Showing papers by "Morimitsu Nishikimi published in 1987"

Structure and function of mitochondria: their organization and disorders.

Abstract: 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 submits, 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.

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

Abstract: 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.

Deficiency of subunits of complex I or IV in mitochondrial myopathies: immunochemical and immunohistochemical study.

Abstract: Mitochondrial DNA codes for 13 polypeptides (Attardi, 1981) out of about 60 subunits in the oxidative phosphorylation enzymes (Capaldi, 1982). Complex I (NADH-ubiquionone oxidoreductase, EC 1.6.99.3) and Complex IV (cytochrome c oxidase, EC 1.9.3.1) have seven and three subunits coded by mitochondrial DNA, respectively (Chomyn et al., 1986), and are frequently implicated in mitochondrial myopathies. We have observed combined deficiency of these complexes in a patient with mitochondrial myopathy (Tanaka et al., 1987a) and in a patient with mitochondrial encephalomyopathy (Tanaka et aI., 1986). However, the etiology of the multiple deficiency of the respiratory complexes is not fully elucidated. This paper reports immunochemical and immunohistochemical investigations on the subunits of respiratory complexes in isolated mitochondria and in frozen sections of muscle from four patients with mitochondrial myopathies. Biochemical aspects of these patients have already been reported (Tanaka et al., 1987b).

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

Abstract: 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.

Characterization of histamine H1-receptor on rat hepatocytes.

Abstract: 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.

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

Abstract: 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.

A stomach oncofetal antigen recognized by monoclonal antibody GC302.

Abstract: 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 beca 40,000 by the Western blot analysis Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate

Medicinal drug containing quinone derivative as active component

Abstract: PURPOSE:To provide a medicinal drug containing a quinone derivative and its salt as active component and having phospholipase-inhibiting action and anti-platelet action CONSTITUTION:A medicinal drug useful as an anti-platelet agent, antithrombotic agent and remedy for various cardiac diseases can be produced by using a quinone compound of formula I [A is group of formula II or formula III (X and Y are OH, methoxy or H); n is 1-5] having carboxyl group at the terminal of polyprenyl chain and its salt It is effective for the remedy and prevention of cerebrovascular disorder such as TIA (transient ischemic attack), cerebral infarction, cerebral arteriosclerosis, etc, various thrombosis, blood flow disorder and various inflammatory diseases such as rheumatic diseases A compound of formula I wherein the group A is group of formula III can be produced by condensing the compound of formula IV with the compound of formula V (R is H or lower alkyl) in the presence of a catalyst

Polypeptides in Mitochondrial Electron-Transfer Complexes

Abstract: We established an isolation method for ubiquinone-binding proteins (QPs) from either mitochondria or electron-transfer particles, and found that at least 29% of the total coenzyme Q in the inner membrane was bound to QPs. The QP from Complex I (QP-I) was identified with a 13-kDa polypeptide, and the QP from Complex III (QP-III) with a 12.4-kDa polypeptide. We also isolated subunits of Complex III: the iron-sulfur protein, cytochrome c 1, cytochrome b, and QP-III, and core proteins in their functional states. In vitro translation of rat liver poly(A)+ RNA followed by immunoprecipitation with antibody directed against QP-III 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. A cDNA for human cytochrome c 1 was cloned using the λgtll expression vector, and the sequence analysis showed that the amino acid sequence deduced from the DNA sequence was quite similar to that of bovine heart cytochrome c 1 determined by amino acid sequence analysis. Together with the structural data from the crystallographic analysis of Complex III, the sequence data would provide a basis for elucidation of structure and function of the complex. Immunoblotting technique gave us information on the molecular level regarding the deficiency of subunits of complexes in patients with mitochondrial cytopathies.