[Biochemical and molecular biological studies on renin-angiotensin system].
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Amino Acid Sequence↗
Biomedical subjects
K Murakami
Publications and source records attributed to K Murakami.
[Developmental biotechnology and hypertension--emphasis on renin-angiotensin system].
The recent remarkable progress in molecular biology and developmental biotechnology has contributed to the evaluation of mechanism of hypertension. The renin-angiotensin system (RAS) is defined as an endocrine substance acting on blood pressure regulation. Accumulating evidence suggests the existence of tissue RAS in various organs, such as the brain, heart, vascular walls, adrenal gland, testis and ovary, in addition to the circulating RAS. However, it remains unclear how the RAS affects those tissues and high blood pressure. The advent of new developmental biotechnology gives an additional dimension to hypertension research. Such approach includes the generation of transgenic animals carrying the component of RAS genes and it should contribute to the elucidation of the RAS action in tissue function. In this manuscript, transgenic animals, produced to date, with emphasis on hypertension is introduced.
Animals↗
Two cases of Goodpasture's syndrome--clinicopathological studies and relapse.
The requirements for a diagnosis of Goodpasture's syndrome (GPS) include: (1) the presence of glomerulonephritis, (2) alveolar bleeding, and (3) the presence of anti-glomerular basement membrane (anti-GBM antibody). Among Japanese case, the 2 patients reported here were rare in that they satisfied all of these requirements. In case 1 (a 40-year-old male), the disease developed with initial signs of proteinuria and hematuria. The patient developed hemoptysis after hospitalization. The interval from onset to hospitalization was 38 days. The serum creatinine (CRN) level was 3.6 mg/dl on admission. The pathological findings were rated as full-circumferential, cellular crescentic nephritis, and the patient did not display oliguria. The renal and pulmonary impairments in this case were markedly improved by glucocorticoid (prednisolone PSL, 60 mg/day), cyclophosphamide therapy (50 mg/day) and plasma exchange (PE 10 times). In case 2 (a 58-year-old male), the initial signs developed as proteinuria and hematuria, followed by rapidly progressive renal functional deterioration and hemoptysis occurred. Compared to Case 1, the interval from onset to hospitalization was longer in Case 2 (125 days) and the CRN level was also higher (10.7 mg/dl). Case 2 was rated as full-circumferential, fibrous crescentic nephritis, and the patient was oliguric. Although the pulmonary impairment was reduced by pulse therapy and 10 PEs, recovery of renal function has not been achieved, still necessitating maintenance hemodialysis at present. In case 1, the disease relapsed at 4.5 years after remission, presenting with aggravated renal function and hemoptysis. In this case, low-dose PSL therapy had been discontinued at 3 months before the relapse.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
[Focal glomerular sclerosis in a sibling].
We described here one sibling with focal glomerular sclerosis. Proteinuria was noticed at the age of five in brother and four in sister. Both of them developed nephrotic syndrome shortly after the discovery of proteinuria. The nephrotic syndrome was resistant to corticosteroid, immunosuppressive agents or the combination of these drugs. Percutaneous renal biopsy in them revealed morphological and immunohistological features compatible to focal glomerular sclerosis. HLA typing in HLA-A, B, C and DR loci was identical to both. This observation suggests that genetic factors is associated with the pathogenesis of focal glomerular sclerosis.
Adolescent↗
[IgA nephropathy with acute exacerbation of idiopathic interstitial pneumonia: an autopsy case].
A 73-year old man was admitted to the hospital because of dyspnea. His proteinuria was pointed out in 1981. He felt dyspnea after exertion and abnormal chest X-ray shadow was pointed out in 1984. He was admitted to our hospital for further examination for the first time in 1988. He was diagnosed as IgA nephropathy by renal biopsy, and idiopathic interstitial pneumonia (IIP) by transbronchial lung biopsy. Then steroid therapy was begun because of progression of renal dysfunction in November 1990. But thereafter dyspnea, hypoxia and fine diffuse reticulonodular shadow on chest X-ray film was exacerbated. And he died because of respiratory failure. In this case, progression of IgA nephropathy seemed associated with exacerbation of IIP.
Aged↗
Inhibition of brain protein kinase C subtypes by lead.
Protein kinase C (PKC) is an important enzyme in mediating cellular signal transduction and neuronal plasticity. Extremely low concentrations (picomolar range) of Pb++ have been reported to activate partially purified PKC from rat brain (Markovac and Goldstein, 1988). However, the lead activation of PKC at such low concentrations is still a matter of discussion (Simons, 1989). To clarify this point, we have examined the lead effect on highly purified PKC subtypes. Pb++ was found to be a potent inhibitor for all three PKC subtypes (types I, II and III) with IC50 of 2 to 10 microM. Characterization of this lead inhibition of PKC suggests that 1) the inhibition is not due to the competition with Ca++, 2) the site of action of lead is on the catalytic domain of PKC, 3) the inhibition is not dependent on the mode of activation (phosphatidylserine/diacylglycerol vs. cis-unsaturated fatty acid) and 4) the inhibition is totally reversible.
Animals↗
c-Ets-1 protein is hyperphosphorylated during mitosis.
The ets-1 and ets-2 proto-oncogene products can serve as transcription factors and become phosphorylated in response to Ca(2+)-mediated signals. We have examined expression of Ets proteins during the cell cycle in cells synchronized by centrifugal elutriation or nocodazole-induced mitotic block. Both methods revealed the presence of a hyperphosphorylated isoform of Ets-1 during the mitotic phase. This isoform showed a characteristic mobility shift and was observed during mitosis in each of four cell lines (three human T-cell lines and a human astrocytoma) that express ets-1. In elutriated cells, only a small portion of the Ets-1 in cells from the G2/M fractions was hyperphosphorylated, while in nocodazole-arrested cells more of the Ets-1 was shifted. When cells were released from nocodazole arrest, this isoform disappeared within 1-2 h as cells completed mitosis and entered G1. This suggests that hyperphosphorylated Ets-1 is present transiently during early mitosis, before or around the time of the metaphase-anaphase transition. Exposure of unsynchronized cells to okadaic acid resulted in a dramatic hyperphosphorylation of virtually all Ets-1, suggesting that changes in cellular phosphatase activity are important for cell cycle regulation of Ets-1. Hyperphosphorylated Ets-1 appears to arise from multiple phosphorylations on serine in the exon 7-encoded domain of the protein and did not appear to alter sequence-specific DNA-binding activity. Although enhanced phosphorylation of Ets-2 was detected in nocodazole-arrested cells, no Ets-2 hyperphosphorylation was seen.
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Transforming growth factor-beta protein and mRNA in glomeruli in normal and diseased human kidneys.
BACKGROUND: Evidence indicates a key role for transforming growth factor-beta (TGF-beta) in the accumulation of pathologic extracellular matrix in experimental glomerular injury. The aim of this study was to elucidate the expression of TGF-beta and its role in human glomerulonephritis. EXPERIMENTAL DESIGN: Expression of TGF-beta 1 in normal and diseased human kidneys was examined by immunohistochemical staining with two antibodies (Ab1 and Ab2), and by in situ hybridization with an oligonucleotide probe. RESULTS: Staining with Ab1, which mainly recognizes mature TGF-beta 1 and the latency-associated peptide (LAP) of natural TGF-beta 1, was linearly positive along the glomerular basement membrane (GBM) and weakly so in the mesangium of normal tissues and those of various glomerular diseases which were pretreated with acid-urea to unmask a hidden epitope. Ab2, which reacts mainly with TGF-beta-LAP, bound to the mesangium and sclerotic areas of the tissues untreated with acid-urea. Immunoelectron microscopy showed that Ab1 was localized to the GBM and the mesangial matrix, and that Ab2 was distributed in subepithelial, or mesangial/paramesangial electron-dense deposits. The presence of mature TGF-beta 1 and TGF-beta-LAP in normal kidneys was confirmed by immunoblotting using guanidine-extracted fractions of glomeruli and GBM isolated from normal human kidneys. Mesangial staining of TGF-beta 1 with Ab2 was significantly correlated with the mesangial matrix increase in mesangial proliferative types of nephritis. In situ hybridization revealed TGF-beta 1 mRNA expression in glomerular cells. Cells with positive mRNA signals were evident in glomeruli that were increased in both mesangial cells and TGF-beta 1 protein expression. The glomerular cells with positive signals were numerous, compared with the number of infiltrating monocyte-macrophages identified with a monoclonal antibody. CONCLUSIONS: These results indicate that mature TGF-beta and TGF-beta-LAP are localized in association with the matrix components of GBM or mesangium, and with immune deposits in human glomeruli. Glomerular expression of TGF-beta is enhanced in human glomerular diseases, and may contribute to the mesangial matrix increase.
Adolescent↗
[Radioimmunodetection of colorectal cancer, using anti-CEA monoclonal antibody CEA102: experience of 20 cases].
The monoclonal antibody CEA 102 against carcinoembryonic antigen was produced by immunization with purified CEA, and used as a radiotracer for the imaging of colorectal cancer. CEA102 was labeled with 131I by the chloramine-T or the Iodogen method, and administered i.v. to 20 patients with liver metastases, local recurrences, and/or primary tumors. Planar scintigraphy was performed during 5 or 6 postinfusion days. Hypersensitivity reactions such as fever and chill occurred in 4 patients, but no side effects were noted after endotoxin was excluded. Overall sensitivity was 71.4% (primary tumor 4/4, liver metastasis 5/6, lymph node metastasis 1/1, local recurrence 5/10), and no false positive was observed. In the cases of local recurrences, this method was useful to distinguish postoperative fibrosis from local recurrence. These results revealed that radiolabeled CEA102 has a great potential in qualitative diagnosis of colorectal cancer.
Adult↗
Dependence of angiotensin production in transgenic mice carrying either the human renin or human angiotensinogen genes on species-specific kinetics of the renin-angiotensin system.
In order to study the functional role of the renin-angiotensin system in the control of blood pressure, projects were initiated using transgenic mice carrying either the human renin gene or the human angiotensinogen gene. To extend the usefulness of the transgenic model system of hypertension research, a two-tiered strategy for generation of transgenic mice with high blood pressure has been developed. This unique system allows the establishment of transgenic lines with a strict species specificity of the renin-angiotensinogen reaction exhibited by transgene products that are regulated in a tissue specific manner. In this report, the strategy concerning the "two-tiered method" is presented and evidence is provided the overproduction of angiotensin occurs only in the combined reaction with human renin and human angiotensinogen expressed in transgenic mice.
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Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family.
We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.
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Levels of androgens and danazol metabolites in serum during danazol therapy.
Thirteen women who had endometriosis were treated with danazol (300 to 400 mg/d). Levels of androgens and danazol metabolites in serum and the influences of danazol metabolites on the assays for serum androgen were investigated during danazol therapy. Serum DHEAS significantly increased (P < 0.05), but serum DHEA slightly decreased. Serum T levels, measured by direct assay, were markedly elevated. However, measured after HPLC separation, the T levels in serum were significantly decreased (P < 0.05). There were considerable cross-reactions between danazol metabolites and androgens (T, DHEA, and A) in RIA. Purification of androgens using column chromatography was necessary to measure serum androgens precisely.
Adult↗
Comparison of ascorbic acid and ascorbic acid 2-O-alpha-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts.
Ascorbic acid 2-O-alpha-glucoside (AA-2G) is a stable ascorbate derivative which has vitamin C activity in vivo and in vitro. We studied whether AA-2G exerts a prooxidant action in cultured fibroblasts from chick embryo and human skin, as does ascorbic acid. At concentrations of 0.1-1.0 mM, ascorbic acid markedly reduced the viable cell number of low density cultures within 24 hr, whereas AA-2G had no such effect. The ascorbate cytotoxicity was dependent on the cell density at the time of its addition and it was characteristic of low density cultures. This cytotoxicity was completely prevented by catalase and partially by an Fe3+ ion chelator, desferrioxamine. In the early culture stage at which a morphological change in the fibroblasts began to occur, intracellular ascorbate concentrations in low density cultures after addition of ascorbic acid were much higher than in high density cultures. However, at the same concentrations, AA-2G did not cause an elevation even in low density cultures and it was also effective on collagen synthesis at high and medium densities. These results suggest that the abnormally accumulated ascorbic acid in the cells cultured at low density possibly amplifies the generation of oxygen radicals through the reduction of Fe3+ ions and subsequent oxidative reactions, leading to cell death. Therefore, it is concluded that AA-2G which supplies an adequate amount of ascorbic acid during culture period is a bioavailable ascorbate source without cytotoxicity.
Animals↗
A carboxyl-terminal truncated version of the activin receptor mediates activin signals in early Xenopus embryos.
The function of a carboxyl-terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of XSTK2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the alpha-actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor.
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Distribution of the piriform cortical terminals to cells in the central segment of the mediodorsal thalamic nucleus of the rat.
A Golgi electron microscopic study was undertaken to investigate the distribution of terminals from the piriform cortex that synapse on identified dendrites of neurons in the central segment of the mediodorsal thalamic nucleus of the rat. The piriform cortical terminals were identified as degenerating terminals following lesions in the cortex. They consisted of two types, i.e., large (LR type) and small (SR type) presynaptic terminals, both of which had round synaptic vesicles and formed asymmetric synaptic contacts. SR boutons terminated preferentially onto distal dendrites and never synapsed on primary dendrites. LR terminals synapsed preferentially on proximal dendrites, but were also found on more distal dendritic segments.
Animals↗
Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli.
Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated. Their defective loci were classified into two groups, AlaA- and AlaB-. The genes that complemented these mutations were cloned. Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E. coli chromosome and encodes glutamate 1-semialdehyde aminotransferase. The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway. Thus, we designated the gene hemM. The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase. However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA. These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.
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[Assessment of a coaxial system accommodated to a 0.035 inch guide wire in superselective hepatic angiography and embolization].
A special coaxial catheter system accommodated to a 0.035 inch guide wire was recently developed. The four-part coaxial system is composed of a central 0.035 inch flexible guide wire, a 4-French inner catheter (straight, 90 cm in length), a 6-French outer catheter (fork-shaped, 60 cm in length), and a hemostatic valve. The 4 French inner catheter is large enough in diameter to allow a larger volume of contrast medium and greater amount of embolic material than the earlier coaxial system. Using this coaxial catheter system, we performed successful superselective hepatic arteriography and embolization in 44 patients with malignant hepatic tumors. Catheterization of the celiac and superior mesenteric arteries with the 6 French outer catheter was easy, and the angiogram obtained was very distinct. The 4 French inner catheter was easily and safely advanced into the segmental hepatic artery, and even common hepatic arteriography with this catheter provided clear images on conventional cut films. Therefore, in most cases, both angiography and embolization can be accomplished using this coaxial system alone.
Aged↗
Renin assay using a fluorogenic substrate and high performance liquid chromatography.
Measurement of renin activity in human fluids using a fluorogenic substrate and high performance liquid chromatography (HPLC) is described. A nine amino acid peptide containing the fluorogenic residue, N-(2-pyridyl) glycine (Pg) is used as a substrate. The peptide sequence is homologous with the cleavage site of human angiotensinogen. This substrate is hydrolyzed by renin to generate fluorogenic and non-fluorogenic products. The amount of fluorogenic product is directly measured by reversed phase HPLC. Optimization of assay conditions and measurement of human serum renin levels are described. Assay results correlated well with those from radioimmunoassay. The method is simple, convenient, highly sensitive and can be used for routine clinical renin assays.
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