders and responders. The distributions of the GSK3B haplotypes (−50T/C and −1727A/T) showed a trend for significant difference between the lithium nonresponders and responders (global P=0.07074). Haplotype 1 (T-A) was associated with a higher lithium response (haplotype-specific P=0.03477), whereas haplotype 2 (C-A) was associated with a lower lithium response (haplotype-specific P=0.03443). The pairwise D′ and r2 values between the 2 SNPs in this study were 1.0 and 0.097, respectively. The 2 SNPs showed weak linkage disequilibrium with each other.

Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body’s immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

Pathological effect of arterial ischaemia and venous congestion on rat testes

Many studies on various organs have concluded that venous congestion (VC) causes severe organ dysfunction with elevation of oxidative stress relative to that of arterial ischaemia (AI). However, a comparison of the pathological effects of AI and VC on the testes has not been conducted. In this study, models of AI and VC and their reperfusion in rat testes, respectively, were developed and analysed. Testicular arteries or veins were interrupted for 6 h, re-perfused and kept for 4 weeks; the effects on the testes were then evaluated. Severe spermatogenic disturbances were observed at 4 weeks after reperfusion in AI but not in VC. At 6 h after blood flow interruption, oxidative stress was significantly increased and germ cells were severely damaged in AI compared with those in VC. RT-PCR analyses revealed that haem oxygenase-1, which exhibits anti-oxidative effects, and vascular endothelial growth factor-A, which exhibits vasculogenic effects, were significantly increased in VC but not in AI. Surprisingly, the results of our experiment in rat testes differed from those of experiments in previous studies performed in other organs. Oxidative stress in testes was more easily elevated by AI than it was by VC, explainable by the different experimental conditions.

Development of heterotopic transplantation of the testis with the epididymis to evaluate an aspect of testicular immunology in rats

Transplantation of testicular cells and tissues has been studied for the investigation of immunology of the testis, which is an immunologically privileged organ. However, reports of transplant of the testis at organ level have been extremely limited because of technical difficulties of the orthotopic testis transplantation (OTT) in experimental animals. In the present study, we developed a new and simple model of the heterotopic testis transplantation (HTT), which is donor testis transplantation into the cervical region of recipients, in a syngeneic model in rats [donor Lewis (LEW) graft to LEW recipient]. The duration of HTT was significantly shorter and success rate higher than that of OTT. To histologically evaluate HTT, the local immune responses were compared among the syngeneic model, an acute rejection allogeneic model [donor Augustus Copenhagen Irish (ACI) graft to LEW recipient] and a chronic rejection allogeneic model (donor F344 graft to LEW recipient) at postoperative day 3. We found that allogeneic ACI grafts resulted in mild and not severe orchitic lesions, whereas immune responses of allogeneic F344 grafts seemed intact and were not significantly different from those of syngeneic LEW grafts. These results suggest that our new operative procedure will be useful in future for the investigation of the testicular immunology.

Association between AUTS2 haplotypes and alcohol dependence in a Japanese population

Objective Recent genome-wide analysis has indicated that the autism susceptibility candidate 2 (AUTS2) gene is involved in the regulation of alcohol consumption. We hypothesised that AUTS2 might be associated with the development of alcohol dependence. Therefore, in this exploratory study, we compared the genotype and allele frequencies of the polymorphisms rs6943555 and rs9886351 in the AUTS2 gene between patients with alcohol dependence and healthy control subjects living in a Japanese provincial prefecture. We also examined whether or not the haplotypes consisting of these polymorphisms are related to alcohol dependence. Methods The subjects of this study consisted of 64 patients with alcohol dependence and 75 unrelated healthy people. The AUTS2 genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. Results No significant differences in the genotype and allele frequencies of the polymorphisms AUTS2 rs6943555 and rs9886351 were found between alcohol dependence and control subjects. On the other hand, the frequencies of the AUTS2 haplotypes were significantly different between them, and the rs6943555 and rs9886351 A-A haplotype was associated with alcohol dependence (p=0.0187). Conclusion This suggests that the rs6943555 and rs9886351 A-A haplotype might affect the vulnerability to alcohol dependence pathogenesis. Further studies are needed to confirm the reproducibility of the results of this study with increased numbers of subjects.

Gosha-Jinki-Gan Recovers Spermatogenesis in Mice with Busulfan-Induced Aspermatogenesis

Busulfan is an anti-cancer chemotherapeutic drug and is often used as conditioning regimens prior to bone marrow transplant for treatment of chronic myelogenous leukemia. Male infertility, including spermatogenesis disturbance, is known to be one of the side effects of anticancer drugs. While hormone preparations and vitamin preparations are used for spermatogenesis disturbance, their therapeutic effects are low. Some traditional herbal medicines have been administered to improve spermatogenesis. In the present study, we administered Gosha-jinki-gan (TJ107; Tsumura Co., Ltd., Tokyo, Japan) to mice suffering from severe aspermatogenesis after busulfan treatment to determine whether TJ107 can recover spermatogenesis. Male 4-week-old C57BL/6J mice were administered a single intraperitoneal injection of busulfan, and they were then fed a normal diet for 60 days and then a TJ107 diet or TJ107-free normal diet for another 60 days. After busulfan treatment, the weight of the testes and the epididymal sperm count progressively decreased in the normal diet group. On the other hand, in the TJ107 group, these variables dramatically recovered at 120 days. These results suggest that busulfan-induced aspermatogenesis is irreversible if appropriate treatment is not administered. Supplementation of TJ107 can completely recover the injured seminiferous epithelium via normalization of the macrophage migration and reduction of the expressions of Tool-like receptor (TLR) 2 and TLR4, suggesting that TJ107 has a therapeutic effect on busulfan-induced aspermatogenesis.

Detection of Protein-DNA Binding in Crude Nuclear Extract Using Biacore Assay

Biacore is a system that is extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. The practical procedures, however, for measurement of protein-DNA association in crude nuclear extract are yet to be defined. In the present study, DNA fragments with the least protein binding activity were identified in database for transcription factors and included in Biacore assay as control, so that the signals from non-specific binding were markedly suppressed. It was known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams showed exponential tendency. Further analysis showed that the interaction between ERα complex from crude nuclear extract and DNA oligos could be fitted to mono- or bi-exponential functions. Discrimination between orders of exponential function was based on the results of several statistical analyses with an average score of more than 95%. As exponential characteristics allow extrapolation of the dissociation, theoretical amount of bound anti-ERα antibodies could thus be evaluated statistically. Our procedures made Biacore a practical technique like Supershift Assay to measure protein-DNA association in crude nuclear extract with reproducible and reliable results.