Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.

STUDY QUESTION: What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? SUMMARY ANSWER: Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. WHAT IS KNOWN ALREADY: Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. STUDY DESIGN, SIZE, DURATION: Systematic mutation screening and genome-wide copy-number analysis of 62 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. LIMITATIONS, REASONS FOR CAUTION: The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. WIDER IMPLICATIONS OF THE FINDINGS: Our data provide evidence that pathogenic mutations can underlie both mild and severe hypospadias and that HSD3B2 mutations cause non-syndromic hypospadias as a sole clinical manifestation. Most importantly, this is the first report documenting possible oligogenicity of non-syndromic hypospadias. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; by the Grant-in-Aid from the Japan Society for the Promotion of Science; by the Grants from the Ministry of Health, Labour and Welfare, from the National Center for Child Health and Development and from the Takeda Foundation. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Lower urinary tract function in spinal cord-injured rats: midthoracic contusion versus transection.

OBJECTIVES: To compare changes in lower urinary tract (LUT) function with modifications in pathways that regulate LUT function using two different animal models (incomplete and complete) of spinal cord injury (SCI). METHODS: Female Sprague-Dawley rats were used. SCI was made at Th8/9 by a contusion injury (contusion, n=9) or a complete transection (transection, n=9). Unoperated rats were used as normal controls (normal, n=6). LUT function was evaluated by micturition behavior in metabolic cages for 24 h and cystometry in awake animals. Immunocytochemical staining at the L6 spinal cord, spinal areas associated with LUT, was performed to identify descending modulatory fibers and dorsal root afferents that project to the L6 spinal cord. RESULTS: Volume/micturition in metabolic cages gradually increased in both contusion and transection groups compared with normals, and operated groups did not differ from each other. Urodynamic parameters from cystometry were significantly different in contusion and transection groups compared with normals, but again there was no significant difference between contusion and transection groups. Immunocytochemical analyses at the L6 spinal cord showed no serotonergic or noradrenergic fibers in transection group, but some descending fibers remained in contusion group, indicating sparing. Small dorsal root afferents were denser in both contusion and transection groups than in normals, indicating sprouting. CONCLUSIONS: Although differences were not found in LUT function in operated animals, supraspinal and dorsal root projections to the L6 spinal cord responded differently to contusion and transection. This suggests that the benefits of pharmacologic treatments may be different in two lesion models.

Impact of laparoscopy for diagnosis and treatment in patients with disorders of sex development.

OBJECTIVE: To review laparoscopy in patients with disorders of sex development (DSD) in order to clarify its usefulness in diagnosis, devising subsequent therapeutic strategies and managing patients with various conditions. PATIENTS AND METHODS: Between April 1992 and December 2012, 29 laparoscopic surgeries were performed in 25 DSD patients. Among them, ten were diagnostic laparoscopy including gonadal biopsy, and 19 were therapeutic laparoscopy. Surgical procedures and complications were evaluated. RESULTS: For diagnostic laparoscopy, laparoscopic gonadal biopsy was performed in three patients. Inspection, with or without open gonadal biopsy, was performed on four out of seven patients with 46XY DSD or mixed gonadal dysgenesis (MGD). Additional surgery was planned and performed based on diagnostic laparoscopic findings in six out of seven patients. In the three patients with ovotesticular DSD, the gonadal pathology was diagnosed as: testis/ovary in one, testis/ovotestis in one and ovary/ovotestis in one--this was from the laparoscopic inspection and/or gonadal biopsy. However, the final diagnoses were bilateral ovotestis in two patients and ovary/ovotestis in one patient. For therapeutic laparoscopy, surgical procedures were: gonadectomy in 17 patients (bilateral in 13, unilateral in three, partial in two); hysterectomy in two patients; orchiopexy in one; and sigmoid vaginoplasty in one patient (included multiple procedures). There were no severe perioperative complications. In the four patients with a history of diagnostic laparoscopy, no severe intra-abdominal adhesions that would disturb therapeutic laparoscopic surgery were observed. CONCLUSION: While diagnostic laparoscopy was helpful in devising a therapeutic surgical strategy in most of the patients with DSD who were suspected as having complex gonadal status or Müllerian duct derivatives, attention must be paid to precisely diagnosing the gonadal status in ovotesticular DSD. On the other hand, therapeutic laparoscopic surgeries were valuable procedures in treating DSD patients, even with a history of previous diagnostic laparoscopy.

Preoperative renal scar as a risk factor of postoperative metabolic acidosis following ileocystoplasty in patients with neurogenic bladder.

OBJECTIVES: We investigated relation of preoperative renal scar to incidence of postoperative metabolic acidosis following ileocystoplasty in patients with neurogenic bladder. PATIENTS: Thirty patients with neurogenic bladder, who underwent ileocystoplasty, were enrolled in the present study. Median age at ileocystoplasty was 13.9 years and median follow-up period after ileocystoplasty was 8.2 years. Metabolic acidosis was defined based on the outlined criteria: base excess (BE) is less than 0 mmol l(-1). Preoperative examination revealed that no apparent renal insufficiency was identified in blood analysis, although preoperative (99m)Tc-DMSA scintigraphy indicated abnormalities such as renal scar in 14 patients (47%). Incidence of postoperative metabolic acidosis was compared between patients with and without preoperative renal scar, which may reflect some extent of renal tubular damage. RESULTS: Postoperative metabolic acidosis was identified in 13 patients (43%). Incidence of postoperative metabolic acidosis was significantly higher in patients with renal scar (11/14, 79%) compared with patients without renal scar (2/16, 13%; P<0.01). Particularly, all eight patients who had bilateral renal scars showed metabolic acidosis postoperatively. Compared with patients without preoperative renal scar, pH (P<0.05) and BE (P<0.01) were significantly lower postoperatively in patients with preoperative renal scar. However, there was no significant difference in PCO2. Hyperchloremia was observed in each patient with or without preoperative renal scar. CONCLUSION: Incidence of postoperative metabolic acidosis was significantly implicated in preoperative renal scar. If renal abnormalities are preoperatively identified in imaging tests, we need to care patients carefully regarding metabolic acidosis and subsequent comorbidities following ileocystoplasty.

Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis.

BACKGROUND: Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs. METHODS: TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using ß-aminopropionitrile (BAPN). RESULTS: LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model. CONCLUSION: LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.

Long-term outcome of single institutional experience with conservative and surgical management for renal artery aneurysm.

BACKGROUND: Spontaneous rupture risk of a renal artery aneurysm (RAA) is extremely low. Indications for surgical repair of RAA remain uncertain. OBJECTIVE: Long-term outcomes of conservative therapy and surgical repair were evaluated. PATIENTS: The study included 58 patients (17 males, 41 females) who were diagnosed with RAA during the last 21 years. Median age at the time of diagnosis was 62 (19-85) years, and the median follow-up 69 months (range 3-216). METHODS: The patients were divided into two groups, conservative group (n = 30) who had been followed with blood pressure control, and treatment group (n = 29), who underwent an intervention. RESULTS: Multiple efferent aneurysmal branches were observed in seven conservative and 16 treatment cases (P = .002). The median maximum diameter of the aneurysm was lower in the conservative than the treatment group (15 versus 25 mm, P = .005). Two conservative group cases showed increases in aneurysm size during follow-up. The hypertensive state showed essentially no change in either group during the follow-up. Renal function decreased with age similarly both in conservative and treatment groups. CONCLUSIONS: Our conservative management criteria for RAA are justifiable and even too strict.

Successful kidney transplantation ameliorates arterial stiffness in end-stage renal disease patients.

PURPOSE: Successful kidney transplantation (KTx) can ameliorate bodily damage caused by end-stage renal disease (ESRD). Arterial stiffness (AS) is one of the critical factors that shorten the survival of patients due to cardiovascular events. KTx may reduce AS as well; however, this has not been investigated well. We therefore conducted a retrospective study using noninvasive pulse wave velocity (PWV), which is a useful index of aortic damage. PATIENTS AND METHODS: Fifty-eight consecutive kidney recipients (34 men, 24 women) were enrolled in this study. Mean age at transplantation was 40.5 ± 12.3 years and the dialysis period was 73.1 ± 95.8 months. The brachial-ankle PWV was measured preoperatively and 6 months postoperatively. First, we investigated the relationship between the PWV and the other parameters related to AS. Second, we studied the pre- to posttransplant change in PWV to evaluate the amelioration of AS after successful KTx. RESULTS: PWV showed significant positive correlations with age, systolic blood pressure (BP), diastolic BP, and abdominal aortic calcification index. After successful KTx, PWV significantly decreased (P < .01). In addition, systolic and diastolic BP significantly decreased (P < .01 and P < .05, respectively). CONCLUSION: Successful KTx ameliorates AS in ESRD patients. This might explain the improved cardiovascular prognosis of ESRD patients who undergo KTx.

Prognostic significance of CD45RO+ memory T cells in renal cell carcinoma.

BACKGROUND: Memory T cells are well known to have a critical role for host defense in humans. However, their role in actual human cancer remains largely unknown. In this study, we tried to reveal the clinical importance of tumour-infiltrating CD45RO+ memory T cells in renal cell carcinoma (RCC). METHODS: We analysed 105 patients with RCC, who received radical or partial nephrectomy. Those were 65 in TNM stage I, 7 in stage II, 15 in stage III, and 18 in stage IV, respectively. CD45RO expression was evaluated by immunohistochemistry. CD4 and CD8 expressions were also systematically assessed in the same manner. RESULTS: Patients with higher TNM stage or high nuclear grade were found to have higher densities of CD45RO. Furthermore, CD45RO status was positively correlated with preoperative C-reactive protein level. In prognostic analysis, CD45RO+lo patients had a significantly better prognosis than CD45RO+hi patients. There was also a significant difference between CD4+lo and CD4+hi groups, whereas no significant difference was observed in CD8 T-cell status. Finally, multivariate analysis revealed that CD45RO+ status was the independent prognostic factor for patient overall survival. CONCLUSION: CD45RO+ memory T-cell status has a significant independent prognostic value, indicating that the adaptive immune response is functionally critical in human RCC.

The incidence and mechanism of sunitinib-induced thyroid atrophy in patients with metastatic renal cell carcinoma.

BACKGROUND: To elucidate the incidence and mechanisms of sunitinib-induced thyroid atrophy, we investigated serial volumetric and functional changes, and evaluated histological changes of the thyroid gland in metastatic renal cell carcinoma patients who received sunitinib. METHODS: Thyroid volume (by computed tomography volumetry) and thyroid function were measured at baseline, during the treatment, and at post-treatment periods. Histological evaluation of the thyroid gland was performed in four autopsied patients. RESULTS: The median reduction rate in thyroid volume at last evaluation during sunitinib treatment was 30% in all 17 patients. The incidence of hypothyroidism during sunitinib treatment was significantly higher in the high reduction rate group (n=8; more than 50% reduction in volume) than in the low reduction rate group (n=9; less than 50% reduction in volume). Half of the patients in the high reduction rate group exhibited a transient thyroid-stimulating hormone suppression, suggesting thyrotoxicosis during sunitinib treatment. Histological evaluation demonstrated atrophy of thyroid follicles and degeneration of follicular epithelial cells without critical diminution of vascular volume in the thyroid gland. CONCLUSION: Thyroid atrophy is frequently observed following sunitinib treatment and may be brought about by sunitinib-induced thyrotoxicosis or the direct effects of sunitinib that lead to degeneration of thyroid follicular cells.

Role of lymph node dissection in the treatment of urothelial carcinoma of the upper urinary tract: multi-institutional relapse analysis and immunohistochemical re-evaluation of negative lymph nodes.

AIM: To determine the role of lymph node dissection (LND) in the treatment of urothelial carcinoma (UC) of the upper urinary tract (UUT). PATIENTS AND METHODS: [Study-1] A retrospective multi-institutional study evaluated 293 patients undergoing predominantly nephroureterectomy for UC of the UUT. Of 293 patients, 267 patients had pure UC and 26 demonstrated other histological components. Regarding the pathological node status, 130 patients had pN0 disease, 141 patients had pNx disease and 22 patients had pN+ disease. The sites of initial recurrence and time to first recurrence were reviewed. The sites of recurrence were classified as locoregional or distant recurrence. The relationship between node status and future recurrence was analyzed. [Study-2] Fifty-one patients treated by nephroureterectomy at Hokkaido University Hospital were included. All had LND and all LNs were negative on hematoxylin and eosin staining. We re-evaluated the presence of micrometastasis in LND specimens by anti-cytokeratin immunohistochemistory. RESULTS: [Study-1] Of 293 patients, 76 developed disease relapse. Regional lymph node recurrence was the most common site (34 patients). On multivariate analyses that adjusted for the effect of tumor stage and tumor grade, pNx (skipping LND) was an adverse factor not only for locoregional recurrence, but also for distant relapse. [Study-2] Immunohistochemistry identified micrometastases in 7 (14%) of 51 patients. Regarding survival, 5 of these 7 patients with micrometastases were alive at last follow-up. CONCLUSIONS: On relapse analysis, skipping LND was an adverse factor not only for locoregional recurrence, but also for distant relapse. Immunohistochemistry detected micrometastases in about 14% of patients previously diagnosed as pN0. These findings further support a potential therapeutic benefit of LND by eliminating micrometastases.

Purified eicosapentaenoic acid induces prolonged survival of cardiac allografts and generates regulatory T cells.

Fish oil, which is rich in eicosapentaenoic acid (EPA), has been found to have immunomodulatory effects. We examined whether administration of purified EPA affected survival of fully mismatched murine cardiac allografts. Hearts from C57BL/10 (H-2(b)) mice were transplanted into CBA (H-2(k)) recipients treated with one intraperitoneal dose of purified EPA the day of transplantation. Untreated CBA recipients and recipients given 0.1 g/kg of EPA rejected C57BL/10 hearts (median survival time [MST], 8 and 13 days, respectively). With a 1.0 g/kg dose of EPA, graft survival was markedly prolonged (MST >100 days). To determine whether regulatory cells were generated, naïve mice (secondary recipients) underwent adoptive transfer of splenocytes from EPA-treated primary recipients and cardiac allograft transplantation. Adoptive transfer of whole, CD4(+) and CD4(+)CD25(+) splenocytes from EPA-treated recipients induced indefinite survival in secondary recipients. Flow cytometry showed that the CD4(+)CD25(+) cells were Foxp3(+). In reverse transcriptase-polymerase chain reaction (RT-PCR) studies, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA was upregulated by EPA treatment. A PPARgamma antagonist abrogated the prolongation of graft survival induced by EPA treatment (MST, 13 days). Thus, in our model, purified EPA induced prolonged survival of fully mismatched cardiac allografts and generated regulatory T cells dependent on PPARgamma activation.

Outcomes of lower urinary and bowel function in meningomyelocele patients with augmentation enterocystoplasty.

OBJECTIVES: We evaluated the outcomes of augmentation ileocystoplasty in video-urodynamics (V-UDS) and questionnaires related to bowel and urinary function. METHODS: In 22 meningomyelocele (MMC) patients (11 male and 11 female, a mean age at surgery 14.4 years) who underwent augmentation ileocystoplasties, V-UDS was performed before and at a median of 15 months (6-120 months) after the surgery and the questionnaires were answered at a median of 7.1 years (1.1-14.5 years) after surgery. RESULTS: Concomitant procedures of urethral sling in four, cecostomy in four and ureteral reimplantation in two were performed at augmentation ileocystoplasty. V-UDS showed a significant improvement after the surgery in bladder capacity, bladder compliance and detrusor overactivity. Vesicoureteral reflux (VUR) disappeared after the surgery in 16 of 19 ureters with VUR. Although questionnaire for urinary condition using International Consultation on Incontinence Questionnaire-Short Form demonstrated that 80% of patients were completely free from urinary incontinence after surgery, more than a half of patients had persistent or deteriorated bowel function such as urgency of bowel movement and fecal incontinence in questionnaires for bowel function. Even with complaints in bowel function, more than 80% of MMC patients had a high level of satisfaction with augmentation enterocystoplasty in overall quality of life (QOL). CONCLUSIONS: Benefits of augmentation ileocystoplasty were maintained in urodynamic parameters as well as urinary continence. Although augmentation ileocystoplasty generally provides high QOL for MMC patients refractory to conservative therapies, an advance of fecal management can make MMC patients' QOL superior to a current state.

Dependence of the photoelectrochemical performance of sensitised ZnO on the crystalline orientation in electrodeposited ZnO thin films.

The influence of the crystal orientation in porous crystalline films of ZnO electrodeposited on the photoelectrochemical characteristics of the films is studied. For differently oriented ZnO thin films following removal of the respective structure-directing agent (SDA) and adsorption of a sensitiser, time-resolved photocurrent measurements, intensity modulated photocurrent spectroscopy (IMPS), intensity modulated photovoltage spectroscopy (IMVS) and current-voltage curves were measured in acetonitrile-based electrolytes containing I(3)(-)/I(-) as the redox electrolyte. The crystal orientation has a significant influence on the charge transport across such films and hence is reflected in the observed electrode kinetics. Films originally grown in the presence of, e.g., Coumarin 343 as a SDA, showed a significantly faster response to illumination. Increased electron diffusion coefficients and diffusion lengths were calculated from the results of IMPS and IMVS, caused by a faster electron movement in the films. Implications of these findings on further improvements of sensitised ZnO films prepared by electrochemical deposition are discussed.

Photoelectrochemical characterisation and optimisation of electrodeposited ZnO thin films sensitised by porphyrins and phthalocyanines.

Hybrid thin films of crystalline ZnO modified by 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato zinc (TSTPPZn) and 2,9,16,23-tetrasulfophthalocyaninatozinc(II) (TSPcZn) were prepared by electrochemical deposition from aqueous zinc salt solutions. A "one-step" process with the sensitisers adsorbed during ZnO deposition represented the most simple approach. ZnO was also grown independently in the presence of Eosin Y as a structure-directing agent, which was then removed and the sensitisers were chemisorbed from solutions ("readsorption" method). The photoelectrochemical characteristics of the electrodes were studied by photocurrent spectra and by time-resolved photocurrent measurements in an acetonitrile-based solution containing I3(-)/I(-) as the redox electrolyte. In films with both sensitisers present, both sensitisers worked in parallel providing panchromatic sensitisation. Recombination of electrons injected into the conduction band of ZnO with remaining holes in the HOMO of the sensitisers was indicated for the one-step films, but was considerably suppressed for the films prepared by the readsorption method. Films prepared by the readsorption method showed a significantly increased efficiency by an increased surface area and suppressed recombination.

Maternal genetic polymorphisms in CYP1A1, GSTM1 and GSTT1 and the risk of hypospadias.

Hypospadias is one of the most common congenital anomalies. Increased exposure to environmental factors (endocrine-disrupting chemicals and smoking) or maternal endogenous estrogen may cause hypospadias because male sexual differentiation is dependent on normal androgen homeostasis. Moreover, interactions between genetic factors and cigarette smoking and other chemicals have been suggested. It has been demonstrated that the CYP1A1 metabolizes not only environmental chemicals but also estrogens, and glutathione-S-transferases (GSTs) are detoxification enzymes that protect cells from toxicants by conjugation with glutathione. In this study, to investigate the association of CYP1A1 (MspI), GSTM1 and GSTT1 polymorphisms with hypospadias, a case-control study of 31 case mothers who had boys with hypospadias and 64 control mothers was performed in Japan. These polymorphisms were investigated by PCR-based methods using DNA from peripheral lymphocytes. We found that the heterozygous CYP1A1 and heterozygous and homozygous CYP1A1 were less frequent in the case mothers than in the control mothers [adjusted odds ratio (OR)=0.17, 95% confidence interval (CI)=0.04-0.74, OR = 0.28, 95% CI = 0.08-0.97, respectively]. We found no effect of maternal smoking on the hypospadias risks among the gene polymorphisms. The results suggest that mothers with the CYP1A1 MspI variant allele may have a decreased risk for hypospadias.

Effect of tacrolimus and cyclosporine on renal microcirculation and nitric oxide production.

OBJECTIVES: Cyclosporine (CSA) and tacrolimus (TAC) frequently induce nephrotoxicity and similar pathologic changes. Acute CSA-induced nephrotoxicity has been reported to be mediated by activation of vasoconstrictors such as endothelin. The purpose of the present study was to investigate the acute effects of TAC and CSA on the renal microcirculation and upon a vasodilator such as nitric oxide (NO) production. METHODS: Renal blood flow (RBF) in the microcirculation was measured by a Laser Doppler flow meter in uninephrectomised rats. RBF, mean arterial pressure (MAP), and renal vascular resistance (RVR) were measured in the following groups: (a) TAC (0.1 to 2.0 mg/kg/h, n = 3 approximately 6); CSA (20 and 50 mg/kg/h, n = 5); (b) L-NAME (10 mg/kg), an NO synthase inhibitor, 8 minutes prior to TAC (0.5 and 1.5 mg/kg/h, n = 5), or CSA (20 and 50 mg/kg/h, n = 5). Stable NO end-products, serum NO(2) and NO(3), were measured by the Griess method (n = 5). RESULTS: None of the parameters were changed by TAC alone, whereas TAC with L-NAME significantly reduced RBF (-28 +/- 7%) and increased RVR (46 +/- 17%) in a dose-dependent manner. CSA alone significantly reduced RBF (-37 +/- 6%) and increased RVR (69 +/- 22%) without any changes in MAP. The effects of CSA were enhanced by L-NAME. Serum concentration of NO(2) + NO(3) was significantly reduced by both L-NAME alone and CSA (50 mg/kg) (P < .05), while there were no changes with TAC (1.5 mg/kg). CONCLUSIONS: Blockade of NO production enhance the vasoconstrictive effect of CSA, and unmasked such an effect of TAC. These results suggest that the nephrotoxicity of CSA and TAC may involve the NO system.

An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis.

To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.

Diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type II diabetic rat model: a possible cause for erectile dysfunction in diabetes.

Erectile dysfunction (ED) is commonly experienced in men with diabetes mellitus. Vascular endothelial growth factor (VEGF) has been extensively documented for its pathogenic significance in different complications of diabetes. We hypothesized that expressions of VEGF, its receptors and its signaling pathway Akt may be drastically altered in diabetic penile tIssues and their alterations may modulate penile expression of the molecules that are believed to play a role in diabetic ED. Otsuka Long-Evans Fatty (OLETF) rats, a type II (non-insulin-dependent) diabetes mellitus, were used at the insulin-resistant stage of type II diabetes (20 weeks of age). We determined protein and mRNA expressions of VEGF, its receptors, Akt, nitric oxide synthase isoforms, and apoptosis-related molecules in the penis using immunohistochemistry, Western blotting, in situ hybridization, and real-time quantitative PCR analyses. The penile sections were also submitted to the Tdt-mediated dUTP nick end labeling assay for apoptosis. OLETF rats showed marked reductions in penile expression of VEGF, its two receptors and Akt. In OLETF rat penises, endothelial and neuronal nitric oxide synthase isoforms were expressed less abundantly. Furthermore, while anti-apoptotic markers, Bcl-2 and phosphorylated Bad, were down-regulated, pro-apoptotic markers, active caspase-3 and Bax, were up-regulated, resulting in the appearance of apoptotic cells in the penile tIssues of OLETF rats. The VEGF signaling system would work less well in diabetic penile tIssues as a result of the reduced expression, leading to diminished endothelial production of nitric oxide and apoptosis-related erectile tIssue damage. We propose that the abnormalities of the VEGF signaling system in the penis may play a role in the pathophysiology of diabetic ED.