Biomedical subjects
Publications and source records attributed to H Hirano.
[reaction: see text] Beginning with D-mannitol, a stereoselective synthesis of the right-half segment of the mycalamides has been accomplished by employing Lewis acid catalyzed intermolecular aldol reaction and oxypalladation as the key steps.
The 20S proteasome is the proteolytic complex that is involved in removing abnormal proteins, and it also has other diverse biological functions. Its structure comprises 28 subunits arranged in four rings of seven subunits, and exists as a hollow cylinder. The two outer rings and two inner rings form an alpha7beta7beta7alpha7 structure, and each subunit, alpha and beta, exists as seven different types, thus giving 14 kinds of subunits. In this study, we report the primary structures of the 14 proteasomal subunit subfamilies in rice (Oryza sativa), representing the first set for all of the subunits from monocots. Amino acid sequence homology within the rice family (alpha-type: 28.9-42.1%; beta-type: 17.2-31. 9%) were lower than those between rice subunits and corresponding orthologs from Arabidopsis and yeast (alpha-type: 49.2-94.5%; beta-type: 34.8-87.7%). Structural features observed in eukaryotic proteasome subunits, i.e., alpha- or beta-type signature at the N-termini, Thr active sites in beta1, beta2 and beta5 subunits, and nuclear localization signal-like sequences in some alpha-type subunits, were shown to be conserved in rice.
We report here the characterization and functional roles of the leptin receptor (ObR) in bovine adrenal medullary cells. The plasma membranes isolated from bovine adrenal medulla showed a single class of specific binding sites of (125)I-leptin with an apparent K(d) of 6.6 nM and B(max) of 62 fmol/mg protein. ObRa but not ObRb mRNA was detected in bovine adrenal medulla by reverse transcriptase-polymerase chain reaction. Incubation of cultured adrenal medullary cells with leptin (3-30 nM) for 20 min resulted in a significant increase in [(14)C]catecholamine synthesis from [(14)C]tyrosine without any change in catecholamine secretion. These findings suggest that leptin stimulates catecholamine synthesis through its receptors in bovine adrenal medullary cells.
N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1 mutant than in the normal strain.
Bone marrow is believed to contain multipotential stromal stem cells which can differentiate into osteoblasts, chondrocytes, adipocytes, and myoblasts (Prockop, D. J. Science 276, 71-74, 1997). Therefore, characterization and identification of the stem-like cell within the stromal cells are important to understand bone marrow function in relation to the hematopoietic microenvironment, and repair/regeneration of tissue defects. TBR31-2 cell, a bone marrow stromal cell line established from bone marrow of transgenic mice harboring temperature-sensitive (ts) simian virus (SV) 40T-antigen gene for immortality, is induced toward both adipocytic and osteogenic cells under conditions of the inactivation of T-antigen (Okuyama, R., Yanai, N., Obinata, M. Exp. Cell Res. 218, 424-429, 1995). In this work, using a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNA expressions of tissue-specific differentiation markers for adipocyte (lipoprotein lipase), osteoblast (type I collagen and osteocalcin), chondrocyte (type II and X collagen), and muscle cell (desmin) were examined during a long-term culture of the cell. In addition, histochemical studies showed the appearance of adipocytic, osteoblastic, chondrocytic, and muscle cells during this long-term culture. Thus, TBR31-2, which has characteristics of an undifferentiated cell, has the potential to express the multipotential cell lineages. These results indicated that a multipotential progenitor cell including potential to differentiate into a muscle cell and which is situated in the mesenchymal cell lineage was first obtained.
Tissue factor (TF) triggers the coagulation cascade reaction in vivo. Overexpression of TF mRNA is one leading cause of disseminated intravascular coagulation and thrombosis-related organ failure. In response to lipopolysaccharide (LPS) stimulation, various cell types can produce TF mRNA in vitro. However, there is currently no agreement on what types of cells in the liver overexpress TF mRNA after LPS treatment. For the first report, we found the increased TF mRNA with reverse transcription-polymerase chain reaction (RT-PCR), and confirmed a fourfold increase (p<0.001 vs. control, t-test) of the TF mRNA level with RT-competitive PCR in the liver of LPS-treated (2.0 mg/kg i.v. injection) rats. There was no significant difference in the glyceraldehyde-3-phosphate dehydrogenase mRNA level between LPS-treated rats and control rats. To clarify the localization and cellular source of LPS-induced TF mRNA, we performed in situ hybridization analysis with [35S]-labeled oligonucleotides probes, which we originally designed. We detected intense signals of TF mRNA in mononuclear cells but not in endothelial cells around the hepatic vein of LPS-treated rats. In this study, we showed that the TF mRNA level induced by LPS treatment, which may indicate mononuclear cells associated, significantly increased in the liver of rats. These results will provide circumstantial support for the therapeutic strategy that mononuclear cell should be one of the target cells to be treated in the early phase of disseminated intravascular coagulation in the liver, and that the need to suppress its overexpression of TF mRNA is essential for preventing hypercoagulable condition.
An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.
AIMS/HYPOTHESIS: Streptozotocin (STZ), a chemically reactive analogue of N-acetylglucosamine, induces necrosis of the beta cells, resulting in diabetes mellitus. Glucose-induced insulin resistance is mediated by increased activity of the hexosamine pathway. We aimed to examine the regulation of O-GlcNAc transferase expression and activity in the normal and streptozotocin diabetic pancreas. METHODS: Rats were made diabetic by an injection of streptozotocin (65 mg/kg). The expression of O-GlcNAc transferase protein was examined by immunoblot analysis. Activity of O-GlcNAc transferase was assayed by the incorporation of [3H]GlcNAc into the synthetic peptide. Localization of O-GlcNAc transferase was done by immunohistochemistry. The change of O-GlcNAc modification of proteins was examined by immunoblot analysis. RESULTS: In the STZ-induced diabetic pancreas, a severe loss of beta cells was observed, whereas alpha cells had increased in number. The diabetic pancreas showed an increase in the expression of O-GlcNAc transferase at the protein level and the O-GlcNAc transferase activity in it was increased significantly (p < 0.05). An increase in the immunostaining intensity in the cytoplasm of islet beta cells was also observed in the diabetic pancreas, whereas exocrine cells and islet cells other than beta cells showed little change in immunostaining intensity. The pancreas of STZ-diabetic rats showed a 3.1-fold increase in total cellular O-GlcNAc-modified proteins. CONCLUSION/INTERPRETATION: These findings indicate that O-GlcNAc transferase plays an important part in the modulation of O-GlcNAc concentrations in the pancreas and suggest that the increase in O-GlcNAc modification of the proteins correlates closely with diabetes.
We report a 63-year-old man who had a rare bilateral thalamic glioma. He complained of difficulty with calculations and had mental deterioration. T1-weighted images revealed bilateral thalamic swelling with homogeneous low signal and no contrast enhancement. The tumour, showing decrease of N-acetylaspartate and the presence of lactate on magnetic resonance spectroscopy, was diagnosed as an astrocytoma by stereotactic biopsy.
OBJECTIVE: This study was undertaken to investigate changes in chondroitin sulfate levels in the cervix and the physiologic role of chondroitin sulfate isomers in the process of cervical ripening. STUDY DESIGN: Uterine cervical mucus samples were obtained from 57 women (7 nonpregnant women, 19 at preterm gestation, 9 at term gestation, 16 during the first stage of term labor, and 6 with threatened premature labor). Chondroitin sulfate isomer (chondroitin 0-sulfate, 4-sulfate, and 6-sulfate) concentrations in cervical mucus were measured by high-performance liquid chromatography. The effect of exogenous chondroitin sulfate on hyaluronidase activity in cervical mucus was evaluated by gel permeation chromatography of fluorolabeled hyaluronic acid. RESULTS: Chondroitin sulfate concentrations in cervical mucus were increased significantly (P <. 05) in the threatened premature labor group compared with the preterm and term groups. However, the same finding was not observed in the first stage of term labor. Exogenous chondroitin sulfate inhibited hyaluronidase activity in mucus. CONCLUSION: Chondroitin sulfate may defend against cervical ripening in threatened premature labor.
OBJECTIVE: The purpose of this study was to investigate the effect of interleukin-6 (IL-6) and interleukin-8 (IL-8), whose concentrations are elevated with chorioamnionitis, on the expression of surfactant apoprotein mRNAs in fetal rat lung. STUDY DESIGN: We employed an animal model in which we were able to administer substances continuously into the cavity between the fetal membranes and endometrium using a miniosmotic pump. Lipopolysaccharide (LPS), IL-6, or IL-8 was administered to timed pregnant rats for 3 days (day 16-19), and fetal lung expression of surfactant apoprotein mRNAs for SP (surfactant apoprotein)-A, SP-B, and SP-C was evaluated by Northern blot hybridization. RESULTS: Continuous administration of LPS increased the expression of each surfactant apoprotein mRNA, but the expression of mRNAs was not dose-dependent. On the other hand, continuous IL-6 or IL-8 administration increased the expression of each surfactant apoprotein mRNA in a dose-dependent manner. CONCLUSION: Fetal lung maturation may be promoted by either IL-6 or IL-8 produced in response to chorioamnionitis.
A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271-283). During scale ridge elongation at 12-15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.
1. In anaesthetized rats, platelet activating factor (PAF; 1 microg kg(-1)) decreased mean arterial blood pressure by around 60 mmHg (n=18). This depressor response was completely blocked by the PAF antagonist, CV-6209 (1 mg kg(-1)), indicating the role of PAF-specific receptor in the response. 2. N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg kg(-1)), an NO synthase inhibitor, profoundly elevated systemic blood pressure (n=19), indicating an important role of NO in the basal blood pressure regulation. The depressor response to PAF (1 microg kg(-1)) normalized against that to sodium nitroprusside (SNP) (10 microg kg(-1)) was not substantially different between rats treated without and with L-NAME (n=4). In contrast, the depressor effect of acetylcholine (0.03 - 1.0 microg kg(-1)) normalized against that of SNP (10 microg kg(-1)) was significantly attenuated by L-NAME (n=5). 3. Charybdotoxin (0.4 mg kg(-1)) plus apamin (0.2 mg kg(-1)) significantly attenuated the depressor response to PAF (1 microg kg(-1)) (n=5) without affecting the blood pressure change due to SNP (1 mg kg(-1)) (n=3). Charybdotoxin (0.4 mg kg(-1)) (n=4) or apamin (0.2 mg kg(-1)) (n=4) alone did not affect the PAF-induced depressor response. 4. These findings suggest that EDHF may make a significant contribution to the depressor response to PAF in rats. Although NO plays the determinant role in the basal blood pressure regulation, its contribution to PAF-produced depressor response seems to be less as compared with that to the depressor response to acetylcholine.
BACKGROUND: Advanced glycation of proteins has been incriminated in the progressive alteration of the peritoneal membrane during chronic peritoneal dialysis (PD). Advanced glycation end products (AGEs) result from a modification of proteins by reactive carbonyl compounds (RCOs). RCOs resulting from glucose breakdown are present in commercial PD fluid. They also accumulate in uremic plasma. The present study was undertaken to evaluate the respective contribution of these two sources of RCOs in the genesis of peritoneal AGEs. METHODS: Three major RCOs formed during heat sterilization of PD fluid, that is, glyoxal, methylglyoxal, and 3-deoxyglucosone, and total RCOs were measured in commercial PD fluid and in PD effluent. The generation of pentosidine, used as a surrogate marker for AGEs, during one-week incubations of PD fluid and effluent samples fortified with bovine serum albumin (BSA) was measured by high-performance liquid chromatography. Peritoneal samples were stained with antibodies specific for two AGEs derived from carbohydrate-dependent RCOs, Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, or for two advanced lipoxidation end products (ALEs) derived from lipid-dependent RCOs, malondialdehyde (MDA)-lysine and 4-hydroxynonenal (HNE)-protein adduct. RESULTS: Glyoxal, methylglyoxal, and 3-deoxyglucosone were identified in commercial PD fluid. Their levels in PD effluents decreased with dwell time probably by diffusion into blood circulation. In contrast, the levels of total RCOs were initially low in commercial PD fluid, increased in PD effluent with dwell time probably by diffusion from circulation into the peritoneal cavity, and after 12 hours, reached values observed in uremic serum. The relevance of the rise in total RCOs for AGE formation is demonstrated by a parallel increase in the generation of pentosidine during incubations of PD effluents. In contrast with RCOs present in glucose-rich PD fluid, RCOs diffusing from uremic circulation originate from both carbohydrates and lipids. Their role in the modification of peritoneal proteins is demonstrated by the immunohistochemical study of peritoneal tissue. Two AGEs and two ALEs increase in parallel in the mesothelial layers and in vascular wall of small arteries in the peritoneum. CONCLUSIONS: Protein modification of the peritoneum is determined not only by RCOs originating in PD fluid, but also by RCOs originating from the uremic circulation. The present data might be relevant to current attempts to improve PD fluid toxicity by lowering its glucose content.
BACKGROUND: Norepinephrine transporters (NETs) terminate the neuronal transmission of norepinephrine, which is released from noradrenergic neurons. To investigate the interaction with NET, the authors examined the effects of short- and long-term treatment with anesthetics on the activity and mRNA level of NET. METHODS: To assay [3H]norepinephrine uptake, bovine adrenal medullary cells in culture were incubated with [3H]norepinephrine in the presence of intravenous anesthetics, including propofol, thiamylal, and diazepam. To study the direct interaction between the anesthetics and NET, the effect of propofol on the binding of [3H]desipramine to the plasma membrane was examined. To study the long-term effect of anesthetics, [3H]norepinephrine uptake by cells pretreated with propofol for 6-24 h and [3H]desipramine binding after pretreatment for 12 h were measured. Simultaneously, we examined the effect of anesthetics on the expression of NET mRNA using the reverse transcriptase-polymerase chain reaction. RESULTS: All of the intravenous anesthetics inhibited [3H]norepinephrine uptake in a concentration-dependent manner. The active concentrations of propofol (1-3 microm) and thiamylal (< or = 30 microm) were similar to those encountered clinically. The kinetic analysis revealed that all the anesthetics noncompetitively inhibited [3H]norepinephrine uptake. Propofol inhibited [3H]desipramine binding with a potency similar to that observed in [3H]norepinephrine uptake. Scatchard analysis showed that propofol competitively inhibited [3H]desipramine binding. On the other hand, long-term treatment of cells with propofol (10 microm) enhanced the NET functional activity and [3H]desipramine binding, and also increased the level of NET mRNA. CONCLUSIONS: These results suggest that intravenous anesthetics have a dual effect on NET; short-term treatment causes inhibition, whereas long-term treatment leads to up-regulation. The interaction of intravenous anesthetics with NET may modulate the neuronal transmission of norepinephrine during anesthesia.
We produced ovalbumin (OVA)-sensitized rats as an animal model of nasal allergy. Intranasal instillation of OVA induced hypertrophic and metaplastic changes of goblet cells in nasal epithelium of OVA- sensitized rats. Intraepithelial mucosubstance in nasal mucosa increased significantly at 24 h after 3 or 7 d of OVA instillation, accompanied by mucosal infiltration of eosinophils. The effects of H1-antagonist (d-chlorpheniramine malate), H2-antagonist (cimetidine), dexamethasone, indomethacin, cysteinyl leukotrienes (cysLTs)-antagonist (ONO1078), and antirat neutrophil antiserum on OVA-induced changes were examined. Mucus production was significantly inhibited by dexamethasone, and ONO1078, whereas eosinophil infiltration was significantly inhibited by H1-antagonist, dexamethasone, and anti-rat neutrophil antiserum. These results indicate that cysLTs (LTs C4, D4, and E4) may play an important role in antigen-induced mucus production, and that eosinophil infiltration does not relate to mucus production. Intranasal instillation of lipopolysaccharide (LPS) also induced intraepithelial mucus production, and it was significantly inhibited by dexamethasone, indomethacin, and antirat neutrophil antiserum; however, cysLTs antagonist had no effect on LPS-induced change. These results indicate that neutrophil and cyclooxygenase products are important in LPS-induced mucus production, and there are different mechanisms of mucus production between allergic inflammation and LPS stimulation.
The Rho family of small GTPases occupies a key position in the control of cell motility and morphology in response to extracellular stimuli. Rho proteins trigger the formation of contractile stress fibers, resulting in regulation of cell motility. We explored the expression and function of RhoA in human endometrium and decidua. RhoA immunoreactivity had a predominantly glandular epithelial distribution in the proliferative phase and midsecretory phase. In decidua, the expression of RhoA was more pronounced in the stromal cells as well as in the glandular epithelium. RhoA protein levels in proliferative phase and midsecretory phase endometrium as well as decidua were evaluated by immunoblotting; a single band of RhoA protein with a molecular mass of 21 kDa was detected in all cell lysates. Cultured human decidual cells were found to have few actin stress fibers. Decidual cells lost their actin stress fibers by the treatment with C3, an exoenzyme produced by Clostridium botulinum, whereas new actin stress fibers appeared in human decidual cells stimulated with lysophosphatidic acid (LPA). Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h. Blastocysts attached to decidual cells exhibited extensive outgrowth after 48 h in culture. Treatment of decidual cells with C3 exoenzyme or LPA did not affect the rates of hatching and attachment of blastocysts, but outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme treatment in a dose-dependent manner. Contrariwise, addition of LPA to decidual cells dose dependently increased the outgrowth of embryos on decidual cells. These findings suggest that RhoA in decidual cells is important for embryonic development and differentiation after attachment.
A 24-year-old male presented with a rare intracranial dural metastasis from a mediastinal germ cell tumor infiltrating the superior vena cava, pericardium, ascending aorta, and lung. Routine chest radiography incidentally revealed the abnormal mediastinum, which was partially resected after median sternotomy, followed by radiotherapy. One year after the radiotherapy, magnetic resonance imaging revealed a right parasagittal tumor mimicking a meningioma. Partial tumor removal was performed. Two cycles of chemotherapy with carboplatin and etoposide were then performed, and the residual tumor almost completely disappeared. The histological diagnosis of both the mediastinal and intracranial tumors was pure seminoma. Sections of metastatic tumor revealed several thick fibrous septa and numerous epithelioid granulomas infiltrated by plasma cells, intermingled between the clusters of tumor cells. Follow up of patients apparently in remission from mediastinal seminoma without evidence of advanced or recurrent disease at other sites is recommended.