Next-generation sequencing for patients with non-obstructive azoospermia: implications for significant roles of monogenic/oligogenic mutations.

Azoospermia affects up to 1% of adult men. Non-obstructive azoospermia is a multifactorial disorder whose molecular basis remains largely unknown. To date, mutations in several genes and multiple submicroscopic copy-number variations (CNVs) have been identified in patients with non-obstructive azoospermia. The aim of this study was to clarify the contribution of nucleotide substitutions in known causative genes and submicroscopic CNVs in the genome to the development of non-obstructive azoospermia. To this end, we conducted sequence analysis of 25 known disease-associated genes using next-generation sequencing and genome-wide copy-number analysis using array-based comparative genomic hybridization. We studied 40 Japanese patients with idiopathic non-obstructive azoospermia. Functional significance of molecular alterations was assessed by in silico analyses. As a result, we identified four putative pathogenic mutations, four rare polymorphisms possibly associated with disease risk, and four probable neutral variants in 10 patients. These sequence alterations included a heterozygous splice site mutation in SOHLH1 and a hemizygous missense substitution in TEX11, which have been reported as causes of non-obstructive azoospermia. Copy-number analysis detected five X chromosomal or autosomal CNVs of unknown clinical significance, in addition to one known pathogenic Y chromosomal microduplication. Five patients carried multiple molecular alterations. The results indicate that monogenic and oligogenic mutations, including those in SOHLH1 and TEX11, account for more than 10% of cases of idiopathic non-obstructive azoospermia. Furthermore, this study suggests possible contributions of substitutions in various genes as well as submicroscopic CNVs on the X chromosome and autosomes to non-obstructive azoospermia, which require further validation.

[Natural history of unruptured cerebral aneurysms of the unoperated and observed cases].

PURPOSE: Natural history of unruptured cerebral aneurysms is still a matter of discussion. In this study, we investigated the prognosis of unruptured cerebral aneurysms of unoperated cases in a prospective design. METHODS: Between September, 1992 and December, 2001, we have encountered a 256 cases of unruptured cerebral aneurysms. Among them, 118 cases were observed and were checked every year for their status. The endpoint was designed as their death and aneurysm rupture. Their rupture rate, mortality due to aneurysm death, and the cause of death other than aneurysm were investigated. Univariate analysis, chi-square test was used as statistics. A p-value less than 0.05 was considered as significant. RESULTS: Annual rupture rate of unoperated unruptured cerebral aneurysms of size below 5 mm, between 5-15 mm, and over 15 mm increased according to the aneurysm size, 0.4%, 3.3% and 9.9% respectively. The sole risk factor for the feasibility of rupture of unruptured aneurysms was their size (p < 0.001). Aneurysm related mortality, however, was high in posterior circulation aneurysms. In patients under 70 years of age, 45% of patients died of cerebral aneurysms, but this rate decreased to 17% for patients over 70 years of age. CONCLUSION: The rupture rate of unruptured cerebral aneurysms over 5 mm in size is not low. Unruptured aneurysms of the posterior circulation may have a much higher risk of rupture, so further investigation is necessary.

Identity of Cypripedium calceolus (Orchidaceae) in Rebun Island: comparative DNA analysis of related species.

Cypripedium calceolus was found in 1980 in Rebun Island off the north coast of Hokkaido, Japan, but the origin of this plant has been a controversial issue. In this study, we have made a comparative study by chloroplast DNA sequencing analysis among C. calceolus which occurs in Rebun Island and populations of C. calceolus from western Europe, China and far eastern Russia (Nakhodka), and also as references, C. macranthos in Japan and other Cypripedium species in North America. A Cypripedium cf. "calceolus", found recently in eastern Hokkaido, was also included in this analysis. The C. calceolus samples analyzed were categorized into three groups, i.e., those from Western Europe, from China and far eastern Russia, and from Rebun Island. The C. calceolus in Rebun Island was clearly different from the others in terms of DNA sequence and morphological features. The C. cf. calceolus from eastern Hokkaido and one sample from Nadhodka, Russia, were also classified into the same group as those from Rebun Island, although some differences in their morphological features were observed. It is concluded that the C. calceolus found in Rebun Island is not identical with those growing in Europe and China. In addition, it was found that it may be possible to classify C. macranthos into two groups, namely groups which include or do not include var. rebunense. An unidentified Cypripedium species found in Rebun Island falls into the same group as var. rebunense.

Triiodobenzoic acid, an auxin polar transport inhibitor, suppresses somatic embryo formation and postembryonic shoot/root development in Eleutherococcus senticosus.

The effect of auxin polar transport inhibitor on somatic embryo development and postembryonic growth in Siberian ginseng (Eleutherococcus senticosus) was examined. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor, embryo formation from embryogenic cells was suppressed, while cell division was not affected. When globular embryos at different stages were transferred onto medium containing TIBA, development of axial and bilateral polarity was suppressed in a stagespecific manner. In abnormal embryos induced by TIBA, further development of shoot and root apical meristems and vascular differentiation was also suppressed. Thus, abnormal development of embryos induced by inhibition of auxin polar transport resulted in plantlets without shoots and roots.

[A case of 9p-syndrome identified chromosome 20 on chromosome 9p by M-FISH].

In this report, we describe one-year-old girl diagnosed with 9p-syndrome. Cytogenetic studies of this patient confirmed a karyotype of 46,XX,add(9) (p24) chromosome, but could not find the additional fragment on 9p22 in one allele. Fluorescence in situ hybridization (FISH) studies could not confirm the fragment in the patient using the LIS1 gene probe which mapped to 9p22. The more recently developed M-FISH method clearly showed that the additional fragment was 20p in this patient. These findings suggest that M-FISH analysis may be a useful method for identifying unknown additional and rearranged chromosomes.

Rice gibberellin-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level.

This paper reports isolation and properties of a rice gene, OsGAI, a putative homolog of the GAI of Arabidopsis thaliana. OsGAI encodes a polypeptide of 625 amino acids, which shows 53-55% identity to GAI and RGA from A. thaliana, and 85% identity to wheat rht-D1a and maize d8. Genomic DNA blot analysis indicated the OsGAI to be a single-copy gene in the rice genome. RNA blot hybridization showed that OsGAI transcripts increased within 6h upon GA(3) but not ABA application. This GA-induced increment in OsGAI transcripts did not require de novo protein synthesis. High levels of OsGAI transcripts were detected in nodes, internodes, leaf sheaths and ears of adult plants and leaf sheaths of young seedlings, where GA enhances cell elongation and division. Transiently expressed OsGAI-GFP fusion protein located to the nucleus in onion epidermal cells. Transactivation assays clearly indicated that OsGAI protein is a transcriptional activator or a coactivator.

Gibberellin-responsive genes: high level of transcript accumulation in leaf sheath meristematic tissue from Zea mays L.

In order to identify genes that are related to the gibberellin (GA) response in maize (Zea mays L.), mRNA species from wild-type and single-gene dwarf mutants, d5 and D8, were compared by fluorescent differential display. The d5 mutant is unable to produce biologically active GA, but recovers its tall stature on exogenous application of GA. D8 is insensitive to GA, despite the accumulation of a high level of endogenous GA, suggesting it to be a receptor mutant or a mutant in signal transduction pathway(s). After screening 7000 cDNA populations, one clone was isolated, for which transcripts were rare in d5 shoots but accumulated within 1 h after GA3 application. This clone, designated as ZmGR1a, encodes a polypeptide with a relative molecular mass of ca. 13 kDa, which shows significant homology to proline-rich proteins from several plant species. A similar experiment with D8 identified a clone, ZmGR2a, with low transcript levels, but accumulation within 6 h after GA3 treatment of d5 shoots. ZmGR2a encodes a polypeptide with a relative molecular mass of ca. 19 kDa, which shows no significant homology with any known protein. Southern blot analysis indicated that ZmGR1a and ZmGR2a form a small multigene family within the maize genome. In situ hybridization with wild-type seedlings showed transcripts on both to be abundant in leaf sheath meristematic tissue, in which GA enhances cell elongation and cell division.

Definitive radiotherapy combined with high-dose-rate brachytherapy for Stage III carcinoma of the uterine cervix: retrospective analysis of prognostic factors concerning patient characteristics and treatment parameters.

PURPOSE: To assess treatment outcome after definitive radiotherapy for Stage III cervical carcinoma retrospective analysis of the clinical data was performed. METHODS AND MATERIALS: We enrolled 265 patients with Stage III cervical carcinoma who were treated with combined external beam radiotherapy [mean +/- standard deviation (SD), 50.3 +/- 1.8 Gy; range 40-70) and intracavitary brachytherapy (ICBT) (mean +/- SD, 19.8 +/- 2.4 Gy; range 10-32) using a high-dose-rate 60Co source. We retrospectively analyzed, as measures of the therapeutic outcome, the overall survival rate (OAS), relapse-free survival rate (RFS), locoregional event-free rate (LREF), distant metastasis (DM), and late complication. RESULTS: The 5-year OAS, RFS, and LREF rates (n = 265) were 50.7%, 57.1%, and 71.2%, respectively. Univariate analysis revealed a significant favorable effect on the OAS rate for complete response (CR) (p = 0.024), maximum tumor diameter < 6 cm (SML) (p = 0.0009), Karnofsky performance score > or =70 (p = 0.046), age <75 years (p = 0.0043), hemoglobin (Hb) concentration of > or =9 g/dl (p = 0.0005), and histopathological diagnosis of squamous cell carcinoma (SCC) (p = 0.0089). In the multivariate analysis, Hb, SCC, SML, and CR remained significant prognostic factors. In both univariate and multivariate analysis, SML, Hb, age, and SCC showed significant effects on the RFS rate. Age > or =60 years (p = 0.017), Hb > or =9 g/dl (p = 0.0039), and SML (p = 0.0046) were significant favorable prognostic factors for the LREF rate identified by univariate analysis. In addition, advanced age, SML, and SCC showed significant beneficial effects on the LREF rate in the multivariate analysis. DM developed in 21.1% of patients, and the groups with Hb < 9 g/dl (p < 0.005), Karnofsky performance score of <70 (p < 0.001), and dose at point A in the ICBT < 16 Gy (p < 0.005) developed a significantly greater incidence of DM than did the groups without. The 5-year incidence was 2.6% for major bladder complication and 8.3% for major rectal complication. The radiation dose in the subgroup with rectal complication was significantly greater than that in the subgroup without complications. CONCLUSION: In patients with Stage III cervical carcinoma, tumor size, concentration of Hb, and histopathological diagnosis are strong prognostic factors for the therapeutic outcome. The ICBT dose in our study was small compared to the reported data, but the outcome was quite comparable. Thus, the dose we used might be the minimum required. However, locoregional control still remains an important problem. It is necessary to attain the optimal dose distribution in both the target volume and in critical organs. In high-risk patients, combined use of chemotherapy is crucial to improve the response to radiation.

Biochemical and serological examination of beta-hemolytic streptococci isolated from slaughtered pigs.

A total of 170 beta-hemolytic streptococci isolated from lesions in slaughtered pigs during 1988 to 1995 were identified by biochemical and serological examinations. Of these, 132 strains (77.6%) were Streptococcus (S.) dysgalactiae and 38 strains (22.4%) were S. porcinus. The largest serological group of streptococci was group C (78 strains, 45.9%), followed by group L (43 strains, 25.3%), group U (14 strains, 8.2%), group G (11 strains, 6.5%), group E (5 strains, 2.9%), and group P (5 strains, 2.9%). Most of isolates from endocarditis (61 strains) and arthritis (25 strains) were group C S. dysgalactiae, but about 33.3% of the isolates from lymphadenitis were group L S. dysgalactiae (28 strains), followed by group C (14 strains, 16.7%), group U S. porcinus (14 strains, 14.3%), and group G (10 strains, 11.9%).

Bacterial isolation from slaughtered pigs associated with endocarditis, especially the isolation of Streptococcus suis.

Bacterial isolation from slaughtered pigs with endocarditis was carried out from 1985 to 1994. A total of 495 (0.025%) out of 2,006,127 pigs were diagnosed as having endocarditis. Though bacteria were significantly isolated from 399 of the 495 pigs, bacteria could not be isolated in 96 pigs (19.4%). In 11 pigs, 2 bacterial species were isolated from heart lesion. Streptococcus suis was isolated from 127 cases (25.7%), Streptococcus dysgalactiae from 75 (15.2%), Erysipelothrix rhusiopathiae from 63 (12.7%), Actinomyces pyogenes from 39 (7.9%), Pasteurella multocida from 11 (2.2%). Staphylococcus aureus from 10 (2.0%), and Streptococcus porcinus from 8 (1.6%). Among the 99 isolates biochemically identified as S. suis, the major serotype was S. suis type 2 (35.4%). The remainder of the typable isolates were identified as serotypes 1/2 (2.0%) and 9 (1.0%). A total of 61 isolates (61.6%) were untypable.

Comparative preparation methods of sialylated capsule antigen from Streptococcus suis type 2 with type specific antigenicity.

The capsular polysaccharide (CPS) of Streptococcus (S.) suis type 2 was isolated from a type strain of S. suis NCTC 10234 by three different preparative methods: (A) lysozyme treatment method, (B) autoclave extraction method, and (C) HCl-extraction method. The structural characteristics of the three CPS (CPS-A, B and C) were examined by gel permeation chromatography, reactivity against rabbit antiserum and proton-nuclear magnetic resonance (1H-NMR). N-Acetylneuraminic acid (NeuAc) residues as sialic acid in CPS-C were partially dissociated or degraded during preparation with a remarkable decrease in the molecular mass and the antigen activity. Although both methods A and B produced intact CPS without releasing NeuAc residues, method B was considered to be a more suitable procedure for preparing the CPS antigen because of time-saving and safety factors. Sugar analysis by high performance liquid chromatography and gas liquid chromatography showed that CPS-B consisted of five kinds of sugars: Rhamnose (Rha), Glucose (Glc). Galactose (Gal), N-acetylglucosamine (GlcNAc) and NeuAc, in a molar ratio of 1.00:0.95:3.68:0.80:1.31. After complete removal of NeuAc residues by mild acid hydrolysis of CPS-B, the reactivity with anti-type 2 serum was not detected. The NeuAc residue in CPS of S. suis type 2 strain was thought to be the antigen epitope portion.

Typing of Rhodococcus equi isolated from submaxillary lymph nodes of pigs in Japan.

Isolation of Rhodococcus equi from the submaxillary lymph nodes of pigs, with or without caseous lymphadenitis, and typing of the isolates by two serological methods were carried out. The rate of isolation of the organisms from the lymph nodes of pigs was 5 times higher in the lymph nodes with caseous lymphadenitis than in those without the lesion. Of 219 isolates, 146 (66.7%) were typable by the method of Prescott, while all the 219 isolates (100%) were typable by the method of Nakazawa et al. The most frequently isolated were serotype 2 of Prescott (identical to serogroup 16 of Nakazawa et al.), and serogroup 3 of Nakazawa et al., which did not correspond with any serotypes of Prescott. Serotypes/serogroups of R. equi from pigs were thus first clarified in Japan.

[Clinical effect of combined chemotherapy with UFT, MMC and OK-432 in recurring or advanced cancer of the digestive organs].

The usefulness of UFTMO therapy (combined chemotherapy with UFT, MMC and OK-432) performed in 40 cases of recurring or advanced cancer of the digestive organs was investigated. According the response criteria by Koyama et al., of 40 eligible cases, the treatment was judged effective in 13, 2 CR and 11 PR cases with a response rate of 32.5%, while of the 35 complete cases, 2 CR and 9 PR cases made for 11 effective cases and a response rate of 31.4%. Side effects were observed in 58.3% of the 36 evaluated cases; of the subjective and objective side effects, however, none were serious enough to require cessation of administration, while stopping administration in the cases of abnormal laboratory findings resulted in rapid recovery. UFTMO therapy, therefore, is considered to be one of the beneficial treatments for recurring or advanced cancer of the digestive organs.

The dominant non-gibberellin-responding dwarf mutant (D8) of maize accumulates native gibberellins.

The endogenous gibberellins (GAs) were examined from young vegetative shoots of the dominant mutant, Dwarf-8, a GA-nonresponder, and normal maize; GA(44), GA(17), GA(19), GA(20), GA(29), GA(1), and GA(8), members of the early-13-hydroxylation pathway, were identified from both kinds of shoots by full-scan mass spectra and Kovats retention indices. In addition, we report the identification of 3-epi-GA(1), GA(3), GA(4), GA(5), GA(7), GA(9), GA(12), GA(15), GA(24), GA(34), and GA(53) by using the same criteria. [1,7,12,18-(14)C(4)]GA(53) and -GA(44), [17-(2)H(2)]GA(19), and [17-(13)C,(3)H(2)]GA(20), -GA(29), -GA(1), -GA(8), and -GA(5) were used as internal standards to determine the endogenous levels of these GAs by measurement of isotope dilution, using capillary gas chromatography and selected ion monitoring. Shoots of Dwarf-8 accumulate relatively high levels of GA(20), GA(1), and GA(8). The accumulation of GA(1) appears to be related to gene dosage. Since Dwarf-8 contains the same pattern of GAs as normals (including GA(1) and GA(3)), the genetic control point probably lies after GA(1) (and GA(3)). Thus Dwarf-8 may be a GA receptor mutant or a mutant that controls a product downstream from the binding of the bioactive GA to a receptor.

[5-FU concentration in tumor tissue and the antitumor effect in patients with gastric cancer after oral administration of UFT].

Serum and tumor tissue concentration of FT, 5-FU and uracil were measured in 23 patients with gastric cancer who were administered UFT preoperatively. The degeneration of cancer cells was evaluated histologically and the correlation between 5-FU concentration in the tumor tissue and the antitumor effect was assessed. The average concentration of 5-FU in tumor tissue (0.122 microgram/g) was significantly higher than that in normal gastric tissues. Serum 5-FU concentration was very low, suggesting no accumulation of 5-FU in blood. A positive correlation between the concentration of 5-FU and uracil in tumor tissues was found. A 5-FU level higher than 0.05 microgram/g was frequently demonstrated in tumor tissue, resulting in moderate degeneration of cancer cells in many cases. When tumors were classified according to histological type, intratumoral 5-FU concentration was not always correlated with degeneration of cancer cells. The above results suggested that UFT was a very effective drug for stomach cancer because of high tumor affinity for 5-FU, but that it is necessary to consider the sensitivity of tumor cells to antitumor drugs in order to obtain an excellent antitumor effect.

Induction of activated natural killer cells from murine spleen cells primed in vivo and subsequently challenged in vitro with the streptococcal preparation OK432.

The present study shows that natural killer cell-mediated cytotoxicity of BALB/c mouse spleen cells to syngeneic tumor cells was augmented by in vivo priming or in vitro stimulation with the streptococcal preparation OK432. The augmentation of spleen cell cytotoxicity to syngeneic tumor cells by in vivo priming alone with OK432 was lower than that obtained by in vitro stimulation alone with OK432. When the murine spleen cells primed in vivo with OK432 were rechallenged in vitro with OK432 at various intervals, the natural cytotoxicity was more strongly enhanced than that seen with in vitro stimulation alone. The cell surface phenotype of killer cells activated with OK432 was Thy 1+ and asialo GM1+, suggesting the activated natural killer cell. Next, mice were transplanted with syngeneic colon adenocarcinoma cells, and primed in vivo with OK432. These spleen cells were subsequently challenged in vitro with OK432. These spleen cells displayed a strong cytotoxic activity not only to the transplanted adenocarcinoma cells but also to other syngeneic tumor cells.