Biomedical subjects
Y Kagawa
Publications and source records attributed to Y Kagawa.
A candidate gene for RNA export carrier protein has two RBDs (RNA binding domain and Ran binding domain).
The human Ras-related nuclear protein Ran/TC4 is the member of a well conserved family of GTPases that can regulate cell-cycle progression, nuclear structure, protein import, and RNA export through nuclear pore complex (NPC). Translocation of RNA through the NPC also needs a RNA-binding protein as a possible mediator of export. By a low-stringency hybridization with a PCR fragment encoding RNA-binding domain as a probe, we obtained a partial human cDNA from skeletal muscle cDNA library. The deduced amino acid sequence indicated that the gene possessed both RNA-binding domain and Ran-binding domain, therefore it is a candidate gene for RNA export carrier protein. RNA blot analysis showed that this gene appeared approximately 8-10 kilo base in length and that the gene expression level in several human tissues is ubiquitous. The function of the gene will be discussed.
Gene structure and cell type-specific expression of the human ATP synthase alpha subunit.
The gene structure of the human ATP synthase alpha subunit (hATP1) was determined by cloning and sequencing. This gene is approximately 14 kbp in length and contains 12 exons interrupted by 11 introns. Mapping of the clones of hATP1 and Southern blot analysis of the genomic gene showed that there were a single copy of bona fide hATP1 gene and two pseudogenes. Primer extension and S1 mapping analysis showed the presence of multiple transcription initiation sites of the hATP1 gene. No TATA box or CAAT box was found near the transcription initiation sites. Comparison with the bovine gene showed that the 5'-flanking region of the hATP1 gene has an unconserved guanine-cytosine (GC) rich region, including several binding motifs of transcriptional factors, such as Sp1, AP-2, and GCF. By functional assay of gene expression, the basal promoter activity was located near the GC rich region. Comparison of the 5'-upstream region of the hATP1 gene with those of the genes for bovine ATP synthase alpha, human beta, and human gamma subunits indicated three common sequences, suggesting that putative cis-elements coordinate the expressions of the three subunit genes for the ATP synthase. The enhancer activities derived from the 5'-deletion mutants of a hATP1-CAT chimeric gene were different in cell lines from four different human tissues, suggesting the existence of cell type-specific gene regulation.
Functional and morphological abnormalities of mitochondria in human cells containing mitochondrial DNA with pathogenic point mutations in tRNA genes.
mtDNA with a point mutation in the tRNA(Ile) gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA(Arg) gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rho zero HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rho zero HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA. Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA, suggesting that the functional abnormality of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.
Transcription of the MRP RNA gene in frog stage I oocytes requires a novel cis-element.
The RNA component of the mitochondrial RNA processing (MRP) enzyme is related to both replication of mitochondrial DNA and processing of 5.8S rRNA, which are accelerated in the frog earliest stage (stage I) of frog oocytes. Microinjection of the deleted genes into the stage I oocytes showed positive cis-elements in the upstream region of the gene. The specific binding of protein(s) to this region was detected in cell extracts from stage I oocytes and liver but not in extracts of stage II-IV oocytes and the concentration of this protein was 40 times higher in the extract of stage I oocytes than that in liver.
Effects of magnesium ions on the relative conformation of nucleotide binding sites of F1-ATPases as studied by electron spin resonance spectroscopy.
Cations like Mg2+ play an important role in the catalytic mechanism of F1-ATPases. In this study we applied ESR spectroscopy and used the ATP analog 2-azido-2',3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP (2-N3-SL-ATP) to investigate the effects of Mg2+ ions on the structure of the nucleotide binding sites of F1-ATPases from beef heart mitochondria (MF1) and from the thermophilic bacterium PS3 (TF1). The results demonstrated that Mg2+ ions not only influenced the binding of the nucleotide analogs to F1 but also altered the structure and geometry of the nucleotide binding sites. We observed that the dipolar interactions that are indicative of the close proximity of enzyme-bound 2-N3-SL-ANP (Vogel, P.D., Nett, J.H., Sauer, H.E., Schmadel, K., Cross, R.L., and Trommer, W.E. (1992) J. Biol. Chem. 267, 11982-11986) were only detectable in MF1-ATPase when the enzyme was preincubated with Mg2+ ions. In the absence of Mg2+, the enzyme exhibited ESR spectra indicative of spin label bound in at least two different environments (binding sites) with no dipolar interactions visible. TF1-ATPase did not exhibit clear dipolar interactions in the presence or absence of Mg2+. The ESR spectra of TF1 in the absence of Mg2+ indicated two different environments of the spin labels. Subsequent addition of Mg2+, however, led to exactly the same spectra as if the enzyme was incubated with the ions, indicating a rearrangement of the nucleotide binding sites. In summary, clear differences in the structures of the nucleotide binding sites of MF1 and TF1 in the presence or absence of Mg2+ were observed. Conformational differences between F1-bound spin-labeled nucleotides were also observed between TF1- and MF1-ATPases.
Comparison of the ATPase activities of bovine heart and liver mitochondrial ATP synthases with different tissue-specific gamma subunit isoforms.
The kinetics of heart and liver mitochondrial ATPase (FoF1) were examined using submitochondrial particles (SMPs) purified from the two tissues to obtain information on the role of gamma subunit isoforms. The F1 portion is mainly composed of the catalytic, common alpha beta subunits and tissue-specific gamma subunits. In contrast to the previous reports on the kinetics and crystallography of various F1's, the Vmax and Km of the two isoforms of FoF1 were identical although the SMPs were prepared from different tissues. Moreover sodium azide inhibited the two equally. The ATPase activity of liver SMP showed slightly steeper pH-dependency than that of heart SMP but the pH optima of the two were the same (pH 8).
Exclusion of an alternatively spliced exon in human ATP synthase gamma-subunit pre-mRNA requires de novo protein synthesis.
Tissue-specific isoforms of the mitochondrial ATP synthase gamma-subunit are generated by alternative splicing, and the heart/skeletal muscle-specific transcript lacks exon 9 in a cassette fashion (Matsuda, C., Endo, H., Hirata, H., Morosawa, H., Nakanishi, M., and Kagawa, Y. (1993) FEBS Lett. 325, 281-284; and Matsuda, C., Endo, H., Ohta, S., and Kagawa, Y. (1993) J. Biol. Chem. 268, 24950-24958). Here, we show that the endogenous heart-type mRNA is cell-specifically induced by the extracellular pH value in the HT1080 (human fibrosarcoma) and KYM-1 (human rhabdomyosarcoma) cell lines. In these cells, a low extracellular pH value induced exclusion of exon 9, and this induction was inhibited by cycloheximide treatment. In contrast, a high extracellular pH value resulted in mRNA transcription of the liver type, including exon 9, and did not require de novo protein synthesis. These results suggest that alternative splicing in the gamma-subunit pre-mRNA is regulated by on-off switching of protein synthesis of a trans-acting factor involved in this exon-excluding step. The signal of low pH value was blocked by the protein kinase inhibitor H-7 or Calphostin C (protein kinase C inhibitor), indicating the involvement of protein kinase C in the alternative splicing. This is a good model system for studies on the induction mechanism of alternative splicing in cultured mammalian cells, in which intracellular factors play a pivotal role for the exon-excluding step in the tissue-specific alternative splicing mechanism.
Purification and reconstitution of an intestinal Na(+)-dependent neutral L-alpha-amino acid transporter.
Using an improved reconstitution method, we have purified an Na(+)-dependent neutral L-alpha-amino acid transporter from rabbit small intestine to apparent homogeneity. The preparation solubilized with octaethylene glycol dodecyl ether (C12E8) was purified by successive chromatographies on DEAE-Toyopearl and lentil lectin-Sepharose 4B columns. The transport activity was assayed by reconstitution of the protein into liposomes. The specific activity of the final preparation was 1364-fold that of brush border membrane vesicles. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the active fractions gave one band of 90 kDa. Kinetic analysis using proteoliposomes reconstituted with the purified fraction showed that alanine transport was mediated by high affinity system with Kt value of 0.19 mM and Jmax value of 2.8 nmol/mg protein/s. Analysis of the amino acid composition of the purified transporter revealed that the transporter is very hydrophobic protein. From its specific activities for transport of individual amino acids this transporter was concluded to possess broad specificity for neutral L-alpha-amino acids. Furthermore, inhibition study of other amino acids allowed us to identify this transport pathway as the intestinal system B.
Nuclear but not mitochondrial genome involvement in human age-related mitochondrial dysfunction. Functional integrity of mitochondrial DNA from aged subjects.
The role of mtDNA and nuclear genome in human aging was examined by their intercellular transfer using skin fibroblasts and mtDNA-less HeLa cells (rho o-HeLa cells). We found in vivo age-related reductions in the activity of cytochrome c oxidase in human skin fibroblasts obtained from 16 donors of various ages (0-97 years). The abnormality in mitochondria of the aged donors was not attributable to either decrease in the copy number of mtDNA molecules or increase in the copy number of deletion mutant mtDNA molecules, but to significant decrease in overall polypeptide synthesis in the mitochondria. However, intercellular mtDNA transfer experiments showed that fibroblast mtDNA from elderly donors is functionally intact. By contrast, intercellular transfer of HeLa nuclei to fibroblasts from aged donors restored cytochrome c oxidase activity, suggesting that the age-related phenotype was nuclear recessive. However, during subsequent cultivation of these hybrids, the activity gradually reduced again, associated with gradual chromosome loss. These observations support the idea that accumulation of nuclear recessive somatic mutations, but not mtDNA mutations, is responsible for the in vivo age-related mitochondrial dysfunction observed in human skin fibroblasts.
Widespread tissue distribution of multiple mitochondrial DNA deletions in familial mitochondrial myopathy.
We used Southern blot analysis and the polymerase chain reaction to analyze the tissue distribution of multiple mitochondrial DNA (mtDNA) deletions in a 45-year-old man with familial mitochondrial myopathy-Southern blots showed two major types of abnormal mtDNA with approximately 4- and 8-kilobase deletions in the skeletal and extraocular muscles. The amount of muscle mtDNA with deletions correlated approximately with the severity of muscle involvement. The polymerase chain reaction showed multiple mtDNA deletions even in clinically asymptomatic tissues, the pattern of which differed with the type of tissue. Nucleotide sequences of several mtDNAs with deletions showed that the deletions were flanked by direct repeats consisting of 3 to 12 nucleotides. Leukocytes from the patient's affected sister and his mother exhibited the same mtDNA deletion pattern. Most of the same deletions were present in leukocytes obtained from the patient's father.
Combined oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate enhances the antitumor effect against P388 ascites tumors.
We investigated the antitumor effect of oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate (C18PCA) against P388 ascites tumors in B6D2F1 mice. Etoposide (25 mg/kg) and C18PCA (5 mg/kg) were given orally on days 1-5 after tumor inoculation. The median life span of the mice treated with etoposide or C18PCA alone was 19.5 and 18 days, respectively. The combination of both drugs significantly extended the median life span to 33 days. To clarify this enhancement of the increase in median life span, we examined intracellular deoxyribonucleoside triphosphate (dNTP) pools, cell-cycle distribution, DNA fragmentation, and the time course of the plasma drug concentration. Etoposide had no effect on intracellular dNTP pools in this experimental system, whereas treatment of cells with C18PCA or with the combination of both drugs resulted in a significant increase in dTTP pools to values ranging from 1.8- to 2.0-fold higher than the control levels. There was a significant increase in cells in the S + G2/M phase when cells had been treated with both etoposide and C18PCA. Agarose-gel electrophoresis of the extracted DNA revealed that C18PCA enhanced the fragmentation of DNA, with a length of about 180 bp being induced by etoposide. The plasma peak levels of etoposide (1000 nM) and ara-C (50 nM) were observed at 20 and 30 min after the simultaneous administration of both drugs, respectively. The plasma etoposide level gradually decreased to 10% of the peak level at 240 min after administration. On the other hand, the plasma concentration of ara-C was maintained at above 20 nM at 240 min. These observations suggest that C18PCA and etoposide act on P388 murine leukemic cells by accumulating cells in the S + G2/M phase. Even if the plasma concentration of ara-C is low, the repair of DNA damage by etoposide may be hindered in the presence of ara-C following an increase in DNA fragmentation.
[Molecular chaperone and its induction].
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[Molecular genetics of mitochondria and diabetes].
The molecular genetics of mitochondria relevant to NIDDM is described, because the mutations in mitochondrial DNA cause diabetes. The non-Mendelian genetics, including maternal inheritance, heteroplasmy, stochastic segregation are characteristic of mitochondrial gene. Since aging causes rapid loss of mitochondrial function, which results in the retardation of insulin secretion via ATP-sensitive K-channel. This loss is not caused by the age-dependent mutation in the mitochondrial DNA, but by a nuclear aging, perhaps accompanied by the shortening of telomere. This was shown by the cybrid experiment. The aged mitochondria in cytoplasts are transferred to immortal rho = cells (cells devoid of mitochondrial DNA) and restored its oxidative and transcriptional activities (J. Biol. Chem. 269:6878, 1994). Thus, the control mechanism of transcription in mitochondria has been analyzed. The genes for mitochondrial transcription factor (mtTF1) an MRP-RNA have been sequenced and their regulatory elements are found (BBRC 194:544, 1993 etc.) The mutations in mitochondrial DNA that cause diabetes have been found, and from the stochastic segregation of the heteroplasmic mutated mitochondrial DNA, we could explain why the MELAS mutation is concentrated in some tissue (J. Neurol. Sci. 120:174, 1993). Although there have been many reports on the mitochondrial mutations found in diabetic patients, we have to be careful on polymorphism (J. Biol. Chem. in press).
Characterization of metabotropic glutamate receptors in cultured Purkinje cells.
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Content of mutant mitochondrial DNA and organ dysfunction in a patient with a MELAS subgroup of mitochondrial encephalomyopathies.
A point mutation of mitochondrial tRNALeu(UUR) gene is responsible for a MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) subgroup of mitochondrial encephalomyopathies. In most cases, the mutant mitochondrial DNA (mtDNA) coexists with normal mtDNA in a heteroplasmic manner. In order to quantify the content of mutant mtDNA, we developed a quantitative method of PCR. Using this method, the distribution of the mutant mtDNA was examined in 32 different tissues among 18 autopsied organs from a patient with MELAS, who had shown hypophyseal dysfunction. The percentage of the mutant mtDNA at nucleotide number 3243 in each tissue was ranged between 22% and 95%. The content of the mutant mtDNA was at the highest (95%) in the hypophysis and higher in the cerebral cortex than in the white matter. This study shows a possible correlation of tissue dysfunction with accumulation of the mutant mtDNA within the brain.
Cerebellar astrocytes specifically support the survival of Purkinje cells in culture.
As a first step toward identifying the factor(s) that is/are produced by astrocytes and support(s) the survival of cerebellar Purkinje cells in dissociated culture, we compared the effect of astrocytes of cerebellar, hippocampal, and cerebral origin. A feeder coverslip of cerebellar astrocytes, which did not have cell-to-cell contact to neuronal culture, increased the percentage of Purkinje cells to about 8-9 fold with no change in the percentage of astrocytes. On the other hand, astrocytes of hippocampal or cerebral origin did not increase the percentage of Purkinje cells at low plating density, whereas they increased the number of astrocytes in neuronal culture. These results indicate that the factor(s), tentatively named as Purkinje-cell survival factor was/were specifically produced by cerebellar astrocytes and affected directly on Purkinje cells.