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Biomedical subjects

Y Hotta

Publications and source records attributed to Y Hotta.

At least 145 records · Page 8Linked to original sources
PubMed1992

Expression of human ornithine aminotransferase (OAT) in OAT-deficient Chinese hamster ovary cells and fibroblasts of gyrate atrophy patient.

Gyrate atrophy is a hereditary chorioretinal degenerative disease caused by a deficiency of the mitochondrial enzyme, ornithine aminotransferase (OAT). Recent investigations have demonstrated the molecular genetic defects of OAT in gyrate atrophy patients. We constructed a eukaryotic expression vector (pcDHOAT) which contains the SV40 promoter and human OAT cDNA. We used OAT(-) Chinese hamster ovary (CHO) cells, which have negligible OAT activity, and fibroblasts from a gyrate atrophy patient (GA35 cell), which have negligible OAT mRNA and enzyme. Incorporation of pcDHOAT and synthesis of human OAT mRNAs and active enzyme were demonstrated in both cell types. The level of expression of human OAT was low in the GA35 cells in comparison to the CHO cells. Despite the limited success, the ability to express active OAT in these OAT-deficient cells using an expression vector offers possibilities of replacement gene therapy for gyrate atrophy.

Animals↗
PubMed1992

High frequency of mitochondrial ND4 gene mutation in Japanese pedigrees with Leber hereditary optic neuropathy.

The association of the ND4 gene mutation (mutation) at nucleotide position 11778 of mitochondrial DNA (mtDNA) was investigated in 14 definitive Japanese pedigrees with Leber hereditary optic neuropathy (LHON). The mutation was detected by SfaNI and MaeIII restriction fragment length polymorphisms of mtDNA amplified by polymerase chain reaction. All 14 LHON pedigrees exhibited the mutation, whereas 10 controls did not. The association of this mutation with LHON was revealed to be significantly higher in Japanese (91.7%) than in 27 reported Caucasian (51.9%) LHON pedigrees, implying genetic heterogeneity. In the tested 14 pedigrees, 28 cases with the mutation comprised 19 affected (17 male and 2 female) and 9 asymptomatic (all female except for one) individuals. Such a predominance of males in the incidence of LHON suggested probable participation of additional pathogenetic factor(s) in the development of optic atrophy in LHON patients.

Adolescent↗
PubMed1992

Clinical features of Japanese family with autosomal dominant retinitis pigmentosa caused by point mutation in codon 347 of rhodopsin gene.

Four members in a Japanese family had autosomal dominant retinitis pigmentosa caused by a single point mutation in codon 347 of the rhodopsin gene. The youngest, an 11-year-old girl, had an abnormal electroretinographic response, although her fundus appeared normal. The other affected family members noticed night blindness in the second decade. Their fundi showed diffuse pigmentation with concentric visual field loss, and there was no recordable electroretinographic response. Cataract developed in the fourth decade in the older patients. Good visual acuity was retained however, even in the fifth decade, after cataract extraction. These clinical features were similar to those of American patients (European family origin) with the same mutation of the rhodopsin gene reported previously.

Adult↗
PubMed1991

Evidence for increased in vitro recombination with insertion of human hepatitis B virus DNA.

Chromosomal translocation, deletion, and inversion/duplication directly linked to hepatitis B virus (HBV) DNA integration occur frequently in host DNA of human hepatocellular carcinomas. To test the possible recombinogenic effect of HBV DNA, we have utilized an in vitro recombination assay. Fragments containing the region spanning DR1, which is believed to be the origin of viral replication and a preferred site in the viral genome for integration, increased the recombination events reproducibly in the presence of extracts from actively dividing cells (e.g., hepatocellular carcinoma) but not resting cells (e.g., normal liver). Moreover, in these extracts we have found a protein(s) that specifically binds to these HBV DNA fragments. These results support the notion that in some instances integrated HBV DNAs cause further genomic instability, possibly involving specific cellular protein(s). The fact that extracts from nondividing, normal liver did not increase recombination events suggests that genomic instability depends upon active cellular growth, a feature more commonly found subsequent to HBV-induced hepatocellular injury than in healthy liver. Our results offer an explanation for the high incidence of liver cancer that accompanies chronic hepatitis and add HBV to the list of agents that can cause genetic recombination.

Animals↗
PubMed1991

A mutation of mitochondrial DNA in Japanese families with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by optic nerve degeneration associated with severe bilateral visual loss in young men and occasionally in women. A mitochondrial DNA (mtDNA) replacement mutation in LHON patient, G to A transition at nucleotide position (nt) 11778 converting the 340th arginine to histidine in the NADH dehydrogenase subunit 4, was detected as SfaNI site polymorphism (Wallace et al., Science, 242: 1427-1430, 1988). To evaluate if the SfaNI site loss can be used to diagnose LHON patients, mtDNAs from peripheral blood of six affected males including five probands from five unrelated Japanese families with LHON, a pair of parents and a normal sister of one of the probands and 4 control persons were analyzed using PCR amplification method. The mutation of leukocyte mtDNA at nt 11778 was identified in all of the affected patients, the normal mother and the sister examined, while the father who is normal and 4 control persons did not show the change. These findings support that the mutation at nt 11778 is also associated with LHON in the Japanese and the test of the SfaNI site loss described here is useful for confirming the clinical diagnosis of LHON patients with the mutation at nt 11778.

Asian People↗
PubMed1991

Heat shock gene activation by mutant actin is independent of myofibril degeneration in Drosophila muscle.

Artificially mutagenized Drosophila Act88F actin genes with triple and double mutations were expressed in the indirect flight muscles of transgenic flies. The triple mutant actin, GD245T (Gly-36----Glu, Glu-83----Asp, and Gly-245----Asp), induced heat shock protein (hsp) synthesis without affecting flight ability. On the other hand, the double mutation, GD245D (Gly-36----Glu and Glu-83----Asp), disrupted myofibrils but induced little hsp synthesis. These results demonstrate that myofibril degeneration is not the primary cause of the anomalous heat shock gene activation by mutant actins.

Actins↗
PubMed1991

Genomic organization of a Drosophila phospholipase C, norpA, and molecular lesions in two temperature-sensitive mutants.

The Drosophila mutant no receptor potential A (norpA) is the phototransduction-defective mutant which lacks phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Recently, norpA cDNA was isolated and its homology to a bovine PI-PLC was demonstrated [Bloomquist, B.T. et al. (1988) Cell 54, 723-733]. On the basis of its cDNA, we determined the genomic organization of the norpA gene and revealed that the norpA gene consists of 13 coding exons spreading over a 15 kb genomic area. Furthermore, we identified the mutational sites of two temperature-sensitive (ts) mutants. The analysis of norpAH52 revealed that the single amino acid change from Ser-551 to Tyr causes the PI-PLC activity to become temperature-sensitive. The other allele, norpAKO50, has two substitutions from Ser-406 to Phe and from Gly-451 to Ser. These three mutations are located in regions highly conserved in other mammalian PI-PLC molecules. This suggests that these regions are important for PI-PLC catalytic activity.

Alleles↗
PubMed1991

Studies on cardiac ingredients of plants. VII: Chemical transformation of proscillaridin by means of the Diels-Alder reaction and biological activities of its derivatives.

The Diels-Alder reactions of a cardiac glycoside, proscillaridin (1), with some dienophiles were investigated. The reaction of 1 with alkenes such as methyl vinyl ketone and methyl acrylate afforded 3-oxo-2-oxabicyclo[2.2.2]oct-7-enes (2-5) and para-substituted benzene derivatives (6 and 7), while 1 reacted with alkynes (3-butyn-2-one, methyl propiolate) to yield para- or meta-substituted benzene derivatives (6-9). The biological activities of the resulting derivatives were evaluated by the use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase (ATPase) preparation from dog kidney. Among the proscillaridin derivatives, compounds 4 and 7 moderately inhibited Na+,K(+)-ATPase activity. Furthermore, the concentration range of 7 over which its positive inotropic effect on guinea-pig papillary muscle preparations, increased from 5% to 95% of maximum was broader than that of 1, i.e., concentration dependency was maintained over a greater range of concentration.

Adenosine Triphosphatases↗
PubMed1991

[Studies on cardiac ingredients of plants. VIII. Preparation of nitrates of proscillaridin and their pharmacological activities].

To reduce the vascular contracting effect of the cardiac glycoside, proscillaridin (1), all kinds of its nitrates were prepared by utilizing effectively an isopropylidene function as a protective group. The pharmacological activities of proscillaridin nitrates were evaluated by the use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase preparations from the dog kidney. Furthermore, the effect for smooth muscle using the helical strips isolated from 13-week old spontaneously hypertensive rat was examined. The positive inotropic effects and Na+, K(+)-adenosine triphosphatase inhibition activities of mononitrates (6, 9, 15) and dinitrates (3, 4, 5) were a little less potent than 1, but those of trinitrate (2) were much reduced. Every nitrate did not exhibited a vascular contracting effect but a relaxing effect. Among them, the vascular relaxing effects of 2',3'-dinitrate (3) and 2',4'-dinitrate (4) were more potent than those of the other nitrates.

Animals↗
PubMed1991

Independence of excision frequency and transposition frequency of P element in Drosophila melanogaster.

Although a family of transposon, P elements, are used as tools for molecular genetics in Drosophila melanogaster, the molecular details and mechanism of their mobilization process have not been studied extensively. In particular, the relationship between excision and transposition is little understood. We have previously produced a transgenic fly with a P element insertion that is nonautonomous (stable without transposase) and is highly-transposable in the presence of transposase. Using this insertion, we traced its mobilizations following introduction of a stable transposase source. We found a strain that has a 26-bp tandem repeat at the end of the original P element insertion. The 26-bp repeat reduced the frequency of excision although the frequency of transposition was not altered. Our results indicate independence of transposition from excision and importance of terminal repeat in excision.

Animals↗
PubMed1991

[A trial of molecular diagnosis in Leber's optic neuropathy].

The high frequency of mitochondrial DNA mutation at the nucleotide position (nt) 11,778 was reported in cases of Leber's hereditary optic neuropathy (LHON) after the first report by Wallace et al.. We already reported that it provided a simple diagnostic test by means of PCR (polymerase chain determined the diagnosis of LHON in a case. No nt 11,778 mutation was found in patients with the other optic nerve diseases and in normal controls. This shows the usefulness of molecular diagnosis in LHON. Problems of genetic counselling for patients and female carriers and the possibilities to clarify the cause of LHON were discussed.

DNA↗
PubMed1990

Purification and partial amino acid sequences of phosphoinositide-specific phospholipase C of Drosophila eye.

To examine whether the norpA (no receptor potential A) gene encodes a phosphoinositide-specific phospholipase C (PLC) in the eye of Drosophila, a major PLC in the extract from normal Drosophila heads, which was absent in the extract from norpA mutant heads, and purified and its partial amino acid sequences were determined. The purification of the major PLC in KCl extract from normal Drosophila heads was achieved by sequential column chromatography on DEAE-Sepharose CL-6B, Mono Q, Superose 12, Mono S, second Mono S, and second Mono Q, followed by column chromatography on Superose 12 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 98,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Interestingly, the calcium and pH requirements for activation of the crude enzyme (KCl extract) were quite different from those of partially purified enzyme (active fraction from second Mono Q column). The maximal activity for PIP2 hydrolysis was observed at calcium concentrations between 10(-7) and 10(-5) M for both the crude and partially purified enzymes. On the other hand, the activity for PI hydrolysis of the crude enzyme increased with increasing calcium concentrations, while that of the partially purified enzyme reached a maximum at calcium concentrations between 10(-6) and 10(-4) M, and decreased at millimollar concentration. The pH dependences for PI hydrolysis of the crude enzyme and the partially purified enzyme were similar. The crude enzyme hydrolyzed PIP2 over a broad pH range from 6 to 8.5, while the activity of the partially purified enzyme monotonously increased with increasing pH. The partial amino acid sequences were determined by treating the purified enzyme with endopeptidase Lys-C; the resultant peptide fragments were purified on a high performance liquid chromatography-reverse phase column and then sequenced with sequencer. The obtained sequences were found to be a part of the deduced amino acid sequences of cDNA which was suggested to be norpA gene.

Amino Acid Sequence↗