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A Ichiyama

Publications and source records attributed to A Ichiyama.

At least 37 records · Page 2Linked to original sources
PubMed1997

Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver.

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

Animals↗
PubMed1997

Morphological analysis of renal cell culture models of calcium phosphate stone formation.

Cell culture models of calcium phosphate renal stone formation were established using the MDCK cell line. Renal microliths were detected within pseudocysts in three-dimensional soft agar cultures, and were also observed in the basal region of cells lining the cell sheet, and immediately beneath domes or blisters in monolayers and collagen gel cultures. Light and scanning electron microscopy indicated that these microliths had a similar lamellated and spherical appearance to those in humans. These microliths were first detected microscopically after 21 days of culture, and were found to be composed of calcium phosphate by X-ray and micro-infrared spectroscopic analyses. These culture models may provide a powerful new tool to study the pathogenesis of renal stone diseases and/or calcium phosphate stone formation in humans and animals.

Animals↗
PubMed1996

Pyridoxal 5'-phosphate binding of a recombinant rat serine: pyruvate/alanine:glyoxylate aminotransferase.

Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at approximately 330 nm in addition to a 278 nm protein peak and a approximately 420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at approximately 420 nm, but still showed the approximately 330 nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of approximately 0.1 microM, resulting in a holo-SPT10 preparation whose specific activity and A approximately 420/A approximately 330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the approximately 22 and approximately 23 kDa pair gave the same sequence, except that Lys was released only from the approximately 22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the approximately 45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore.

Alanine Transaminase↗
PubMed1996

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Fidelity of translation initiation of mRNA for the precursor of rat mitochondrial serine:pyruvate/alanine:glyoxylate aminotransferase.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) of rat liver is localized in both mitochondria and peroxisomes. The rat SPT/AGT gene is single, but there are two species of mRNA which differ at their 5' termini due to transcription from two alternative initiation sites. The longer mRNA is translated from the first AUG codon and thereby directs synthesis of the 45 kDa precursor of mitochondrial SPT/AGT, which includes a mitochondria-targeting N-terminal signal sequence. Peroxisomal SPT/AGT is synthesized as a product of mature size (43 kDa) from the shorter mRNA, which starts 3' to the first AUG codon and thus is translated from a downstream AUG codon. In our previous immunocytochemical study, SPT/AGT was found to be localized only in peroxisomes, when a cDNA encoding 43 kDa SPT/AGT was expressed in COS cells. When a cDNA encoding the 45 kDa precursor was expressed, on the other hand, SPT/AGT was localized mostly in mitochondria, but a small number of peroxisomes were also positively stained [Yokota, S., Funai, T., and Ichiyama, A. (1991) Biomed. Res. 12, 53-59]. We show in this paper that 43 kDa SPT/AGT is also synthesized from the longer mRNA in an in vitro translation system through a leaky scanning mechanism. Although the first AUG initiator codon is in a suboptimal context, the amount of 43 kDa SPT/AGT synthesized from the longer mRNA was small, probably because a downstream stem-loop structure facilitates recognition of the first AUG initiator codon.

Alanine Transaminase↗
PubMed1994

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Amino Acid Sequence↗
PubMed1994

Regulation by glucagon of serine: pyruvate/alanine: glyoxylate aminotransferase gene expression in cultured rat hepatocytes.

The effects of glucagon on serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene expression were studied in primary cultured rat hepatocytes. When hepatocytes had been precultured for 16-18 h under serum- and hormone-free conditions, the addition of glucagon caused (after a lag period of about 2 h) a remarkable increase in the cellular level of SPT/AGT mRNA by 4 h in a time- and dose-dependent manner. The induced mRNA was that for mitochondrial SPT/AGT, as judged by ribonuclease protection analysis. A nuclear run-on assay revealed that activation of transcription is responsible for the increase in mitochondrial SPT/AGT mRNA and that the maximal rate of transcription occurs 1.5 h after glucagon addition. The effect of glucagon was mimicked by 8-bromo-cAMP and suppressed by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (protein kinase A), while both 12-O-tetradecanoylphorbol-13-acetate and A23187 were without effect in elevating the SPT/AGT mRNA level, suggesting that the cAMP/protein kinase A system is involved in the regulation of SPT/AGT gene expression. In hepatocytes precultured for 16-18 h under serum- and hormone-free conditions, the glucagon-induced transcription was severely inhibited by cycloheximide. When the preculture was for 2 h, on the other hand, the activation of transcription by glucagon was more rapid, and the inhibition by cycloheximide was less than that observed with cells precultured for 16-18 h, suggesting that a short-lived protein factor is involved in the hormonal regulation. The glucagon-induced expression of the SPT/AGT gene was also turned off by dexamethasone.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Mutations in the glucokinase gene are not a major cause of late-onset type 2 (non-insulin-dependent) diabetes mellitus in Japanese subjects.

The role of the glucokinase gene in the development of diabetes in a group of 349 Japanese subjects with late-onset Type 2 diabetes was examined. These diabetic subjects and 197 non-diabetic controls were typed at two simple tandem repeat DNA polymorphisms in the glucokinase gene termed GCK2 and GCK3. Six and five alleles were evident in Japanese subjects at GCK2 and GCK3, respectively. There were no significant differences in allele, genotype or haplotype frequencies between diabetic and normal groups. In addition, the glucokinase gene of 340 diabetic and 170 non-diabetic Japanese subjects was screened for mutations using single strand conformation polymorphism analysis. Four nucleotide substitutions were identified: a silent substitution in exon 4 in the codon for proline 145 (CCC-->CCG), and A-->T, C-->G, and C-->A substitutions in introns 1b, 3, and 5, respectively. There were no significant differences in the frequencies of these nucleotide substitutions between diabetic and non-diabetic groups. These results suggest that glucokinase gene defects are not a major cause of late-onset Type 2 diabetes in Japanese subjects.

Amino Acid Sequence↗
PubMed1994

Regulation of glucokinase gene expression in cultured rat islet cells: the inhibitory effects of T3 and glucagon, and the stimulatory effect of glibenclamide.

Hormonal and non-hormonal regulation of glucokinase gene expression was investigsted in cultured rat islet cells. To measure glucokinase mRNA in pancreatic islet cells, the competitive PCR method was adopted. With this method, GKmRNA levels can be measured using only 0.1-1.0 microgram of total RNA isolated from cultured rat islet cells. Following 24 h preculture with 5.5 mM glucose, islet cells were cultured for 24 or 8 h with hormonal or non-hormonal factors. Glucokinase mRNA levels tended to increase, but not significantly, at 16.7 mM glucose compared to those at 5.5 mM glucose. Treatment with either 1 microM T3 or 1 microM glucagon resulted in a decrease in the glucokinase mRNA level with 16.7 mM glucose, whereas 1 microM insulin had no effect on glucokinase mRNA. Five mM dibutyryl cyclic AMP decreased the glucokinase mRNA level with 16.7 mM glucose, but cycloheximide did not block this inhibitory effect, suggesting that the effect of glucagon may be mediated by cyclic AMP and that protein synthesis is not involved in the response. Furthermore, the islet glucokinase mRNA level increased in response to 1 microM glibenclamide with 5.5 mM glucose and the response was abolished by cycloheximide, which indicates the involvement of protein synthesis in the glibenclamide-induced mRNA change. An 8-bromo-cyclic GMP (1 microM) and vanadate (1 microM) did not affect the islet GKmRNA level. These findings suggested that thyroid hormone and glucagon-cyclic AMP suppress, and glibenclamide increases the GKmRNA level in cultured rat islet cells, and that insulin, cyclic GMP and vanadate differentially affect glucokinase gene expression in pancreatic islet cells and in the liver.

Animals↗
PubMed1993

Characterization and sequence analysis of rat serine:pyruvate/alanine:glyoxylate aminotransferase gene.

We have reported the isolation of genomic clones encoding serine:pyruvate aminotransferase (SPT; also named alanine:glyoxylate aminotransferase, AGT) (T. Oda, T. Funai, and A. Ichiyama, 1990, J. Biol. Chem. 265: 7513-7519). These clones contained the entire SPT/AGT gene of 10 kb. In this work, we characterized this gene. The SPT/AGT gene consists of 11 exons, and the exon-intron boundaries have typical splice donor and acceptor sequences. Determination of the nucleotide sequence up to -1.25 kb from the transcription initiation site revealed the presence of many putative cis elements, some of which may explain the transcriptional regulation of the SPT/AGT gene by glucagon and glucocorticoid. The nucleotide sequence around the 5' flanking region of the rat SPT/AGT gene and the whole gene organization were compared with those of the human SPT/AGT gene. No obvious similarities were observed in the 5' flanking region up to -1.25 kb from the initiation site of the gene, but exons 2 to 10 of the rat and human genes have identical sizes and show high similarities.

Alanine Transaminase↗
PubMed1993

ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case.

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half-life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.

Adenosine Triphosphate↗
PubMed1992

A single serine:pyruvate aminotransferase gene on rat chromosome 9q34-q36.

It was found in our previous study (Oda et al., 1990. J. Biol. Chem. 265: 7513-7519) that in the rat two mRNAs encoding mitochondrial and peroxisomal serine:pyruvate aminotransferase (SPT/AGT) are formed from a single SPT/AGT gene through alternative transcription initiation in exon 1. In an attempt to analyze the mechanisms underlying this unique phenomenon, we have isolated genomic clones harboring the entire rat SPT/AGT gene. In the present study, the location of the rat SPT/AGT gene was determined to be in the q34-q36 region of chromosome 9 by fluorescence in situ hybridization. Southern blot analysis of rat genomic DNA revealed an allelic BamHI restriction fragment length polymorphism among three different inbred rat strains. These results indicated that a single copy SPT/AGT gene is located on chromosome 9q34-q36 in the rat genome. This locus has been assigned the gene symbol Spat.

Animals↗
PubMed1992

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. METHODS: We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. RESULTS: RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. CONCLUSION: We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Animals↗
PubMed1991

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene.

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5'-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.

Adult↗
PubMed1991

Possible involvement of free radicals and antioxidants in the early stages of the development of cardiomyopathy in BIO 14.6 Syrian Hamster.

The BIO 14.6 cardiomyopathic Syrian hamster is a well-known animal model of congestive cardiomyopathy. To evaluate the role of free radicals and antioxidant protection in the pathogenesis of cardiomyopathy in this animal, we studied the concentration of heart mitochondrial free radicals, the activities of glutathione peroxidase (GSHPx) and superoxide dismutase (SOD), and the effect of alpha-tocopherol on the early stage of myocardial damage (up to 90 days). The GSHPx activity in BIO 14.6 hamsters was found to be twice that in the normal control hamsters at 30 days of age, while SOD activity was unchanged at 30 and 90 days of age. The concentrations of mitochondrial free radicals in BIO 14.6 hamsters at 40 and 90 days of age were significantly higher than those in the normal control hamsters. A protective effect of alpha-tocopherol therapy was shown in BIO 14.6 hamsters treated during the early stage of cardiomyopathy (up to 90 days). These results show the role of free radicals and antioxidant protection in the pathogenesis of hamster cardiomyopathy. We suspect that an increase in the GSHPx activity in BIO 14.6 hamsters may be due to a compensatory mechanism to counteract oxidative stress, but antioxidant reserve was not sufficient to protect the heart from the toxic effects of increased free radicals in the early stage of cardiomyopathy.

Animals↗
PubMed1991

Gastrointestinal serotonin: depletion due to tetrahydrobiopterin deficiency induced by 2,4-diamino-6-hydroxypyrimidine administration.

To study the role of serotonin in gastrointestinal function in mice, a peripheral deficiency was induced by administration of 2,4-diamino-6-hydroxypyrimidine, an inhibitor of tetrahydrobiopterin (6-[dihydroxypropyl-(L-erthro)-5,6,7,8,-tetrahydropterin) (BH4) biosynthesis. BH4 is an essential cofactor for tryptophan hydroxylase, the rate limiting enzyme in the biosynthesis of serotonin. When 2,4-diamino-6-hydroxypyrimidine (DAHP) was administered for 4 days at the dose of 3 g/kg/day, serotonin decreased in the duodenum and colon to approximately 46 and 40% of the control levels, respectively. These mice showed visual signs of gastrointestinal dysfunction in addition to a remarkable decrease in uptake of a p.o. glucose load. The decrease in serotonin was prevented and prevention of the apparent dysfunction of digestive tract was observed by simultaneous administration of (6R)BH4. Serotonin levels in the brain and blood were also decreased by treatment with p-chlorophenylalanine, an irreversible inhibitor of tryptophan hydroxylase. In contrast, DAHP had no effect on brain serotonin levels, presumably due to poor brain penetration. DAHP caused a rapid decrease (T1/2 of less than 12 hr) in the BH4 levels in all tissues examined, except in the brain. The BH4 level returned to within the normal range less than 24 hr after cessation of the administration of DAHP, a finding which suggests that the BH4 level is in a dynamic steady state maintained by rapid local biosynthesis. Thus, the local biosynthesis of BH4 supports normal digestive functions, presumably by controlling normal serotonin biosynthesis.

5-Hydroxytryptophan↗