Michio Tsuda

A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes

Much attention has focused on the aetiology of oxidative damagein cellular and organismal ageing. Especially toxic arethe reactive oxygen byproducts of respiration and other biological processes. A mev-1 (kn1 ) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations,. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60% (ref. 7). Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type,. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b , which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlonys double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Enhancement of Oxidative Damage to Cultured Cells and Caenorhabditis elegans by Mitochondrial Electron Transport Inhibitors

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans ( C. elegans ) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans . In addition the cell damage is different dependent on cell type and organism.

Clinical and Biochemical Aspects of Thiamine Treatment for Metabolic Acidosis During Total Parenteral Nutrition

We encountered six cases of total parenteral nutrition (TPN)-associated lactic acidosis during the 6-y period of 1988-1993. The patients were characterized by severe disease of the digestive organs, minimal food intake before surgery, and postoperative TPN with no food intake and with no vitamin supplements. Within 4 wk of TPN, they developed hypotension (< or = 80/60 mmHg), Kussmauls respiration, and clouding of consciousness, as well as abdominal pain not directly related to the underlying disease. Routine laboratory examinations revealed no acute aggravation in hepatic, renal, or pancreatic functions. Arterial blood gas analysis showed pH < or = 7.134 and base excess < or = -17.5 mmol/L. Additional laboratory examinations revealed serum lactate > or = 10.9 mmol/L, serum pyruvate > or = 159 mumol/L, and lactate/pyruvate ratio > or = 0.029. None of the patients responded to sodium bicarbonate or other conventional emergency treatments for shock and lactic acidosis. After the first case, we suspected that thiamine deficiency might be responsible for this pathologic condition, Serum thiamine was proved to be < or = 196 nmol/L in 5 patients. Thiamine replenishment at intravenous doses of 100 mg every 12 h resolved lactic acidosis and improved the clinical condition in 3 patients. This article includes a review of 11 relevant reports published from 1982-1992 and a discussion of the biochemical mechanism of onset of thiamine deficiency-associated lactic acidosis. We emphasize the needs (1) to supplement TPN with thiamine-containing vitamins for the patients whose food intake does not meet nutritional requirements; (2) to monitor the patients routinely measuring serum thiamine concentration and erythrocyte transketolase activity during TPN; and (3) to intravenously replenish using high-dose thiamine simultaneously with the manifestation of signs and symptoms of lactic acidosis.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

Complication of Parenteral Nutrition Composed of Essential Amino Acids and Histidine in Adults With Renal Failure

This is a case report on six patients with hyperammonemia that developed while they were receiving total parenteral nutrition (TPN) as a component of renal failure therapy. Clinically, the hyperammonemia presented as mental status changes in all six cases. Four of the six patients with renal failure initially received 400 mL Amiyu in 1400 mL 17% glucose (total = 1800 mL TPN-A) administered over each 24-hour period. Two patients had been placed on 400 mL complete amino acid in 1400 mL 17% glucose (total = 1800 mL TPN-C over each 24-hour period) prior to therapy with TPN-A. Approximately 3 weeks after initiation of TPN therapy with TPN-A, episodes of mental status changes of increasing duration and paroxysms were documented in five of the six patients. In one of the patients receiving TPN-C prior to TPN-A therapy, toxicity was clinically evident only 4 days after initiation of TPN-A. Serum ammonia levels were obtained and found to be elevated in the acute (ie, presenting) stage in all patients. With the discontinuance of TPN-A, ammonia levels normalized uniformly. Mental status also improved in all cases except for the patient with rapid clinical presentation who died 2 weeks after first evidence of clinical toxicity. In cases 1, 2, and 6, serum amino acid analysis in the acute phase showed reduced levels of ornithine and citrulline, the substrate and product, respectively, of condensation with carbamyl phosphate at its entry into the urea cycle. Moreover, levels of arginine, precursor to ornithine, were found to be elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of a serum DNA binding protein (64DP) with a molecular weight of 64,000 and its diagnostic significance in malignant diseases

Abstract A DNA binding protein with a molecular weight of 64,000(64DP) has been purified to homogeneity from human serum, and its quantitative assay has been developed. The average level of serum 64DP in 30 normal controls was 41.4 μg/ml, whereas it was 175 μg/ml in 87 patients with untreated malignant disease. Furthermore it was found to be elevated in all tested patients, 8 cases, with carcinoma in early stages. Serum 64DP has been found to be different from C3DP, CEA # or α-FP # , and it appears that this protein might prove to be a useful tumor marker in malignant diseases.

Role of argininosuccinate synthetase in the regulation of urea synthesis in the rat and argininosuccinate synthetase-associated metabolic disorder in man

Abstract Regulation of urea synthesis and the role of ASS were studied in rats subjected to acute dietary transitions from high to low protein or vice versa and in rats injected with an ammonium salt. Significant increase/decrease in urea excretion and urea in the liver preceded increase/decrease of ASS during the dietary transitions. Shortly after the switch of diet from high to low protein, the ratios of the rate of urea synthesis from ammonium chloride to the activity of ASS in the perfused liver decreased to much lower values than those of rats fed on the low protein diet. In contrast, during the acute transition from low to high protein diet, the ratios increased above the values of rats fed on the high protein diet. The results indicated that regulatory factors other than the activity of ASS may influence urea synthesis. The concentrations of ornithine and acetylglutamate capable of stimulating urea synthesis markedly changed shortly after the dietary switch. The fact that fluctuations of ornithine and acetylglutamate concentrations were greater and occurred prior to the activity changes of ASS strongly suggests a possible regulatory function of these amino acids in urea synthesis. ASS may be rate-limiting only during the dietary transition from low to high protein. Intraperitoneal injection of an ammonium salt caused an increase in ammonia and urea in the liver of fed or starved rats. The increase in urea was accompanied by increases in acetylglutamate, ornithine and citrulline. In perfused liver, the addition of an ammonium salt to the perfusate caused increases in acetyl-glutamate and citrulline as observed in vivo, while the concentration of ornithine, one-fifth of that in vivo, did not respond to the addition. From these results, we conclude that ammonia, a direct substrate for urea synthesis, may control the concentrations of both ornithine and acetylglutamate in the liver, directly or indirectly, although perfused liver lacks some factors that keep the concentration of ornithine at the in vivo level and cause its increase in response to ammonia. In another part of the work, enzymological analysis in the liver of 4 cases of so-called adult-type citrullinemia with neuro-psychiatric symptoms of late-onset (24–48 years old) caused by ASS abnormalities was performed. One patient in whose family there was a hereditary tendency, judging from their serum citrulline levels, had an abnormal ASS with very high Km values for citrulline and aspartate. This was similar to the cases of neonatal-type citrullinemia summarized by Shih (13). We also observed another type of citrullinemia: decreased ASS activity in the liver of three patients was associated with the decrease in the enzyme protein. ASS activity in the kidney, however, was not decreased at all. The kinetic properties of the enzymes of the patients were quite similar to those of healthy control. No hereditary tendency was observed in these families. Thus, we assume that adult-type citrullinemia results from an epigenetic abnormality as well as a genetic one and heterogeneity in the cause may explain the higher incidence of citrullinemia in Japan.

Congenitally abnormal plasminogen in juvenile ischemic cerebrovascular disease.

Background and Purpose Congenitally abnormal plasminogen is characterized by markedly decreased fibrinolytic activity and has been reported mainly in association with venous occlusive disease. Case Description We found three young adult patients (34, 45, and 27 years old at onset) with ischemic cerebrovascular disease, all of whom had congenital plasminogen abnormalities but no other known risk factors. Hemostatic tests of all three patients revealed plasma plasminogen activities at almost one half of the normal level despite normal plasma plasminogen antigen levels. They were found to be heterozygotes with abnormal plasminogen (normal Ala-601[GCT] to abnormal Thr-601[ACT]) by DNA sequence analysis after polymerase chain reaction. Conclusions Congenital plasminogen abnormalities could be one of the risk factors of juvenile ischemic cerebrovascular disease of the arterial as well as venous type.

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human MhcDRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.