Systematic characterization of human testis-specific actin capping protein ß3 as a possible biomarker for male infertility.

STUDY QUESTION: Is actin capping protein (CP) ß3 involved in human spermatogenesis and male infertility? SUMMARY ANSWER: Human CPß3 (hCPß3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. WHAT IS KNOWN ALREADY: The testis-specific α subunit of CP (CPα3) was previously identified in human, and mutations in the cpα3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CPß3, which is considered to be a heterodimeric counterpart of CPα3, has been neither characterized in human nor reported in association with male infertility. STUDY DESIGN, SIZE, DURATION: To confirm the existence of CPß3 in human testis, fresh semen samples from proven fertile men were analyzed. To investigate protein expression during spermatogenesis, cryopreserved testis obtained from men with obstructive azoospermia were examined by immunofluorescent analysis. To assess the association of CP with male infertility, we compared protein expression of human CPα3 (hCPα3) and hCPß3 using immunofluorescent analysis of cryopreserved sperm between men with normozoospermia (volunteers: Normo group, n = 20) and infertile men with oligozoospermia and/or asthenozoospermia (O + A group, n = 21). PARTICIPANTS/MATERIALS, SETTING, METHODS: The tissue-specific expression of hCPß3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCPα3 and hCPß3 form a heterodimer, a tandem expression vector containing hcpα3 tagged with monomeric red fluorescent protein 1 and hcpß3 tagged with enhanced green fluorescent protein in a single plasmid was constructed and analyzed by co-immunoprecipitation (Co-IP) assay. The protein expression profiles of hCPα3 and hCPß3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCPα3 and hCPß3 in sperm were compared between the Normo and O + A groups by immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: RT-PCR showed that mRNA of hcpß3 was expressed exclusively in testis. Western blot analysis detected hCPß3 with anti-bovine CPß3 antibody. Co-IP assay with recombinant protein showed that hCPα3 and hCPß3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCPß3 changed dynamically. In spermatogonia, hCPß3 showed a slight signal in cytoplasm. hCPß3 expression was conspicuous mainly from spermatocytes, and hCPß3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCPß3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCPα3 localization was identical to hCPß3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCPα3 and hCPß3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCPα3 or hCPß3 (abnormal staining) (P < 0.001). The percentage of abnormal staining was higher in the O + A group (52.4 ± 3.0%) than in the Normo group (31.2 ± 2.5%). Even by confining the observations to morphologically normal spermatozoa selected in accordance with David's criteria, the percentage of abnormal staining was still higher in the O + A group (39.9 ± 2.9%) versus the Normo group (22.5 ± 2.1%) (P < 0.001). hCPß3 in conjunction with hCPα3 seemed to play an important role in spermatogenesis and may be associated with male infertility. LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: Owing to the difficulty of collecting fresh samples of human testis, we used cryopreserved samples from testicular sperm extraction. To examine the interaction of spermatogenic cells or localization in seminiferous tubules, fresh testis sample of healthy males are ideal. WIDER IMPLICATIONS OF THE FINDINGS: The altered expression of hCPα3 and hCPß3 may not only be a cause of male infertility but also a prognostic factor for the results of ART. They may be useful biomarkers to determine the fertilization ability of human sperm in ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JP16K20133). The authors declare no competing interests.

Dietary salt with nitric oxide deficiency induces nocturnal polyuria in mice via hyperactivation of intrarenal angiotensin II-SPAK-NCC pathway.

Nocturnal polyuria is the most frequent cause of nocturia, a common disease associated with a compromised quality of life and increased mortality. Its pathogenesis is complex, and the detailed underlying mechanism remains unknown. Herein, we report that concomitant intake of a high-salt diet and reduced nitric oxide (NO) production achieved through Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) administration in mice resulted in nocturnal polyuria recapitulating the clinical features in humans. High salt intake under reduced NO production overactivated the angiotensin II-SPAK (STE20/SPS1-related proline-alanine-rich protein kinase)-NCC (sodium chloride co-transporter) pathway in the kidney, resulting in the insufficient excretion of sodium during the day and its excessive excretion at night. Excessive Na excretion at night in turn leads to nocturnal polyuria due to osmotic diuresis. Our study identified a central role for the intrarenal angiotensin II-SPAK-NCC pathway in the pathophysiology of nocturnal polyuria, highlighting its potential as a promising therapeutic target.

Alkaline phosphatase activity in the human promyelocytic leukemia cell line, HL-60, induced by retinoic acid and recombinant human granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) not only enhanced the growth of HL-60 cells, but also significantly increased NBT-reducing ability and alkaline phosphatase (ALP) activity of the cells, which were enhanced by the treatment with retinoic acid (RA). Protein kinase C inhibitors (H-7 and staurosporine) significantly suppressed this induction of ALP. The pretreatment with RA followed by rhG-CSF treatment showed almost the same degree of ALP activity as that induced by the simultaneous treatment with RA and rhG-CSF. This study suggests that RA and rhG-CSF are the potent inducers of ALP activity of HL-60 cells and protein kinase C is supposed to have a role in this induction of ALP.

Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana.

In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.

Peplomycin sulfate and pulmonary fibrosis: hydroxyproline, uronic acid, proline hydroxylase and glucosamine 6-phosphate synthetase in lungs of hamsters treated with peplomycin.

Effect of peplomycin sulfate (PLM) on pulmonary fibrosis was examined. Hydroxyproline, uronic acid, proline hydroxylase (EC 1.14.11.2) and glucosamine 6-phosphate synthetase (EC 2.6.1.16) in lungs of hamsters treated with PLM were studied and compared with those of hamsters treated with bleomycin (BLM). PLM, when administered intraperitoneally, one injection daily for 10 consecutive days, at either a high- (5 mg/kg) or low- (2.8 mg/kg) dosage-level, caused no significant increase of lung hydroxyproline and uronic acid as compared with controls. BLM on the other hand effected a significant increase in lung hydroxyproline on the high-dosage level (5 mg/kg) but not on the low-dosage level (2.8 mg/kg). In contrast, when administering PLM intratracheally, the concentrations of hydroxyproline in lungs increased 20% over the control levels. A transient increase of proline hydroxylase and glucosamine 6-phosphate synthetase also occurred shortly after the instillation. These increases were also observed in the corresponding groups treated with BLM, which confirmed the previous observations by other investigators. However, the magnitude of the increase was relatively lower in those values of PLM as compared with those of BLM. These data suggested that (1) PLM, when administered with multiple dosages intraperitoneally, showed no significant effect on the elevation of lung hydroxyproline; (2) PLM, when administered with a dose intratracheally, induced pulmonary fibrosis similar to that caused by BLM. However, the hydroxyproline accumulation in lungs of PLM-treated hamsters was less than in those of the BLM-treated; (3) The fibrotic effect on the lungs caused by either PLM or BLM was probably attributed to acceleration of the syntheses of collagen and acidic glycosaminoglycans.

Effect of rush immunotherapy in house-dust-mite (HDM)-sensitive adult bronchial asthma: changes in in vivo and in vitro responses to HDM.

An open study was conducted to evaluate the changes in in vivo and in vitro responses to house-dust-mite (HDM) after rush immunotherapy (RI). A 7-day RI protocol using an extract containing HDM allergen was administered to 12 subjects with HDM-sensitive asthma, and the effects on bronchial responsiveness and serum antibody levels were evaluated up to 16 or 20 weeks after RI. The levels of HDM-specific IgG, IgG1 and IgG4 antibodies were significantly elevated from 4 or 8 weeks after RI. Provocative doses causing a 20% fall in FEV1 (PD20) by allergen inhalation were elevated in all subjects at 16 to 20 weeks after RI. There was a high correlation between the increase in log-PD20 and the increase in the ratio of HDM-specific IgG4 to IgG1 (r = 0.68, p < 0.05). The results suggest that RI elicits the improvement of allergen-specific bronchial responsiveness and the increase in serum antibody levels within a relatively short period.

Identification and characterization of a plaque forming avian rotavirus isolated from a wild bird in Japan.

From fresh faeces of a wild bird (Melanitta fusca), a virus that showed granular cytopathic effects (CPE) on chicken kidney cell (CKC) cultures was isolated. By indirect immunofluorescence analyses (IFA), this isolate reacted with an antiserum against a bovine rotavirus. The isolate produced clear plaques on CKC by conventional techniques, without trypsin. Three virus plaques were selected by plaque size (small, medium, and large) and cloned by three successive plaque cloning. In the SDS-PAGE analyses, dsRNA bands showed a typical profile of avian rotavirus and quite different from that of avian reovirus. With dsRNA patterns, IFA results, CPE, and a morphological property, the clones were identified as avian rotaviruses of group A rotavirus. The clones killed chicken embryos, when they were inoculated to yolk sac.

[Contamination of endoscopes and endoscope washers by atypical mycobacteria].

Contamination of endoscopes and endoscope washers by atypical mycobacteria was studied. Large amounts of atypical mycobacteria were detected with high frequency inside endoscopes and endoscope washers. The species of atypical mycobacteria was Mycobacterium chelonae subsp. abscessus. Antibacterial-effects of glutaraldehyde against isolated atypical mycobacteria were checked. Sufficient antibacterial-effect was not obtained by 2% glutaraldehyde solution for endoscope sterilization. However, after frequent manual washing and brushing of endoscopes, by using 3% glutaraldehyde solution and 70% alcohol, all endoscopic instruments were completely decontaminated. We must pay attention to contamination of endoscopes and endoscope washer at least once a month.

[Clinical studies on pivmecillinam in urinary tract infections (author's transl)].

Pivmecillinam (PMPC), semisynthetic penicillin for oral use, was studied clinically and following results were obtained. 1) Twenty-eight patients with acute simple cystitis treated with the drug 200 mg/day for 4 days, clinical results were excellent in 23 cases and moderate in 5 cases. Six patients with chronic simple cystitis treated with the drug 400 mg/day for 7 days, clinical results were excellent in 4 cases, moderate in 1 case and poor in 1 case. Eight patients with complicated urinary tract infections treated with the drug 400 mg/day for 7 days, clinical results were excellent in 2 cases, moderate in 2 cases and poor in 4 cases. Overall effectiveness amounted to 88.1%. 2) Pivmecillinam was clinically effective in patients infected by Gram-negative bacteria except Pseudomonas aeruginosa. 3) No side effects were observed in all cases. The effectiveness of PMPC for acute simple cystitis was compared with that of talampicillin (TAPC), ampicillin (ABPC), amoxicillin (AMPC) and pivampicillin (PVPC) reported previously and PMPC was assessed as the most useful agent in these. Then, pivmecillinam should be chosen firstly as a chemotherapeutic agent for acute simple cystitis.

[Hematological study in 27 patients with myelodysplastic syndrome (MDS)].

The clinical, histologic, and cytogenetic features of myelodysplastic syndrome (MDS) were analyzed in 27 patients consisting of 21 males and 6 females (M: F ratio = 3.5:1) whose median age was 60.9 years. Analysis of the peripheral blood of the patients revealed pancytopenia (8 patients), bicytopenia (12), and cytopenia in one cell lineage (5). The patients had high serum iron value and low UIBC value. More than 20% of all erythroid cells in the bone marrow consisted of ringed sideroblasts in not only RARS patients but also RAEB, CMMoL, and RAEB-T patients. Morphological changes of the bone marrow cells were observed in patients with all types of MDS. Eighty percent and 60% of RAEB and RAEB-T patients, respectively, showed trilineage myelodysplasia. Chromosomal abnormalities were observed in 52% of MDS patients. The rate of leukemic transformation was high in RAEB, CMMoL, and RAEB-T patients.

[The effects of retinoic acid and recombinant human granulocyte colony-stimulating factor on alkaline phosphatase activity of HL-60 cells].

We examined alkaline phosphatase (ALP) activity in the HL-60 cell induced by retinoic acid (RA) and recombinant human granulocyte colony-stimulating factor (rhG-CSF). rhG-CSF induced a small but significant increase of NBT-reducing ability and ALP activity of the HL-60 cells. Among various inducers of cell differentiation, 1,25(OH)2D3 and dimethylsulfoxide (DMSO) caused the increase of the NBT-reducing ability and the suppression of ALP activity induced by rhG-CSF, while RA enhanced both of them. Protein kinase C inhibitors (H-7 and staurosporine) but not a protein kinase A inhibitor (HA1004) significantly suppressed the ALP activity induced by the simultaneous treatment with RA and rhG-CSF.

[A case of clinically multifocal renal cell carcinoma].

A 61-year-old woman was diagnosed with a renal tumor of the left kidney by ultrasound sonography during a health check-up. Computerized tomography (CT) and colored Doppler ultrasound sonography demonstrated two hypervascular tumors as typical renal cell carcinomas. A radically nephrectomized specimen was step-sectioned. Four tumor nodules were detected macroscopically, and 47 small nodules were detected microscopically, showing the clear cell type and alveolar growth pattern. Then all nodules including the 47 small nodules were diagnosed renal cell carcinoma.

[Improvement of intractable nocturnal asthma treated by therapeutic awakening].

A 33-year-old female was admitted to our hospital because of intractable asthma which had not been improved by intensive treatment at another hospital. Her asthmatic attacks were improved within a week by treatments including systemic corticosteroids and mechanical ventilation. However, in a few weeks nocturnal asthma recurred frequently, and it was resistant to various bronchodilators and steroids. Therapeutic awakening, that is, awakening the patient quietly at 3 a.m. with inhalation of 2.5 mg salbutamol was started. Her symptoms and physical findings rapidly improved thereafter. It was concluded that therapeutic awakening may be effective for the treatment of refractory morning dipping of asthma.

[The long-term effect of high-dose beclomethasone dipropionate on the pituitary-adrenal function].

The pituitary-adrenal function was studied in seven asthmatic subjects who had been received daily inhalations of 800 to 1,000 micrograms of beclomethasone dipropionate (BDP) over a year. None of the subjects had taken oral corticosteroids for at least six months prior to the study. As indices of pituitary-adrenal function, 1) circadian rhythm of plasma ACTH and cortisol, 2) urine 17-OHCS and 17-KS, and 3) the response of cortisol in rapid ACTH test were examined. All subjects showed normal circadian rhythms of plasma ACTH and cortisol levels. Urinary 17-OHCS and 17-KS excretions over a 24-hour period were also within the normal range. Plasma cortisol levels in the rapid ACTH test were significantly increased and judged as normal responses in all subjects. These results indicate that long-term treatment with BDP ranging from 800 to 1,000 micrograms/day induces no suppressive effect on the pituitary-adrenal function.

[Combination chemotherapy with bronchial arterial infusion (BAI) and systemic chemotherapy in advanced non-small cell lung cancer].

Five patients of advanced non-small cell lung cancer with atelectasis and/or poor performance status were treated with combination MVP therapy consisting of bronchial arterial infusion (BAI) using cisplatin (50 mg) and simultaneous systemic MVP therapy. The tumor size reduced more than 50% in three patients, and less than 25% in one patient. In another patient the tumor size was not measurable. Four of five patients showed disappearance of atelectasis, and three of four patients showed improvement of performance status. Average time taken was 13 and 17 days, respectively. We expect that combination MVP therapy has beneficial effects not only for relieving local symptoms and improving performance status within shorter period, but also achieving a better prognosis.