Biomedical subjects

A Ichihara

Publications and source records attributed to A Ichihara.

At least 19 recordsLinked to original sources

PubMed1996

Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma.

Interferon (IFN) gamma induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair of proteasome subunits expressed reciprocally in response to IFN-gamma. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-gamma, showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-gamma. Thus, IFN-gamma induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, beta-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-gamma may be responsible for acquisition of its functional change.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of two types of cDNA encoding subunit RC6-I of rat proteasomes.

A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27,783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the alpha-type subfamily of the proteasome gene family, which shows similarity to the alpha-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other alpha-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions.

Amino Acid Sequence↗

PubMed1995

Long-term maintenance of functional rat hepatocytes in primary culture by additions of pyruvate and various hormones.

Mature adult rat hepatocytes were cultured as monolayers in serum-free Williams medium E containing 10(-7) M each of insulin (Ins), dexamethasone (Dex) and triiodothyronine (T3) and 30 mM pyruvate. The hepatocytes remained morphologically intact for at least 14 days, during which period they maintained normal liver functions such as the expressions of cytochrome P-450 mRNA and glucokinase and secretion of albumin. They also retained the ability to resume proliferation. Cells cultured with pyruvate had a much higher ATP level than those without pyruvate, suggesting that pyruvate can sustain functional hepatocytes for a long period in culture in the presence of Ins, Dex and T3, probably by producing enough energy for their maintenance.

Adenosine Triphosphate↗

PubMed1995

Roles of proteasomes in cell growth.

Proteasomes are large, unique protein complexes catalyzing energy- and ubiquitin-dependent proteolysis. Recent studies have revealed that these complexes are involved in two important cellular functions. One is to make antigen fragments for major histo-compatibility complex (MHC) class I-restricted antigen presentation and the other is to regulate the cell cycle by proteolysis. Here we review only the latter function of proteasomes. Proteasomes are widely distributed in eukaryotic cells, but their levels have been shown to be particularly high in various immature cells, such as cancerous, fetal and lymphoblastic cells, and agents including cell differentiation were found to suppress their expression. These conditions also regulate the expression of ubiquitin genes in a similar way, suggesting that proteasomes act ubiquitin-dependently in their 26S form in immature cells. High levels of proteasomes were found immunochemically in the nuclei of rapidly growing cells, indicating that proteasomes are important for eukaryotic cell growth. Indeed, gene disruptions of most subunits of proteasomes in yeast resulted in total suppression of cell growth and cell death. Short-lived regulatory factors of the cell cycle, such as Fos, p53, Mos, and cyclins are degraded by the proteasome-ubiquitin pathway under phosphorylated or dephosphorylated conditions. Ornithine decarboxylase, which is also a short-lived enzyme and is involved in the early phase of cell growth, is quickly degraded by proteasomes with antizyme, but without ubiquitination. Recently, we found that one of the regulatory factors of 26S proteasomes, p31, is a homologue of Nin1p, whose mutation caused inhibition of the cell cycle in yeast. These results indicate that proteasomes play important roles in regulation of the cell cycle in eukaryotes.

Amino Acid Sequence↗

PubMed1995

Status of patients who underwent uninephrectomy in adulthood more than 20 years ago.

We investigated the status of patients without systemic diseases who had undergone uninephrectomy for unilateral renal diseases in adulthood more than 20 years ago at Tokyo Denryoku Hospital. There were 21 participants (mean age +/- SD, 58.6 +/- 8.0 years) who fulfilled these criteria. The average interval since nephrectomy was 27.9 +/- 6.2 years. The mean current creatinine clearance was 88.5 +/- 21.2 mL/min/1.73 m2, which is 92.9% of that in healthy age- and sex-matched controls with two kidneys. The 24-hour urine protein excretion in these patients was only slightly higher than in the controls (214 +/- 190 mg v 119 +/- 62 mg, P = NS). Age at nephrectomy, length of time with a single kidney, or sex had little effect on the remnant renal functions. There was a positive correlation between current mean arterial pressure and serum creatinine (r = 0.44, P < 0.05). Patients who developed hypertension after uninephrectomy had a family history of hypertension more frequently than those with normotension (86% v 29%, P < 0.05). We conclude that (1) renal function after compensatory hyperfiltration of more than 20 years due to uninephrectomy for unilateral renal diseases in adulthood is well maintained, although hypertension has a considerable effect on the renal functions, and that (2) family history of hypertension plays a key role in determining the incidence of hypertension even in the uninephrectomized patients.

Adult↗

PubMed1995

Ordered structure of the crystallized bovine 20S proteasome.

Eukaryotic proteasomes are multicatalytic proteinase complexes with a molecular weight of 750 kDa, containing, respectively, two copies of a hetero-heptamer of alpha-type subunits and one of beta-type subunits, (alpha 1-7 beta 1-7)2. Proteasome was purified from bovine liver and crystallized into a hexagonal system with cell dimensions of a = b = 121.83(2) A, c = 930.68(6) A. A cylindrical particle size of 122 A diameter and 155 A height was determined from the molecular packing in a unit cell. The crystal gave diffraction spots up to at least 4.4 A resolution, which was the minimum spacing of the camera used. The overall temperature factor of the enzyme was estimated to be in the range of 36.2 to 25.8 A2. These results imply that the enzyme complex has a unique ordered structure comprising multisubunits with two types of hetero-heptamer. This ordered structure may facilitate highly organized cooperation of individual functions of subunits within the enzyme complex.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of the IS3-like element from Thermus aquaticus.

We have cloned and characterized a genetic element (1187 bp) that is responsible for the induction of thermotolerance as well as ompC expression in E. coli. This element (ISLtaq1) was isolated from Thermus aquaticus. DNA and protein data bases were searched with this element (ISLtaq1), which suggested it to be very similar to IS150 belonging to the IS3 family. ORF1, found on ISLtaq1, which encodes 100 amino acids (aa), had a DNA-binding motif: a helix-turn-helix and a leucine zipper. In fact, when the ORF1 protein was overproduced in E. coli, thermotolerance as well as ompC expression was induced.

Adaptation, Biological↗

PubMed1995

Modulation of angiotensin II type 1 receptor mRNA expression in human blood cells: comparison of platelets and mononuclear leucocytes.

The objective of the present study was to quantify the expression of angiotensin II type 1 (AT1) receptor transcripts in human blood cells--platelets and mononuclear leukocytes--from 10 normal healthy volunteers during the alterations in the renin-angiotensin system. A quantitative assay employing reverse transcription-polymerase chain reaction (RT-PCR) was utilized. Oral administration of furosemide, 40 mg for 2 days, under mild salt restriction (50 mEq NaCl/day) for 6 days stimulated the renin-angiotensin system resulting in significant increases in plasma renin activity (PRA) (1.84 +/- 0.12 vs. 1.05 +/- 0.17 ng/l/s; P < 0.01), plasma angiotensin II concentration, and plasma aldosterone concentration (PAC). The ratio of AT1 receptor mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in mononuclear leucocytes was significantly (P < 0.05) increased from the basal level (0.49 +/- 0.05 vs. 0.29 +/- 0.03) (P < 0.01), while in platelets these changes were opposite (0.11 +/- 0.05 vs. 0.25 +/- 0.05) (P < 0.01). Compared to these significant changes, salt loading (200 mEq NaCl/day) for 6 days decreased PRA(0.49 +/- 0.10 vs. 1.05 +/- 0.17 ng/l/s; P < 0.01) and induced the opposite changes in the ratio of AT1 receptor/GAPDH mRNA. These data suggest that AT1 receptors in human blood cells may be of two different types--platelets and mononuclear leucocytes.

Adult↗

PubMed1995

Interactions between angiotensin II and norepinephrine on renin release by juxtaglomerular cells.

While the interactions between angiotensin II (Ang II) and norepinephrine (NE) on cardiovascular responses are well known, their effects on renin responses are not. We determined the renin secretion rate (RSR) and intracellular calcium level in juxtaglomerular cells harvested from Sprague-Dawley rats using a radioimmunoassay and a two-dimensional calcium analyzer. The effect of Ang II and NE was inhibitory on RSR and stimulatory on intracellular calcium. The NE-induced RSR response was amplified in the presence of Ang II (20 nmol/l). The NE-induced intracellular calcium response was also potentiated by the Ang II. There was a significant correlation (r = 0.994, p < 0.0001) between the changes in the RSR and those in intracellular calcium levels. Losartan (0.1 mumol/l). an Ang II type 1 receptor antagonist, blocked the Ang II threshold RSR responses and completely abolished the Ang II-related enhancements. The exclusion of calcium from the buffer reduced the maximal RSR response to NE but did not prevent the enhancement, suggesting the importance of the mobilization of intracellular calcium in the mechanism. The Ang II-induced RSR was amplified in the presence of NE (0.2 mumol/l). The Ang II-induced intracellular calcium response was also potentiated by the NE. A significant correlation (r = 0.996, p < 0.0001) between the changes in the RSR and the changes in intracellular calcium levels was also noted. Prazosin (1 mumol/l), an alpha 1-adrenoceptor antagonist, blocked the NE threshold RSR responses and abolished the agonist-related enhancements. The calcium-free buffer diminished this amplication with a slight decrease in the maximum RSR response to Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1994

Isolation and characterization of alpha-type HC3 and beta-type HC5 subunit genes of human proteasomes.

Eukaryotic proteasomes from an evolutionarily conserved multi-gene family and are thought to have originated from a common ancestral gene and diverged into alpha-type and beta-type subgroups. To understand the molecular basis of the proteasome genes, we isolated and characterized two human proteasome genes econding the alpha-type HC3 and beta-type HC5 subunit. The functional genes for HC3 and HC5 are similar in being approximately 15 kb in length, but differ in having exon numbers of 9 and 6, respectively. Analyses of about 2.5 to 3.0 kb of the 5'-flanking regions of these two genes revealed the absence of TATA and CAAT promoter elements. However, two or three GC boxes were found. By analysis of the transcriptional regulatory activities in the 5'-flanking regions of the two genes, these GC boxes were found to function coordinately as promoters of the two genes. Interestingly, the HC3 gene possesses an additional silencer element in the 5'-upstream region near the first exon. This element is also able to repress the promoter activities of other genes, such as the HC5 and the type 1 glucose transporter genes, irrespective of whether it has a sense or antisense orientation, indicating that it acts as a general transcriptional silencer. The HC5 gene does not have this silence element, and its promoter activity is five to ten times that of HC3. These results show that the human proteasomal HC3 and HC5 genes differ not only in their genomic structures, such as their numbers of exons and their exon-intron organizations, but also in the mechanisms regulating their transcription, suggesting that they diverged at an early stage of evolution.

Animals↗

PubMed1994

Expression of GATA-binding transcription factors in rat hepatocytes.

Recently, we demonstrated that a DNA-binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA-binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA-1. Rat GATA-1 expressed as a GST-fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA-1 and GATA-4 mRNAs are expressed in fetal hepatocytes.

Amino Acid Sequence↗

PubMed1994

Sequence analyses and inter-species comparisons of three novel human proteasomal subunits, HsN3, HsC7-I and HsC10-II, confine potential proteolytic active-site residues.

Proteasomes play a major role in non-lysosomal pathways of protein turnover mediated by distinct multiple proteolytic activities. Identification of their active-site residues is important for elucidating their catalytic mechanisms. Here we report the nucleotide sequences of three human proteasomal subunits, HsN3, HsC7-I and HsC10-II, coding for proteins with 264, 201 and 205 amino acid residues with calculated molecular weights of 29,192, 22,836 and 22,931, respectively. Sequence comparison showed that all three proteins belong to the beta-type superfamily and that they are the human counterparts of subunits reported to participate in the peptidyl-glutamyl-peptide hydrolyzing, chymotrypsin-like and trypsin-like activity of this complex. Alignments of the putative catalytically active subunits of various species revealed several family-specifically conserved serinyl residues within highly conserved amino acid stretches. Based on localization and hydrophobicity, the roles of these amino acid residues as active site and substrate binding site candidates are discussed.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of the 26 S proteasome from spinach leaves.

The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellulose, gel filtration on Biogel A-1.5, and glycerol density gradient centrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiquitin. By nondenaturing electrophoresis, the purified enzyme was separated into two distinct forms of the 26 S complex, named 26 S alpha and 26 S beta proteasomes, with different electrophoretic mobilities. The 26 S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115 kDa, which were thought to be those of a 20 S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and deubiquitinating activities, respectively. The subunit multiplicity of the spinach 26 S proteasome closely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multicomponent polypeptide complex. Electron microscopy showed that the 26 S proteasome complex had a "caterpillar"-like shape, consisting of four central protein layers, assumed to be the 20 S proteasome, with asymmetric V-shaped layers at each end. These structural and functional characteristics of the spinach 26 S proteasome showed marked similarity to those of the mammalian 26 S proteasomes reported recently, suggesting that the 26 S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.

Adenosine Triphosphate↗

PubMed1994

cDNA cloning and interferon gamma down-regulation of proteasomal subunits X and Y.

Proteasomes are the proteolytic complex responsible for major histocompatibility complex (MHC) class I-restricted antigen presentation. Interferon gamma treatment increases expression MHC-encoded LMP2 and LMP7 subunits of the proteasome and decreases expression of two proteasome subunits, named X and Y, which alters the proteolytic specificity of proteasomes. Molecular cloning of complementary DNAs encoding X and Y showed that their proteins are proteasomal subunits with high amino acid similarity to LMP7 and LMP2, respectively. Thus, interferon gamma may induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing proteasomes perhaps more appropriate for the immunological processing of endogenous antigens.

Amino Acid Sequence↗

PubMed1994

ATP- and antizyme-dependent endoproteolysis of ornithine decarboxylase to oligopeptides by the 26 S proteasome.

Previously we reported that ornithine decarboxylase (ODC) is degraded ATP-dependently by the 26 S proteasome in the presence of antizyme (AZ), an ODC inhibitor (Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599). Here we examined the cleavage of ODC by the 26 S proteasome. When ODC purified from ODC-overproducing cells was incubated with the 26 S proteasome and with AZ fused with maltose-binding protein (MBP) in the presence of ATP, ODC was degraded specifically without appreciable breakdown of MBP-AZ. The major degradation products of ODC, which were separated by high performance liquid chromatography on a reverse-phase column, were identified by N-terminal amino acid sequencing. The 26 S proteasome generated a variety of short peptides of 5-11 amino acid residues derived from regions throughout the ODC sequence. No detectable amounts of free amino acid residues were produced, indicating endoproteolytic degradation of ODC by the 26 S proteasome. Their major sites for cleavage of ODC by the 26 S proteasome were on the carboxyl sides of neutral/hydrophobic amino acid residues, but a few were on those of acidic or basic amino acid residues. These results demonstrate that the 26 S proteasome causes exhaustive endoproteolysis of the naturally occurring short-lived protein ODC in a multicatalytic and ATP-dependent manner.

Adenosine Triphosphate↗

PubMed1994

Functional regions of ornithine decarboxylase antizyme.

Degradation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is accelerated by the binding of antizyme, an ornithine decarboxylase inhibitory protein induced by polyamines. In the present study, we examined the effects of a series of deletion mutants of rat antizyme. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for its binding to ornithine decarboxylase and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization.

Animals↗

PubMed1994

Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing.

Proteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-gamma) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-gamma appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens.

Amino Acid Sequence↗

PubMed1994

Developmental regulation of rat serine dehydratase gene expression: evidence for the presence of a repressor in fetal hepatocytes.

To determine why the rat serine dehydratase gene becomes transcriptionally activated just after birth, we examined the interactions of DNA binding proteins of fetal and adult rat livers with the serine dehydratase gene promoter by DNase I protection analysis and gel mobility shift assay. Several binding regions of nuclear proteins were found to be common to fetal and adult livers and interaction of factors with the characteristics of Sp1 or NF-Y was suggested. Two additional regions, named regions B and I, were specific to fetal liver. These regions contain GATA-like sequences and competition experiments by gel mobility shift assay suggested that the fetal liver-enriched factor binds to the GATA-like sequences. The function of the regions B and I in transcription regulation was investigated in fetal and adult hepatocytes by transient DNA transfer experiments with serine dehydratase-chloramphenicol acetyltransferase fusions. These experiments showed that these regions functioned as negative cis-acting elements in fetal hepatocytes, but not in adult hepatocytes.

Animals↗