Ovary and ovulation: Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles

To study the relationship between follicular atresia, apoptosis,and nitric oxide (NO) generation in follicular development,steroidogenesis, NO levels in follicular fluid and apoptosiswere analysed in the various sized follicles of women receivingovarian stimulation with human meno-pausal gonadotrophin (HMG)—humanchorionic gonado-trophin (HCG) treatments for in-vitro fertilization(IVF)-embryo transfer. The follicles were divided into threegroups by diameter: large follicle, 18 mm; medium follicle,12 and 15 mm; small follicle, 10 mm. Follicular fluid was obtainedfrom 20 women 34 h after HCG administration, and the concentrationsof oestradiol, progesterone and testosterone, and nitrite, nitrate,arginine and citrulline were measured. Granulosa cells obtainedfrom each group of follicular fluid were stained with Hoechstdye, and nuclear morphology was examined by a fluorescence microscopy.Oestradiol and progesterone concentrations in large follicleswere significantly (P < 0.01) higher than those in mediumor small follicles, and testosterone concentrations in smallfollicles were significantly (P < 0.01) higher than thosein large follicles. There were no significant differences inthe concentrations of nitrite, nitrate, arginine and citrullineamong three groups. The percentage of apoptotic cells with nuclearfragmentation was significantly (P < 0.01) higher in smallfollicles than in large follicles. The present results suggestedthat small follicles with poor response to HMG may undergo atresiathrough apoptosis. No significant difference in the follicularNO level between large and small follicles led us to speculateon a different responsiveness to NO in these two types of follicles.     

Expression and function of Fas antigen on activated murine B cells

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.     

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.     

Characterization of adenocarcinoma of the lung in a familial adenomatous polyposis patient

The incidence of several extracolonic tumors, such as duodenal carcinoma, is higher in familial adenomatous polyposis (FAP) patients than in the general population, but there is little information about lung carcinoma in FAP. A 43-year-old woman presented with a lung tumor 17 years after total colectomy for FAP. Pathohistological analysis of the lung tumor demonstrated mixed adenocarcinoma consisting of a papillary adenocarcinoma component and a bronchioloalveolar carcinoma component. Sequencing analysis indicated a germline APC mutation from TCA to TGA (stop) at codon 1110, but no pathogenic germline MYH mutations. The other APC allele in the lung carcinoma was not inactivated by somatic mutations, promoter methylation, or chromosomal deletion. No somatic mutations in any of the coding regions of the p53 gene or in the mutation hot spot regions of the K-ras or EGFR genes were detected in the carcinoma. Amplification, however, of three chromosome regions, 5p, 8q, and 12q14-12q21, was identified in the carcinoma on genome-wide high-resolution single-nucleotide polymorphism (SNP) microarray. The present results suggest that the chromosomal copy number alterations detected on SNP microarray were involved in the carcinogenesis of the adenocarcinoma of the lung in the present FAP patient.     

Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection

The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre‐S1 84 (P = 0.042), pre‐S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log‐rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre‐S1 and pre‐S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection. J. Med. Virol. 84:1360–1368, 2012. © 2012 Wiley Periodicals, Inc.     

A Novel Characterization Method for Graft‐Polymer Structures Chemically Attached on Thermally Stable Polymer Films

Most polymer grafts in grafted polymer films obtained by radiation‐induced solid graft polymerization are not analyzed in detail due to difficulties in isolation of the grafts without structural decomposition. Herein, a novel structural and molecular weight characterization method is reported for polymer grafts that are chemically attached to thermally and chemically stable polymer substrates, based on a swelling‐induced graft detachment in hot water. Polymer grafts prepared by the radiation‐induced polymerization of alkyl acrylate into poly(ethylene‐co‐tetrafluoroethylene) (ETFE) followed by a sulfonation reaction are found to have a ternary copolymer structure whose monomer units contain –COOH or –SO₃H groups, or both.     

Tissue-specific functions based on information content of gene ontology using cap analysis gene expression

Gene expressions differ depending on tissue types and developmental stages. Analyzing how each gene is expressed is thus important. One way of analyzing gene expression patterns is to identify tissue-specific functions. This is useful for understanding how vital activities are performed. DNA microarray has been widely used to observe gene expressions exhaustively. However, comparing the expression value of a gene to that of other genes is impossible, as the gene expression value of a condition is measured as a proportion of that for the same gene under a control condition. We therefore could not determine whether one gene is more expressed than other genes. Cap analysis gene expression (CAGE) allows high-throughput analysis of gene expressions by counting the number of cDNAs of expressed genes. CAGE enables comparison of the expression value of the gene to that of other genes in the same tissue. In this study, we propose a method for exploring tissue-specific functions using data from CAGE. To identify tissue-specificity, one of the simplest ways is to assume that the function of the most expressed gene is regarded as the most tissue-specific. However, the most expressed gene in a tissue might highly express in all tissues, as seen with housekeeping genes. Functions of such genes cannot be tissue-specific. To remove these from consideration, we propose measuring tissue specificity of functions based on information content of gene ontology terms. We applied our method to data from 16 human tissues and 22 mouse tissues. The results from liver and prostate gland indicated that well-known functions of these tissues, such as functions related to signaling and muscle in prostate gland and immune function in liver, displayed high rank.     

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.     

Radiofrequency ablation of hepatocellular carcinoma: therapeutic response using contrast-enhanced coded phase-inversion harmonic sonography

OBJECTIVE: This study was performed to evaluate the usefulness of contrast-enhanced coded phase-inversion harmonic sonography in assessing the therapeutic response of percutaneous radiofrequency ablation in patients with hepatocellular carcinoma. SUBJECTS AND METHODS: Sixty-seven patients with a total of 107 examinations on 91 hepatocellular carcinoma nodules underwent coded harmonic angio, a technique of coded phase-inversion harmonic sonography, using the IV microbubble contrast agent Levovist before and after percutaneous radiofrequency ablation. The intratumoral blood vessels and tumor parenchymal stain were detected in the early arterial phase and the late vascular phase, respectively. The results of contrast-enhanced imaging with coded harmonic angio were compared with those of three-phase dynamic CT. RESULTS: Before treatment, all examined 107 hepatocellular carcinoma nodules were found to be hypervascular on contrast-enhanced imaging with coded harmonic angio. After radiofrequency ablation, contrast-enhanced coded harmonic angio detected persistent signal enhancement in 41 examined nodules (38.3%), whereas this technique showed no intratumoral enhancement in the remaining 66 (61.7%) examined nodules. Compared with dynamic CT, the sensitivity, specificity, and diagnostic accuracy of contrast-enhanced coded harmonic angio were 95.3%, 100%, and 98.1%, respectively. With contrast-enhanced coded harmonic angio, we found that it was difficult to identify the safety margin that can be detected on dynamic CT. CONCLUSION: Contrast-enhanced imaging with coded harmonic angio may provide an alternative approach that has high diagnostic agreement with dynamic CT in assessing the therapeutic effect of radiofrequency ablation in hypervascular hepatocellular carcinomas, in spite of having limitations in identifying the safety margin.     

Transcatheter arterial chemoembolization of hepatocellular carcinoma: usefulness of coded phase-inversion harmonic sonography

OBJECTIVE: The purpose of our study was to assess the value of coded phase-inversion harmonic sonography performed approximately 1 week after the patients had undergone transcatheter arterial chemoembolization with iodized oil for hepatocellular carcinoma. SUBJECTS AND METHODS: We studied 40 patients with 44 nodules measuring 1.5-11.0 cm in diameter (mean +/- SD, 3.9 +/- 2.0 cm) who underwent transcatheter arterial chemoembolization. Coded phase-inversion harmonic sonography, a technique based on a combination of phase-inversion harmonics and coded technology, was performed with a contrast agent approximately 1 week after chemoembolization. The results were compared with those obtained using dynamic CT (n = 44 lesions) and dynamic MR imaging (n = 20 lesions). We also evaluated the recurrence of hepatocellular carcinoma during clinical follow-up in 17 patients who did not undergo additional local therapy. RESULTS: The detection rates of intratumoral vascularity of coded phase-inversion harmonic sonography, dynamic CT, and dynamic MR imaging were, respectively, 38 (86%) of 44 lesions, 19 (43%) of 44 lesions, and 10 (50%) of 20 lesions. Of 19 nodules of hepatocellular carcinoma treated only by transcatheter arterial chemoembolization, 17 nodules showed enhancement on coded phase-inversion harmonic sonography, suggesting incomplete responses. In all 17 nodules, apparent recurrence was noted on dynamic CT during clinical follow-up, even in nodules that had been observed to be completely filled with iodized oil 1 week after the chemoembolization. CONCLUSION: We found coded phase-inversion harmonic sonography to be highly sensitive and accurate for evaluating the treatment response in patients with hepatocellular carcinoma even shortly after treatment. Consequently, it allows early recognition of the need for additional local ablation therapy and estimation of the risk of hepatocellular carcinoma recurrence.