Biomedical subjects

Y Fujisawa

Publications and source records attributed to Y Fujisawa.

At least 19 recordsLinked to original sources

PubMed1996

Characterization and crystallization of recombinant human cathepsin L.

Human procathepsin L (25mg) was highly purified from the culture filtrate (4L) of mouse myeloma cells (Sp-HCL/HE14) transformed with human procathepsin L cDNA. The procathepsin L was almost completely converted to the mature form (18mg) under the acidic condition. Some properties of the mature cathepsin L were found to be different from those of the human liver-derived enzyme. In addition, we first produced crystals of mature human cathepsin L with E-64 using polyethylene glycol 6000 as the precipitant. The crystal was orthorhombic and belonged to the space group P2(1)2(1)2(1). The unit cell dimensions were: a = 49.8 A, b = 103.9 A, c = 47.8 A. The cell volume (2.47 x 10(5) A3) and calculated molecular mass (24.6 kDa) gave a volume/mass ratio of 2.5 A3/Da, which indicates that the asymmetric unit contains one molecule of enzyme.

Animals↗

PubMed1996

Development of radioimmunoassay for human leptin.

Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma leptin-LI concentration in nonobese subjects (17.6 <body mass index (BMI)<23.0) was 5.6 +/- 1.3 (mean +/-SE) ng/ml, while that in obese subjects (29.0 <BMI) was 43.0 +/- 9.4 (mean +/- SE) ng/ml. In gel permeation chromatographic analyses, leptin-LI in culture media and plasma consisted of single component emerging at the elution position of recombinant human leptin. These findings indicate that leptin is secreted from the adipose tissue into the circulating blood as a large molecular form corresponding to recombinant leptin. The RIA developed in the present study will be a powerful tool to investigate the physiological and pathophysiological significance of leptin in human.

Adipose Tissue↗

PubMed1996

Inhibitory mechanism of intestinal ethanol absorption induced by high acetaldehyde concentrations: effect of intestinal blood flow and substance specificity.

This study describes the effects of high blood acetaldehyde concentrations on the intestinal absorption of ethanol and 2-butanone using an in situ single-pass perfusion technique on the rat intestine and the colored microsphere method to measure intestinal blood flow. We found that high blood acetaldehyde concentrations inhibit intestinal ethanol absorption in an inverse proportion to peak acetaldehyde concentrations, decrease intestinal blood flow, and inhibit intestinal absorption of 2-butanone. The decrease of the intestinal blood flow, induced by high blood acetaldehyde concentrations, is a major mechanism inhibiting intestinal ethanol absorption, but other mechanisms are also thought to inhibit absorption. Therefore, inhibition by high acetaldehyde concentrations is not the only factor affecting ethanol absorption.

Acetaldehyde↗

PubMed1996

Receptor binding and antagonist properties of a novel endothelin receptor antagonist, TAK-044 [cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl) carbonyl]-L-alanyl-L-alpha-aspartyl-D-2-(2-thienyl) glycyl-L-leucyl-D-tryptophyl]disodium salt], in human endothelinA and endothelinB receptors.

Receptor binding and antagonist properties of an endothelin (ET) receptor antagonist, TAK-044 ¿cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl) carbonyl]-L-alanyl-L-alpha-aspartylD-2-(2-thienyl) glycyl-L-leucyl-D-tryptophyl]disodium salt¿, were investigated using recombinant human ETA and ETB receptors expressed in Chinese hamster ovary cells. The membranous ETA receptor was shown to be heterogeneous in ET-3 binding affinity (Hill coefficient = 0.54, Kd1 = 390 pM and Kd2 = 8.1 nM). This heterogeneity disappeared upon the addition of guanosine-5'-O-3-thiotriphosphate (Hill coefficient = 0.95, Kd = 7.8 nM). The Kd (from a computer program LIGAND analysis) and Ki (from Dixon plot analysis) values of TAK-044 were 95 and 120 pM for the membranous ETA receptor and 41 and 60 nM for the ETB receptor, respectively. The Kd values of TAK-044 for the ETA receptor was comparable to that of ET-1. The Ki values of TAK-044 for the cellular ETA and ETB receptors were 130 pM and 130 nM at 5,000 cells/well and 1.3 and 590 nM at 50,000 cells/well, respectively. Dixon plot analysis indicated that TAK-044 is a competitive inhibitor of ET-1 binding. TAK-044 inhibited ET-1-induced phosphatidylinositol hydrolysis and arachidonic acid release at 50,000 cells/well in a competitive manner with respective pA2 values of 8.5 and 8.7 in the ETA-expressing cells and 7.4 and 6.6 in the ETB-expressing cells. TAK-044 suppressed ET-1-induced transient increase in intracellular Ca+2 concentration in the ETA- and ETB-expressing cells with respective IC50 values of 2.8 and 230 nM. TAK-044 is a potent and competitive ETA receptor antagonist which simultaneously exhibits definite antagonist activity at the ETB receptor.

Animals↗

PubMed1995

Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer.

We have purified and characterized recombinant Xenopus bone morphogenetic proteins (xBMPs): homodimers of xBMP-4, 7 and heterodimers (xBMP-4/7) produced by a baculovirus expression system. Highly purified xBMPs had homogeneous NH2-termini predicted from a consensus motif, Arg-X-X-Arg, while they possessed diverse sugar chains. Implantation of xBMPs together with pure collagen carrier in rats induced new bone formation in a dose-dependent manner. The xBMP-4/7 heterodimer showed the strongest activity, with an effective dose of 1-30 micrograms, while more than 10 micrograms of xBMP-4 or 7 homodimer was required for a significant effect. Histological examination revealed that xBMP-4/7 implants showed intramembranous ossification without chondrogenesis. In primary cultures of rat bone marrow stromal cells, xBMP-4/7 induced alkaline phosphatase 3-fold more strongly than xBMP-7 and 20-fold more than xBMP-4. These results suggest that the heterodimeric form of BMP would generate the strongest signal triggering osteogenic differentiation of osteoprogenitor cells in adult tissues.

Alkaline Phosphatase↗

PubMed1995

Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system.

Recombinant baculoviruses as expression vectors for Xenopus bone morphogenetic protein (xBMP)-2, 4 and 7 were generated. The conditioned medium of insect cells infected with the virus for xBMP-2 or 4 showed strong alkaline phosphatase-inducing activity in a mouse osteoblastic cell line, MC3T3-E1, although a large portion of the activity remained in the infected cells. In contrast, xBMP-7 was preferentially secreted into the medium, but had only weak activity. Conditioned media following simultaneous inoculation with the viruses for xBMP-7 and for xBMP-2 or 4 showed a remarkably increased level of activity. The increased activity was clearly separated from other peaks derived from single infection on a cation-exchange column and was found to arise from the disulfide-linked heterodimer consisting of xBMP-4 and 7 subunits by immunoblot analysis. The heterodimer also augmented osteocalcin production and parathyroid hormone-sensitivity more strongly than the homodimers. These results suggest that our expression system provides a convenient source of heterodimeric BMP with high osteogenic differentiation-inducing activity.

3T3 Cells↗

PubMed1995

Effects of a synthetic rat adrenomedullin on regional hemodynamics in rats.

The effects of rat adrenomedullin, a novel vasorelaxant peptide, on systemic and regional hemodynamics were examined in conscious Sprague Dawley (SD) rats and spontaneously hypertensive rats (SHR). The intravenous infusion of adrenomedullin at rates of 1.67 and 5 micrograms/kg per min decreased the mean arterial pressure in a dose-dependent fashion in both types of rats. Adrenomedullin at a rate of 5 micrograms/kg per min increased the heart rate and cardiac output. As a result, the total peripheral resistance significantly decreased. With regards to the regional hemodynamics, adrenomedullin significantly increased the flow rates in the lungs, heart, spleen, kidneys, adrenal glands and small intestine of SHR. The flow rates in the brain and skin did not change and the flow rates in the skeletal muscle and testis were decreased. These regional hemodynamic changes were also observed in SD rats and there was no qualitative difference in the regional responses to adrenomedullin between SHR and SD rats. Thus, adrenomedullin predominantly increased the flow rates in organs in which adrenomedullin gene was highly expressed. It therefore seems that adrenomedullin may act as a local vasodilatory hormone rather than as a circulatory hormone.

Adrenomedullin↗

PubMed1995

Binding properties of fibrinogen receptor GPIIb-IIIa purified from human erythroleukemia cells.

Large amounts (2.3 mg) of an inactive form of the glycoprotein GPIIb-IIIa were obtained in highly purified form from 7-liter cultures of human erythroleukemia (HEL) cells. The purified GPIIb-IIIa was converted to an activated form after its immobilization to 96-well plastic plates. The binding of fibrinogen to the activated GPIIb-IIIa was investigated using 24 kinds of RGD peptides and showed that the IC50 values of these peptides correlated with those obtained with activated platelets. These findings indicated that the binding properties of the activated GPIIb-IIIa from HEL cells are quite similar to those of the platelets.

Amino Acid Sequence↗

PubMed1995

A suppressor mutation which suppresses adenylyl cyclase mutations in Neurospora crassa.

A spontaneous suppressor mutant, hah, which suppressed the colonial growth of adenylyl cyclase mutants (cr-1) was isolated. The morphology of cr-1 hah was filamentous, but slightly different from that of wild type on solid medium. The hah strain formed many high aerial hyphae, but did not form any conidia. The expression levels of an adenylyl cyclase gene, nac, in both hah and cr-1 hah were much higher than those in wild type or in cr-1. The level of cAMP in cr-1 was very low but returned to close to the wild-type level in the cr-1 hah suppressed strain, whereas that in the strain carrying hah alone was similar to or a little higher than that in wild type. The hah gene was located 13.3 map units from in1 on the right arm of the linkage group V. The hah mutation was recessive and allele specificity of hah for the suppression of cr-1 mutations was weak.

Adenylyl Cyclases↗

PubMed1995

Treatment of advanced hormone-refractory prostate carcinoma with a combination of etoposide, pirarubicin and cisplatin.

A total of 20 patients with hormone-refractory prostate carcinoma entered a pilot study of combination chemotherapy based on the EAP (etoposide, Adriamycin and cisplatin) regimen, in which Adriamycin was replaced by pirarubicin, a less cardiotoxic derivative of Adriamycin. The response was assessed by criteria modified from those of the National Prostatic Cancer Project: prostate-specific antigen was employed instead of acid phosphatase. Of 18 evaluable patients, 6 achieved a partial response, 5 had stable disease, and in 7 the disease had progressed during therapy; thus, the overall response rate was 33.3% [95% confidence interval (CI) 11.5-55.1%]. Significant pain alleviation and performance status improvement were obtained in 5 of 12 patients (41.7%; CI 13.8-69.6%) and 3 of 13 patients (23.1%; CI 0.2-46.0%), respectively. Although myelosuppression was moderate to severe, no chemotherapy-related deaths or bacteriologically documented sepsis occurred; nor was there any clinical cardiotoxicity. All the responding patients received maintenance chemotherapy with etoposide thereafter. At present, the median duration of response is 33 weeks (range: 23-91 weeks) and the median survival period for all patients is 42 weeks (range: 27(+)-136 weeks), with 12 deaths. In spite of the small number of patients treated, these results suggest that this chemotherapy regimen is active in advanced hormone-refractory prostate carcinoma.

Adenocarcinoma↗

PubMed1995

Chondromatosis within a meniscal cyst of the knee.

A 32-year-old man complained of left gonalgia for 2 years and noticed a soft part tumor on the lateral side of his left knee. Roentgenograms showed some small calcified shadows at the same site of the tumor. Arthroscopy revealed a lateral meniscus to be an incomplete discoid with degenerative tears. At the operation, a multilocular soft part tumor was noticed in continuity with the lateral meniscus macroscopically. Small, loose bodies and gelatinous fluid were found in the cavity of the tumor. Histologically loose bodies were chondroma and the soft part tumor was meniscal cyst. The meniscal cyst wall contained hyaline cartilagenous tissue. Therefore it was thought that chondroma originated from the cyst wall.

Adult↗

PubMed1995

Severe mitochondrial cardiomyopathy and extra-neuromuscular abnormalities in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS).

An autopsy case of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) in a 56 yr-old woman is reported. Histopathologic abnormalities are shown widely in the heart, liver, kidney, pancreas and thyroid gland, other than the central nervous system and skeletal muscles that had been so far emphasized in the cases of MELAS. Particularly in the myocardium, focal fibrosis and a disarray of myofibrils were demonstrated, which closely resembled that seen in idiopathic hypertrophic cardiomyopathy. In addition, unique mitochondrial abnormality exhibiting a central glycogen aggregation with surrounding multiple radiating cristae was noted in some cardiomyocytes, which has never been reported in previous cases of MELAS. Thus, in MELAS, various histopathological abnormalities including the mitochondrial abnormalities may involve tissues other than those of the neuromuscular system.

Abnormalities, Multiple↗

PubMed1995

Polymyositis associated with thymoma and the subsequent development of pure red cell aplasia.

A 53-year-old woman with polymyositis associated with thymoma subsequently developed pure red cell aplasia (PRCA). She was hospitalized because of fever and muscle weakness, and diagnosed as having polymyositis by muscle biopsy. Remarkable clinical improvement followed administration of prednisolone. Progressive anemia became evident, however, while prednisolone was being tapered. Erythroid aplasia and the presence of thymoma confirmed the diagnosis of PRCA. Further examinations revealed that cytotoxic T cells may play an important role in the pathogenesis of this case.

Cytotoxicity, Immunologic↗

PubMed1995

Relation between age-related changes in hyper-emotionality and serotonergic neuronal activities in the rat limbic system.

To examine the relationship between higher concentrations of 5-HT and 5-HIAA in the aged rat brain and their hyper-emotionality, behavioral and neurochemical studies were performed. In the behavioral studies, under novel circumstances, the aged rats showed an significant increase in defecation. Furthermore, their locomotor activity was much less than that of young rats, and the numbers of head-dips of aged rats were significantly decreased compared to those of young rats. In the neurochemical studies, the concentrations of 5-HT and 5-HIAA in the limbic system (e.g., hippocampus, amygdala and septal area) of aged rats were much higher than those of young rats (P < 0.001) under normal conditions. The possibility that the hyper-emotionality of aged rats might be related to the hyper-activities of serotonergic neurons in their limbic system were taken into consideration when the results were analyzed.

Aging↗

PubMed1995

A hemidesmosomal transmembrane collagenous molecule, the 180-kDa bullous pemphigoid antigen (BPA II), is phosphorylated with 12-O-tetradecanoylphorbol-13-acetate in a human squamous cell carcinoma cell line (DJM-1).

We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.

Amino Acids↗

PubMed1995

The prevalence of diabetic complication of elderly diabetics in Himeji.

Diabetes mellitus has recently markedly increased among elderly patient's diseases. There are no recent epidemiological reports on the relative number of male and female diabetic patients. So, an epidemiological study was performed on 746 Non-Insulin-Dependent Diabetes Mellitus patients, whose data were obtained from members of the Himeji Internal Medicine Association, divided into six groups according to sex and duration of illness. The following results were obtained. 1) The number of male patients was greater by about 20% than that of female patients, while elderly patients accounted for a larger proportion, nd age at onset of disease was about ten years higher in female than in male patients. 2) All indicators of diabetes mellitus became worse with longer duration of illness. 3) There was a correlation between the prevalence of complications and the duration of illness: The prevalence of complications increased in parallel with increasing duration of illness, and this tendency was more marked in female than in male patients. 4) Female patients had a more marked tendency to develop hypertension, hyperlipidemia and obesity than male patients. 5) Microangiopathy generally manifested itself earlier than macroangiopathy, and the increase in the prevalence of angiopathy in accordance with prolonged duration of illness was more marked for microangiopathy than for macroangiopathy. Clinical features of Japanese diabetics are found to be similar to those of Europeans, especially dominant in females. This might be due to the changing life style in japan.

Aged↗

PubMed1994

Novel subtype of human angiotensin II type 1 receptor: cDNA cloning and expression.

Angiotensin II (AII) plays a major role in regulation of cardiovascular function and fluid homeostasis through the action of an AII type 1 receptor (AT1R). The cDNA encoding a novel subtype of human AT1R (AT1bR) was cloned from a human placental cDNA library. The full-length cDNA clone (1563 bp) encoded a polypeptide that consists of 359 amino acid (aa) residues with 97.2% aa identity to the human AT1aR. All the aa replacements between two human AT1Rs reside within the C-terminal half region of AT1R molecule. The 2.4-knt AT1bR mRNA is expressed in the lung, placenta and liver, and differs from AT1aR mRNA in its tissue distribution. The AT1bR expressed in COS-7 cells is pharmacologically distinct from the human AT1aR.

Amino Acid Sequence↗

PubMed1994

Novel subtype of human angiotensin II type 1 receptor: analysis of signal transduction mechanism in transfected Chinese hamster ovary cells.

We examined the intracellular signal transduction of two subtypes of human angiotensin II type 1 receptor (AT1aR and AT1bR) by means of the stable expression of each receptor cDNA in Chinese hamster ovary cells. Both receptors showed a rapid stimulation of phosphatidylinositol hydrolysis, a transient increase of intracellular Ca2+ concentration and an inhibitory action on the forskolin-induced cyclic AMP formation. Interestingly, at high AII concentrations (> 1 microM), these AT1bR-mediated responses were inhibited, whereas the AT1aR-mediated responses were not. Thus, the two AT1Rs are considered to be coupled to the same signal transduction cascades, but to be regulated differently on the post-translational level (presumably desensitization).

Angiotensin II↗