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Biomedical subjects

M Nishikimi

Publications and source records attributed to M Nishikimi.

At least 73 records · Page 4Linked to original sources
PubMed1988

Familial mitochondrial myopathy associated with peripheral neuropathy: partial deficiencies of complex I and complex IV.

Two brothers, 46 and 48 years old, presented with optic atrophy and blepharoptosis since childhood, and later developed muscle wasting and weakness of the extremities, and glove and stocking type sensory impairment. Biopsies of muscles and sural nerves clearly showed mitochondrial myopathy with many ragged-red fibers and peripheral neuropathy with onion-bulb formation. Biochemical studies of muscles disclosed partial deficiencies of complexes I and IV of the mitochondrial respiratory chain in both cases. Since the parents were first cousins, this mitochondrial disorder seemed to be transmitted as an autosomal recessive trait.

Cytochrome-c Oxidase Deficiency↗
PubMed1988

Extensive defects of mitochondrial electron-transfer chain in muscular cytochrome c oxidase deficiency.

This study was undertaken to estimate the extent of molecular defects in the mitochondrial electron-transfer chain of a patient with mitochondrial myopathy. Biochemical and immunochemical studies were performed on the skeletal muscle mitochondria. Spectrophotometry and enzyme activity measurements localized a definite defect at the segment of cytochrome c oxidase (complex IV) of the electron-transfer chain. Immunoblotting and immunoprecipitation studies using the anti-complex IV antibody revealed that the contents of subunits 1, 4, 5, 6, and 7 of complex IV were markedly diminished and that subunit 2 was almost absent. Immunohistochemistry of the muscle tissue revealed a considerable accumulation of immunoreactive materials of complex IV in the ragged-red fibers. The immunoblots using the anti-NADH-ubiquinone oxidoreductase antibody demonstrated that the contents of NADH-ubiquinone oxidoreductase subunits were 47% of control and that the contents of three subunits were considerably decreased. The contents of ubiquinol-cytochrome c oxidoreductase subunits were also somewhat low (77% of control) and one of the minor contaminants detected in the control was completely absent. High-resolution one-dimensional sodium dodecyl sulfate-urea-gel electrophoresis disclosed that six additional unidentified polypeptides in the control were markedly diminished or completely missing. These results demonstrate that the molecular defects in the mitochondrial electron-transfer chain are more extensive than would be expected from either spectral analysis or enzyme activity measurements alone, and involve not only complex IV but also NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase and several unidentified mitochondrial proteins.

Adult↗
PubMed1988

Assignment of the human cytochrome c1 gene to chromosome 8.

We previously isolated a cDNA clone for human cytochrome c1. The insert DNA of approximately 950 bp from this clone was used as a probe to identify the cytochrome c1 gene. High molecular weight DNAs extracted from a panel of 14 independent human-mouse somatic cell hybrids were digested with BamHI and analyzed by Southern blot hybridization. The results indicated that the gene for human cytochrome c1 is located on chromosome 8.

Animals↗
PubMed1988

Cytotoxic activity of leukotoxin, a neutrophil-derived fatty acid epoxide, on cultured human cells.

Leukotoxin (LX: 9,10-epoxy-12-octadecenoate) which was previously found to be biosynthesized by neutrophils, exhibited a dominant cytotoxic activity toward human tumor cells as well as toward normal cells. At the concentration of 50-100 micrograms LX/ml cell culture medium, LX completely blocked the growth of the cells of several transformed cell lines (HeLa, Hep-2, MG-63, Chang liver, Wish, FL-Amnion) as well as the normal (Flow 2000 and Flow 13000). From the results, a part of inhibition of tumor growth by neutrophils can be attributed to LX.

Cytotoxicity, Immunologic↗
PubMed1988

L-gulono-gamma-lactone oxidase deficiency in rats with osteogenic disorder: enzymological and immunochemical studies.

A strain of Wistar rats with L-gulono-gamma-lactone oxidase deficiency has recently been established. The activity of L-gulono-gamma-lactone oxidase in hepatic microsomes of the mutant rats was not detectable, while that of heterozygous rats was about half that of normal rats. These results were confirmed by immunological quantitation using antibody directed against this enzyme. Thus, it appears that these mutant rats either possess no enzyme at all or a very aberrant form of the enzyme in the liver. Aldonolactonase, another enzyme participating in the biosynthesis of L-ascorbic acid, was found to be present in normal amount in the mutant strain.

Animals↗
PubMed1987

Isolation of a cDNA clone for human cytochrome c1 from a lambda gt11 expression library.

Antiserum directed against a purified preparation of beef heart cytochrome bc1 complex has been used to screen a human liver cDNA expression library in lambda gt11. The inserts of two recombinants, which gave strong signals, were found to represent cytochrome c1 by epitope selection using nitrocellulose filters containing the expressed proteins. The amino acid sequence deduced from the nucleotide sequence of an insert DNA revealed a high degree of homology with the sequence of bovine cytochrome c1.

Amino Acid Sequence↗
PubMed1987

A stomach oncofetal antigen recognized by monoclonal antibody GC302.

A monoclonal antibody, GC302, was established by fusing murine myeloma NS/1 cells with the splenocytes of a BALB/c mouse immunized with a human gastric cancer cell line, NU-GC-3. The serological specificity of GC302 was analyzed by an anti-mouse Ig mixed-hemadsorption (MHA) test on a panel of human cell lines, and an immunoperoxidase method using the frozen sections of tumors and normal tissues of adult and fetus. GC302 reacted with cancers of the stomach and colorectum but did not react with hepatocellular carcinomas, melanomas, or astrocytomas in the MHA tests. By the immunoperoxidase method, GC302 was found not to react with normal adult gastric mucosa, but to react with the mucosa in the fetal stomach, intestinal metaplasia, and almost all of the cancer of the stomach. GC302 also reacted with the normal mucosa of the intestine, colon, and rectum as well as with cancers of these origins. In normal liver sections, the antibody reacted with the bile ducts, but not with the hepatic cells. These results indicate that the antigen detected by GC302 is characterized as an oncofetal antigen in the stomach, and also as a differentiation antigen whose localization discriminates between the gastrointestinal tracts of the forgut origin and those of the midgut and hindgut origin. The molecular weight of the GC302 antigen was estimated to be ca. 40,000 by the Western blot analysis. Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate.

Animals↗
PubMed1987

Structure and function of mitochondria: their organization and disorders.

In this lecture, recent advances in studies on the structure and function of mitochondria were reviewed. In particular, in order to understand the etiology of mitochondrial myopathies, the mechanism of the biogenesis of the mitochondrial structure with proteins synthesized in mitochondria and in the cytoplasm was discussed; namely, how proteins encoded by mitochondrial DNA are biosynthesized, and how nuclealy encoded proteins are targeted into the appropriate compartments inside the mitochondria. Recent advances in mitochondriology have made it possible to isolate and purify the enzyme complexes and their subunits, which are involved in mitochondrial oxidative phosphorylation. Immunochemical analyses using a specific antibody against each complex or subunit enabled us to detect defects in individual subunits in mitochondria isolated from a small amount of biopsied material. Several examples of molecular defects revealed by these methods in patients with mitochondrial myopathies were presented, and the principles of their therapy are discussed on the basis of the pattern of the defect. Specific antibodies are also a powerful tool for the cloning of the human cDNAs for the subunits in the mitochondrial energy-transducing machinery. This approach will hopefully facilitate elucidation of the genetic defects underlying these disorders.

Animals↗
PubMed1987

Characterization of histamine H1-receptor on rat hepatocytes.

The binding sites for [3H]pyrilamine in isolated rat hepatocytes were characterized. Scatchard analysis revealed two kinds of binding sites in hepatocytes, a high-affinity site and a low-affinity one. The rates of binding of the radioligand with the high-affinity binding site and its dissociation were rapid. The specificity of the sites for various histamine antagonists indicated that the high-affinity [3H]pyrilamine binding site is representative of the histamine H1 receptor. Treatment of hepatocytes with protease or phospholipase A2 significantly decreased the maximum binding capacity of the high-affinity site without affecting its dissociation constant, suggesting that the binding site is proteinaceous and is sensitive to a change in the lipid moiety of the membrane. Hepatocytic cyclic AMP and cyclic GMP were not significantly modulated by incubating hepatocytes with histamine. Thus, the action of histamine on hepatocytes might not be mediated by the cyclic nucleotides.

Animals↗
PubMed1987

Partial deficiency of subunits in complex I or IV of patients with mitochondrial myopathies.

Subunits of Complexes I, III, IV, and V from skeletal muscle mitochondria isolated from four patients with mitochondrial myopathies were analyzed by immunoblotting. A deficiency was observed in Complex I or IV, or in both. The patterns of deficiency of subunits of each complex were similar both in patients with deficiency of a single complex and in patients with combined deficiency of the two complexes. The deficiency of any of these subunits was only partial, and no abnormalities in the electrophoretic mobilities of the subunits were observed. These results suggest that the deficiency of Complex I or IV in these patients are based on similar molecular defects which lead to decreased levels of these subunits.

Adolescent↗
PubMed1987

Variation in the levels of complex I subunits among tissues in a patient with mitochondrial encephalomyopathy and renal dysfunction.

Enzymic activity and the levels of immunochemically detectable subunits of NADH-ubiquinone oxidoreductase (Complex I) were measured in the mitochondria from various tissues of a patient with mitochondrial encephalomyopathy and renal dysfunction. Rotenone-sensitive NADH-cytochrome c reductase activity was decreased in all the tissues examined, but the degree of deficiency varied from tissue to tissue. The levels of subunits in Complex I were decreased roughly in parallel with the activity in each tissue. These results indicate that the apparently tissue-specific manifestation of symptoms depends mainly on the levels of subunits in Complex I.

Adolescent↗
PubMed1987

In vitro synthesis of L-gulono-gamma-lactone oxidase by rabbit reticulocyte lysate.

In vitro synthesis of L-gulono-gamma-lactone oxidase [L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8], one of the microsomal flavin enzymes, was performed with poly(A)+ RNA of rat liver using the rabbit reticulocyte lysate system in order to study the biosynthesis of the enzyme. The apparent molecular weight of the synthesized enzyme protein was almost the same as that of the purified L-gulono-gamma-lactone oxidase from rat liver. It was demonstrated that the enzyme protein was not detectable when guinea pig poly(A)+ RNA was used for the translation, indicating that the mRNA for the enzyme is absent in the guinea pig or, if it exists, is not translatable.

Animals↗
PubMed1987

Stabilization of ascorbate solution by chelating agents that block redox cycling of metal ions.

Among the seven chelating agents tested, ethylenediamine di(o-hydroxyphenylacetic acid) and diethylenetriamine pentaacetic acid were found to almost completely inhibit ascorbate oxidation catalyzed by iron ions. The inhibition with the former chelator is due to the prevention of the reduction of Fe3+ by ascorbate, while the inhibition with the latter is caused by the strong inhibition of both this reductive reaction and the oxidation of Fe2+ by O2. These chelators almost completely inhibit ascorbate oxidation catalyzed by copper ions as well. These results indicate that the blocking of redox cycling of metal ions is important to prevent the oxidation of ascorbate.

Ascorbic Acid↗
PubMed1986

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy.

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.

Adolescent↗
PubMed1986

Cell-free synthesis of ubiquinone-binding protein of mitochondrial cytochrome bc1 complex.

In vitro synthesis of the ubiquinone-binding protein of mitochondrial cytochrome bc1 complex has been studied. In vitro translation of rat liver poly(A)+ RNA followed by immunoprecipitation with antibody directed against bovine ubiquinone-binding protein yielded a polypeptide with a molecular weight that was the same as that of the mature protein. It was also found that this polypeptide was imported into mitochondria and became resistant to trypsin treatment, and that the import required the energized state of the inner membrane.

Animals↗
PubMed1986

Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction.

Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins.

Cytochrome-c Oxidase Deficiency↗