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Biomedical subjects

H Hirano

Publications and source records attributed to H Hirano.

At least 307 records · Page 17Linked to original sources
PubMed1992

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.

Acetylation↗
PubMed1992

Involvement of actin filaments in mouse testicular cord organization in vivo and in vitro.

Involvement of actin filaments in mouse fetal testicular differentiation was examined in vivo and in vitro. During testicular cord formation in vivo, actin filaments accumulated in the basal cytoplasm of Sertoli cells. Addition of cytochalasin D (CD) to organ cultures of undifferentiated gonadal primordia significantly inhibited testicular cord formation. In brief, treatment with 25 ng/ml CD induced the formation of slender testicular cords, and treatment with 50 ng/ml largely inhibited cord formation in the explants. However, development and growth of the testicular parenchyma and Leydig cell differentiation occurred in the presence of CD. By electron microscopic and immunohistochemical examinations, it became clear that CD also affected formation of the basal lamina and accumulation of vimentin filaments in Sertoli cells. On the other hand, treatment with colcemid at 12.5 or 15 ng/ml prevented growth of the testicular parenchyma and development of interstitial regions. Interestingly, testicular cords formed under this condition. These results indicate that the basal actin filaments of Sertoli cells may play an important role in testicular cord formation, especially Sertoli cell polarization. Cell mitosis and/or microtubules, on the other hand, may not be directly involved in this process.

Actins↗
PubMed1992

Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin.

The production of extracellular matrix components such as laminin, Type IV collagen, fibronectin, and tenascin during the formation of basement membrane in cultured epidermis-dermis recombinant skin of 13-day-old chick embryo was analyzed immunohistochemically. The epidermis and dermis were separated from each other by treatment with EDTA and/or dispase. The basal lamina of the basement membrane was thus removed from both epidermis and dermis. The isolated epidermis was overlaid onto the isolated dermis, i.e., recombined, and then cultured for 1-7 days in a chemically defined medium (BGJb) on a Millipore filter. Immunofluorescence labeling was used for light microscopy and HRP or colloidal gold labeling for electron microscopy. In specimens from 2-day cultures, positive sites of anti-laminin and anti-fibronectin reaction were observed light microscopically as patches which, at the electron microscopic level, corresponded to fragments of the basal lamina located immediately beneath and in the vicinity of the attachment plaques of the hemidesmosomes. The staining pattern became continuous 7 days after recombination. Fluorescence labeling of laminin and fibronectin appeared somewhat earlier than that of Type IV collagen and tenascin. All of the four components were found localized primarily in the basal lamina. Furthermore, fibronectin and tenascin were also distributed in the extracellular matrix of the dermis. The expression of tenascin, which does not exist in the basement membrane of 13-day-old intact embryonic skin, was induced in vitro. These results suggest that hemidesmosomes may play an important role in the reconstruction of the basement membrane and that various components of the basement membrane appeared at different times during the reconstruction.

Animals↗
PubMed1992

80 kDa mouse sperm protein as a substrate of protein kinase C.

Calcium and phospholipid-dependent protein kinase (PKC) activity was detected mainly in the cytosol of the mouse sperm. The PKC in the cytosol fraction was partially purified by ion-exchange chromatography. Using the partially purified PKC, the phosphorylation of PKC substrates was examined in vitro. The phosphorylation of the 80 kDa protein was enhanced by phorbol ester treatment in vitro as well as in vivo. The partial amino acid sequence of this protein was homologous with that of guanosine 5'-cyclic monophosphate (cGMP)-dependent protein kinase and myosin light chain kinase, both of which are related to ligand-receptor-transduction. The present data suggest that the activation of PKC and subsequent specific protein phosphorylation might be involved in the regulation of the zona pellucida-induced acrosome reaction.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of equinatoxins from the sea anemone Actinia equina L.

Equinatoxins were purified from the tentacles and bodies of the sea anemone Actinia equina by the use of acetone precipitation, as well as column chromatographies on Sephadex G-50 and CM-cellulose according to the modified method of Macek and Lebez (1988). The equinatoxins obtained, equinatoxin 1 and 2, were hemolytic glycoproteins with a relative molecular mass of 20000. Equinatoxin 2 was found to be rich in glycine, alanine and valine. The amino acid sequences of equinatoxins 1 and 2 were partially determined. A portion of the N-terminal amino acid sequence of equinatoxin 2 was similar to those of pyruvate kinase and sialidase.

Amino Acid Sequence↗
PubMed1992

Changes in intracellular and cell surface localization of Le(x) epitope during germ cell differentiation in fetal mice.

Changes in the intracellular and cell surface localization of Lewis x (Le(x)) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le(x) monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le(x) MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le(x) determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.

Animals↗
PubMed1992

The usefulness of step sectioning of transbronchial lung biopsy specimen in diagnosing sarcoidosis.

To evaluate the usefulness of step sectioning of the transbronchial lung biopsy (TBLB) specimens obtained from patients with suspected sarcoidosis, we examined all TBLB specimens obtained from 132 patients who were diagnosed clinically as having sarcoidosis at our institute. When routine sections of TBLB specimens did not show sarcoid granuloma, we prepared additional serial sections from each block and stained every fifth section with hematoxylineosin (step sectioning). All step sections were examined histologically. With the aid of step sectioning, the diagnostic yield of sarcoidosis increased from 38 percent (18/47) to 47 percent (22/47) in stage 1 patients, and from 57 percent (37/65) to 82 percent (53/65) in stage 2 patients. All of the newly detected granulomas were identified between the first and seventh sections. We conclude that step sectioning is useful method in diagnosing sarcoidosis.

Aged↗
PubMed1992

In vitro monitoring activation by the ligands and specific DNA-binding of the glucocorticoid receptors.

The glucocorticoid receptor is a member of the steroid and thyroid hormone receptor superfamily and acts as a ligand-activated transcription factor. To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits glucocorticoid-induced activation of the latent cytosolic glucocorticoid receptor to an active DNA-binding species. We demonstrate here that cytosol from a rat hepatoma cell, M1.19, contains glucocorticoid receptor-specific immunoreactivities and target DNA-binding activities. Moreover, specific DNA-binding activities of M1.19 cytosol were dose-dependently induced by dexamethasone treatment, and linearly correlated with the hormonal induction of chloramphenicol acetyltransferase activity at the corresponding concentrations. These results indicate that the cytosolic glucocorticoid receptor could be converted in a DNA-binding form under cell-free conditions and the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

Animals↗
PubMed1992

Simultaneous measurements of adenosine deaminase activity and tuberculostearic acid in pleural effusions for the diagnosis of tuberculous pleuritis.

Adenosine deaminase (ADA) activity and tuberculostearic acid (TSA) levels in pleural effusions were measured in 18 patients with active tuberculous pleuritis, 16 patients suspected of having tuberculous pleuritis, 14 patients with carcinomatous pleuritis, and 19 patients suffering from pleuritis of non-malignant and non-tuberculous etiology. In the patients with active tuberculous pleuritis, ADA was elevated in 56% and TSA was positive in 78%. In 83% of these patients, either ADA was elevated or TSA was positive. ADA was elevated together with a positive TSA in 50%. In contrast, TSA was positive in only 6% and ADA was elevated in 24% of the patients with non-tuberculous pleuritis, and none of these patients showed the combination of an elevation of ADA and a positive TSA. These results suggest that simultaneous measurements of both ADA and TSA in pleural effusions are useful for the diagnosis of tuberculous pleuritis.

Adenosine Deaminase↗
PubMed1992

Emergency cervical cerclage using a metreurynter in patients with bulging membranes.

Emergency cervical cerclage was performed in 26 women between 18 and 32 weeks of gestation who had fetal membranes bulging through a widely dilated cervix. A bulging bag was pushed into the uterine cavity by a new method, using a metreurynter. The average prolongation of pregnancy after the operation in the 26 women was 41 days (ranging from 2 to 115 days). All pregnancies resulted in the live birth of altogether 28 babies. The neonatal survival rates at gestational ages of less than or equal to 26 (including two sets of twins), 27-29, and greater than or equal to 30 weeks at the time of cervical cerclage were 80.0 (12/15), 85.7 (6/7), and 100% (6/6), respectively. Ten (83.3%) of the 12 infants who survived from patients receiving cerclage at a gestational age of less than or equal to 26 weeks are alive and well. The average duration of pregnancy after cerclage was 60 days when cervical dilation was less than or equal to 5 cm at operation, in contrast to 5.4 days in patients with cervical dilation of greater than or equal to 6 cm, suggesting that emergency cervical cerclage could be suitable in the presence of a cervical dilation of 5 cm or less.

Emergencies↗
PubMed1992

[Complete remission with MEC regimen of acute myeloid leukemia (M4) secondary to 5-year treatment of non-Hodgkin lymphoma].

A 32-year-old woman was admitted to our hospital with pyrexia and general lymphadenopathy in July 1984. She was diagnosed as having malignant lymphoma (follicular, small cleaved cell), stage IV based on the histological findings of lymph nodes in the neck and bone marrow specimen. She was treated with melphalan orally for 3 years, followed by MACOP-B. She attained partial remission with MACOP-B. Thereafter, she received melphalan or Endoxan orally as maintenance therapy. She developed fever and swelling in the gingivae in October 1989. Peripheral blood showed WBC 80,200/microliters with 7.5% myeloblasts and 85.5% monocytes. Bone marrow aspirate revealed hypercellularity with 47.9% myeloblasts, 46.5% monoblasts and monocytes, which were positive for peroxidase and NSE stains. The karyotype of bone marrow cells showed a 46,XX,t(9;11). The lysozyme in serum was elevated. She was diagnosed having AML (M4). DCMP regimen was initiated but failed to achieve CR. Consequently she received MEC regimen and obtained complete remission, lasting for 6 months. Patients with second leukemia have a low probability of achieving complete remission using conventional chemotherapy. The MEC regimen is thought to be one of the most promising treatments for secondary leukemia.

Adult↗
PubMed1992

Treatment of diffuse non-Hodgkin's lymphoma with combined chemotherapy using methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin (MACOP-B).

Forty-nine previously untreated adult patients with diffuse non-Hodgkin's lymphoma were treated with MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin) between December 1986 and December 1990. Forty patients (82%) achieved a complete response (CR), three (6%) a partial response (PR), while four (8%) had either no response or progression of disease, one (2%) patient ceased MACOP-B therapy and received other chemotherapy because of sustained neutropenia, and one patient (2%) died of sepsis during therapy. The factors that adversely affected the CR rate were by stage IV, the presence of B symptoms, the presence of a large mass (greater than 5 cm), and low serum total protein level. The 4-year survival for all 49 patients was 70% and the 4-year disease-free survival (DFS) for the 40 CR patients was 77%. Relapses were higher in patients whose initial serum lactic dehydrogenase (LDH) level was higher than 660 IU/1 (DSF 89% vs. 49%). Toxicity was substantial but acceptable, with neutropenia and mucositis proving to be the most frequent severe side-effects. These preliminary results confirmed the effectiveness of MACOP-B therapy for diffuse non-Hodgkin's lymphoma.

Adolescent↗
PubMed1992

[Thrombosis-inducing activity (TIA) in plasma of a patient with lung cancer, which disappeared after radiotherapy].

A 58-year-old man had been suffering from persistent right shoulder pain was admitted following detection of a Pancoast tumor in the right lung. On admission, there was elevation of peripheral platelet count, plasma FDP and fibrinogen levels and thrombosis-inducing activity (TIA) was detected in his plasma. After receiving 30 Gy of Liniack, the mass shadow and the shoulder pain disappeared, which was accompanied by the normalization of laboratory data and the disappearance of TIA from plasma. However, plasma TIA became positive again one month later, in association with reappearance of shoulder pain and elevation of FDP and fibrinogen levels, which were considered due to recurrence of the tumor, 30 Gy of Liniack together with thermotherapy was again given. There was no subsequent recurrence of tumor, nor TIA has been detected for more than 3 years.

Blood Coagulation Factors↗
PubMed1992

Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT1) in cells of the blood-retinal barrier in the rat.

The blood-retinal barrier is part of the blood-ocular barrier. Retinal pigment epithelial cells connected by tight junctions serve as an outer blood-retinal barrier, and the nonfenestrated endothelial cells of blood vessels sealed by tight junctions serve as an inner blood-retinal barrier. Using antibodies specific for the erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of facilitated-diffusion glucose transporters, it was found, by ultrastructural cytochemical examination, that GLUT1 in the rat was localized at both the apical and basolateral plasma membranes of retinal pigment epithelial cells. The fenestrated endothelial cells of the underlying choriocapillaries were negative for GLUT1. In the inner retina, GLUT1 was found at both the luminal and contraluminal plasma membranes of endothelial cells. These observations show that GLUT1 is concentrated at the critical plasma membranes of the blood-retinal barrier and may serve as the machinery for glucose transport across the barrier.

Animals↗
PubMed1991

The role of N-glycosylation of GLUT1 for glucose transport activity.

To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.

Affinity Labels↗