Accumulation of grisorixin caused by treating a nigericin-producing strain with a P-450 inhibitor.
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Cytochrome P-450 Enzyme Inhibitors↗
Biomedical subjects
A Ichihara
Publications and source records attributed to A Ichihara.
Proteasomes: protein and gene structures.
Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides. These particles have a latent multicatalytic proteinase activity. Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin. This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells. We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution. Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene. Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell. In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.
Amino Acid Sequence↗
Alterations in sex hormones and sexual function of patients with renal failure treated with recombinant human erythropoietin.
Since it has been reported that correction of anemia in long-term hemodialysis patients by using human recombinant erythropoietin (r-HuEPO) is associated with improve sexual function, we conducted the present study to evaluate the changes in sex hormones as well as sexual function after r-HuEPO administration (1500 to 4500 IU per dialysis) for a year in patients on regular hemodialysis. Thirteen patients receiving regular hemodialysis entered this study. Their median age was 43 years. Along with correction of anemia (the hematocrit increased from 20 to 28%), testosterone (T) increased from 2.4 +/- 0.1 to 2.6 +/- 0.2 ng/ml, follicular stimulating hormone (FSH) increased (29 +/- 5 to 73 +/- 7 mIU/ml), luteinizing hormone increased (69 +/- 14 to 160 +/- 21 IU/ml) and prolactin decreased (all changes are significant at p less than 0.05). However, the improvement of sexual function was not remarkable. Only 25% of the uremic patients treated with r-HuEPO showed amelioration of this function. From the present data, it does not seem likely that therapy with r-HuEPO induces directly amelioration of sexual function through changes in sex hormones.
Adult↗
Changes in expressions of proteasome and ubiquitin genes in human renal cancer cells.
Proteasomes and ubiquitin (Ub) are essential components of the energy-dependent, nonlysosomal proteolytic pathway. To clarify the physiological role of this proteasome/Ub-dependent pathway, we meaured the levels of expressions of proteasomes and Ub in human renal cancers by Northern blot and immunochemical analyses. The mRNAs for two of the multiple subunits of proteasomes, C2 and C9, were expressed at abnormally high levels in most neoplastic lesions of patients with various primary renal cell carcinomas and in all renal cancer cell lines examined. However, no significant difference was found by enzyme immunoassay in the proteasomal contents of cancerous and normal parts of the kidney. The levels of mRNAs for the subunits of proteasomes were high in rapidly proliferating renal cells and appeared to be correlated with the activities of these cells for proteasome synthesis, but the cellular contents of proteasomes in these cells were normal, suggesting rapid turnover of proteasomes in rapidly proliferating cancer cells. Consistent with the increased expressions of proteasomal mRNAs, the expressions of three Ub genes, mono-UbA80, mono-UbA52, and poly-UbC, were found to be greatly increased in these renal cancer cells. Immunohistochemical staining of normal kidney showed that the levels of both proteasomes and Ub were high in cells of renal tubules and collecting ducts, but low in the glomerulus. The levels of both proteins appeared to be considerably increased in the nuclei of granular and clear carcinoma cells of the kidney. Moreover, the profiles of cellular proteins conjugated with Ub in normal kidney tissues were different from those in cancerous parts of the kidney and in established renal cancer cells. These results suggest that the proteasome- and ubiquitin-mediated system is functionally involved in the cancerous state in human kidney.
Adult↗
Electron microscopy of 26 S complex containing 20 S proteasome.
A high molecular weight protease complex (26 S complex) involved in the intracellular protein degradation of ubiquitinated proteins was purified from rat liver and studied by electron microscopy. The most prevalent molecular species with best preserved symmetrical morphology had two large rectangular terminal structures attached to a thinner central one having four protein layers. We concluded that they were the closest representation of the 26 S complex so far reported. The central structure was identified as 20 S proteasome and the terminal one as recognition units for ubiquitinated proteins.
Animals↗
Improved method for preparation of ubiquitin-ligated lysozyme as substrate of ATP-dependent proteolysis.
A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat-denatured 125I-labeled lysozyme was highly ubiquitinated by incubation at pH 9.0 with a ubiquitin-protein ligase system consisting of E1, E2 and E3 that had been partially purified from rabbit reticulocytes by affinity chromatography with ubiquitin as a ligand. The resulting conjugates were separated from free lysozyme and other proteins by successive chromatographies on anion and cation ion-exchange resins. The ubiquitinated 125I-lysozymes recovered in the fraction not adsorbed to either resin served as an efficient substrate for ATP-dependent proteolysis in a reticulocyte lysate or with a purified 26 S protease complex. By the present method, 125I-lysozyme-Ub conjugates can be prepared in 3 h with a high yield of 15-20%.
Adenosine Triphosphate↗
Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle.
The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.
Aging↗
Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.
The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.
Amino Acid Sequence↗
Molecular cloning of the ecotin gene in Escherichia coli.
The nucleotide sequence of a 876 bp region in E. coli chromosome that encodes Ecotin was determined. The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18,192 Da. The deduced primary sequence of Ecotin includes a 20-residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16,099 Da. Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor.
Amino Acid Sequence↗
Cell-density dependent expression of the c-myc gene in primary cultured rat hepatocytes.
During culture of mature rat hepatocytes as monolayers, c-myc mRNA was found to be expressed transiently within 2 h, decreasing rapidly to the basal level at 10 h. Then its level increased again to over 10-fold the basal level at 24 h, and remained at this high level during culture. The increase of c-myc mRNA in the second phase was shown by nuclear run-off experiments to be due to an increase of its transcription. The second, but not the first, increase in c-myc expression was inversely proportional to the cell density in culture. The expression of c-myc mRNA was not affected by various hormones including growth factors. These results indicate that hepatocytes in culture at lower cell density tend to move from the Go phase to the G1 phase, but remain in the Go phase when cultured at high cell density.
Animals↗
Molecular cloning of cDNA for the import precursor of human coupling factor 6 of H(+)-ATP synthase in mitochondria.
The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat factor 6 as a probe. The sequence was composed of 466 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions on the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame were found to consist of 108 and 76 amino acid residues with molecular weights of 12,596 and 8,969, respectively. The presequence of 32 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.
Adenosine Triphosphatases↗
ATP-dependent reversible association of proteasomes with multiple protein components to form 26S complexes that degrade ubiquitinated proteins in human HL-60 cells.
The role of proteasomes in ubiquitin (Ub)-dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL-60 cells by glycerol density gradient centrifugation. High succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide hydrolyzing activity was found in the 26S fraction, whereas the 20S fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26S, but not the 20S fraction catalyzed ATP-dependent degradation of [125I]lysozyme-Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26S complex to the latent 20S form, whereas in the lysate prepared from ATP-depleted cells, ATP converted 20S proteasomes to 26S complexes. The immunoprecipitated 26S complexes were found to consist of proteasomes and 13-15 other proteins ranging in size from 35 to 110 kDa. We conclude that in the lysate, latent proteasomes undergo reversible, ATP-dependent association with multiple protein components to form 26S complexes that catalyze ATP-dependent degradation of Ub-protein conjugates.
Adenosine Triphosphate↗
Molecular cloning and sequence analysis of cDNAs for five major subunits of human proteasomes (multi-catalytic proteinase complexes).
Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components. Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes. The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes. The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low. These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.
Amino Acid Sequence↗
Protease Do is essential for survival of Escherichia coli at high temperatures: its identity with the htrA gene product.
The DNA encoding protease Do was isolated from an E. coli genomic DNA library in lambda gt11, and cloned into a Bluescript plasmid. The cells transformed with the recombinant plasmid were able to overproduce protease Do and grew normally. A mutant lacking the protease activity was also isolated by interrupting the chromosomal DNA with the kan gene. The mutant showed a prolonged lag period and reduced ability to degrade cell proteins as compared to its wild type. Moreover, they were unable to survive at high temperatures, similarly to the htrA mutants. These results suggest that protease Do may play an important role in the intracellular protein breakdown and is essential for survival at high temperatures. Identity of protease Do with the htrA gene product is discussed.
Bacterial Proteins↗
Identification of glucocorticoid- and cyclic AMP-responsive elements of the rat serine dehydratase gene: difference in responses of the transfected and chromosomal genes.
Transcription of the gene coding for serine dehydratase (SDH, EC 4.2.1.13) in rat liver is induced 3-4 fold by glucocorticoids plus glucagon, but not by either hormone alone. For identification of the DNA elements mediating the glucocorticoid- and cyclic AMP-regulated expression of the SDH gene, primary cultures of adult rat hepatocytes were transfected with a fusion gene consisting of the 2.15 kb 5'-flanking sequence of the SDH gene linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). CAT assay demonstrated that transient expression of the SDH-CAT fusion gene was inducible by either dexamethasone or dibutyryl cyclic AMP, but that the effects of these inducers were not additive or synergistic. These results suggest that some structural organization of the DNA influences the hormonal actions in regulation of gene expression.
Animals↗
Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells.
Hepatocyte growth factor (HGF), which is a potent growth factor of adult rat hepatocytes in primary culture, also strongly stimulated DNA synthesis of rabbit renal tubular epithelial cells in secondary culture. Its mitogenic activity was dose-dependent, being detectable at 3 ng/ml and maximal at 30 ng/ml. Over 20% of the cells were shifted to the S-phase by HGF alone, judging by the labeling index. HGF had additive effects with EGF, acidic fibroblast growth factor (a-FGF), and insulin. Transforming growth factor-beta 1 (TGF-beta 1) strongly inhibited DNA synthesis of renal tubular cells stimulated by HGF. The growth of renal tubular epithelial cells was also regulated by cell density: DNA synthesis stimulated by HGF was high at lower cell density and was strongly suppressed at high cell density. These results suggest that HGF may act as a renotropic factor in compensatory renal growth or renal regeneration in vivo.
Animals↗
Mechanisms controlling growth of hepatocytes in primary culture.
Mature hepatocytes in primary culture express most of the functions and hormonal responsiveness seen in normal liver studied in vivo. The growth of hepatocytes in culture is regulated by various growth factors. We have identified a hepatocyte growth factor that is isolated from rat platelets. We found that rat platelets also contain a growth inhibitor, transforming growth factor-beta which is secreted as a latent molecule. Its latency is due to its binding with a masking protein. Growth of hepatocytes is also suppressed by interleukin-1 (IL-1) and IL-6. Moreover, the growth and functions of liver cells in culture are regulated reciprocally by cell density: at higher cell density liver-specific functions are expressed and growth is suppressed, whereas the opposite situation is observed at lower cell density. In contrast, neonatal hepatocytes in culture grow autonomously without a requirement for added hormones. This autonomous growth is due to an autocrine mechanism in which the cells secrete one or more growth factors into the culture medium. However, this autonomous growth ceases one week after birth at a time when the cells begin to express differentiated characteristics. Based upon these data, the mechanisms of liver regeneration, differentiation, and hepatocarcinogenesis are discussed.
Animals↗
Effects of L-arginine on systemic and renal haemodynamics in conscious dogs.
1. The effects of L-arginine on systemic and renal haemodynamics were investigated in conscious dogs. L-Arginine was administered intravenously at doses of 15 and 75 mumol min-1 kg-1 for 20 min. 2. Mean arterial blood pressure, heart rate and cardiac output were not changed significantly by L-arginine infusion. However, L-arginine infusion induced a significant elevation of renal blood flow from 50 +/- 3 to 94 +/- 12 ml/min (means +/- SEM, P less than 0.01). 3. Simultaneous infusion of NG-monomethyl-L-arginine (0.5 mumol min-1 kg-1) significantly inhibited the increase in renal blood flow produced by L-arginine (15 mumol min-1 kg-1) without significant changes in mean arterial blood pressure or heart rate. 4. Pretreatment with atropine completely inhibited the L-arginine-induced increase in renal blood flow, whereas pretreatment with indomethacin attenuated it (63 +/- 4 versus 82 +/- 10 ml/min, P less than 0.05). 5. A continuous infusion of L-arginine increased renal blood flow in the intact kidney (55 +/- 3 versus 85 +/- 9 ml/min, P less than 0.05), but not in the contralateral denervated kidney (58 +/- 3 versus 56 +/- 4 ml/min, P greater than 0.05). 6. These results suggest that intravenously administered L-arginine produces an elevation of renal blood flow, which may be mediated by facilitation of endogenous acetylcholine-induced release of endothelium-derived relaxing factor and vasodilatory prostaglandins.
Animals↗