Tomotsugu Yasuda

The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein.

Citrullinaemia (CTLN) is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase (ASS). Adult-onset type II citrullinaemia (CTLN2) is characterized by a liver-specific ASS deficiency with no abnormalities in hepatic ASS mRNA or the gene ASS (refs 1–17). CTLN2 patients (1/100,000 in Japan) suffer from a disturbance of consciousness and coma, and most die with cerebral edema within a few years of onset. CTLN2 differs from classical citrullinaemia (CTLN1, OMIM 215700) in that CTLN1 is neonatal or infantile in onset, with ASS enzyme defects (in all tissues) arising due to mutations in ASS on chromosome 9q34 (refs 18–21). We collected 118 CTLN2 families, and localized the CTLN2 locus to chromosome 7q21.3 by homozygosity mapping analysis of individuals from 18 consanguineous unions. Using positional cloning we identified a novel gene, SLC25A13, and found five different DNA sequence alterations that account for mutations in all consanguineous patients examined. SLC25A13 encodes a 3.4-kb transcript expressed most abundantly in liver. The protein encoded by SLC25A13, named citrin, is bipartite in structure, containing a mitochondrial carrier motif and four EF-hand domains, suggesting it is a calcium-dependent mitochondrial solute transporter with a role in urea cycle function.

Developmental changes in the Ca2+-regulated mitochondrial aspartate–glutamate carrier aralar1 in brain and prominent expression in the spinal cord

Aralar1 and citrin are two isoforms of the mitochondrial carrier of aspartate-glutamate (AGC), a calcium regulated carrier, which is important in the malate-aspartate NADH shuttle. The expression and cell distribution of aralar1 and citrin in brain cells has been studied during development in vitro and in vivo. Aralar1 is the only isoform expressed in neurons and its levels undergo a marked increase during in vitro maturation, which is higher than the increase in mitochondrial DNA in the same time window. The enrichment in aralar1 per mitochondria during neuronal maturation is associated with a prominent rise in the function of the malate-aspartate NADH shuttle. Paradoxically, during in vivo development of rat or mouse brain there is very little postnatal increase in total aralar1 levels per mitochondria. This is explained by the fact that astrocytes develop postnatally, have aralar1 levels much lower than neurons, and their increase masks that of aralar1. Aralar1 mRNA and protein are widely expressed throughout neuron-rich areas in adult mouse CNS with clear enrichments in sets of neuronal nuclei in the brainstem and, particularly, in the ventral horn of the spinal cord. These aralar1-rich neurons represent a subset of the cytochrome oxidase-rich neurons in the same areas. The presence of aralar1 could reflect a tonic activity of these neurons, which is met by the combination of high malate-aspartate NADH shuttle and respiratory chain activities.

Identification of two novel mutations in the SLC25A13 gene and detection of seven mutations in 102 patients with adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific deficiency of argininosuccinate synthetase (ASS) protein. We have recently identified the gene responsible for CTLN2, viz., SLC25A13, which encodes a calcium-binding mitochondrial carrier protein, designated citrin, and found five mutations of the SLC25A13 gene in CTLN2 patients. In the present study, we have identified two novel mutations, 1800ins1 and R605X, in SLC25A13 mRNA and the SLC25A13 gene. Diagnostic analysis for the seven mutations in 103 CTLN2 patients diagnosed by biochemical and enzymatic studies has revealed that 102 patients had one or two of the seven mutations and 93 patients were homozygotes or compound heterozygotes. These results indicate that CTLN2 is caused by an abnormality in the SLC25A13 gene, and that our criteria for CTLN2 before DNA diagnosis are correct. Five of 22 patients from consanguineous unions have been shown to be compound heterozygotes, suggesting a high frequency of the mutated genes. The frequency of homozygotes is calculated to be more than 1 in 20,000 from carrier detection (6 in 400 individuals tested) in the Japanese population. We have detected no cross-reactive immune materials in the liver of CTLN2 patients with any of the seven mutations by Western blot analysis with anti-human citrin antibody. From these findings, we hypothesize that CTLN2 is caused by a complete deletion of citrin, although the mechanism of ASS deficiency is still unknown.

Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism

Introduction Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. Methods Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. Results Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. Conclusions Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Infantile citrullinemia caused by citrin deficiency with increased dibasic amino acids

In an infant who suffered from prolonged icterus and hepatocellular dysfunction we detected an increase of citrulline and dibasic amino acids in plasma and urine. The amino acid levels along with all the abnormal liver tests normalized upon replacing breast-milk by formula feeding; there was no relapse after human milk was tentatively reintroduced. A novel mutation, a approximately 9.5-kb genomic duplication, was identified in the citrin gene (SLC25A13) resulting in the insertion of exon 15. No mutation was detected in the CAT2A specific exon of the SLC7A2 gene which encodes for the liver transporter of cationic amino acids. This is the first report of infantile citrin deficiency in non-Asian patients.

Sepsis-induced myocardial dysfunction: pathophysiology and management

Sepsis is aggravated by an inappropriate immune response to invading microorganisms, which occasionally leads to multiple organ failure. Several lines of evidence suggest that the ventricular myocardium is depressed during sepsis with features of diastolic dysfunction. Potential candidates responsible for septic cardiomyopathy include pathogen-associated molecular patterns (PAMPs), cytokines, and nitric oxide. Extracellular histones and high-mobility group box 1 that function as endogenous damage-associated molecular patterns (DAMPs) also contribute to the myocardial dysfunction associated with sepsis. If untreated, persistent shock causes cellular injury and the liberation of further DAMPs. Like PAMPs, DAMPs have the potential to activate inflammation, creating a vicious circle. Early infection control with adequate antibiotic care is important during septic shock to decrease PAMPs arising from invasive microorganisms. Early aggressive fluid resuscitation as well as the administration of vasopressors and inotropes is also important to reduce DAMPs generated by damaged cells although excessive volume loading, and prolonged administration of catecholamines might be harmful. This review delineates some features of septic myocardial dysfunction, assesses its most common underlying mechanisms, and briefly outlines current therapeutic strategies and potential future approaches.

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice.

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.

Type II Citrullinemia (Citrin Deficiency): A Mysterious Disease caused by a Defect of Calcium-Binding Mitochondrial Carrier Protein

Citrullinemia (OMIM 215700) (McKusick, 1998) is an autosomal recessive disease that is caused by a deficiency of argininosuccinate synthetase (ASS; EC 6.3.4.5). The clinical, biochemical and molecular aspects of citrullinemia have been reviewed elsewhere (Walser, 1983; Saheki et al., 1987a; McKusick, 1998). So far, we have analyzed almost 200 patients with citrullinemia in our laboratory and have classified them into three types according to enzyme abnormality and into two forms according to pathogenesis (Figure 1) (Saheki et al., 1981, 1985a, 1987a, b; Kobayashi et al., 1993, 1999). The first form is the classical form (CTLN1) found in most patients with neonatal/infantile-onset citrullinemia (type I and type III), first described by (1962); the second form is the adult-onset type II citrullinemia (CTLN2) caused by a liver-specific ASS deficiency. In CTLN1, the enzyme defect is found in all tissues and cells in which ASS is expressed (Saheki et al., 1980, 1981, 1982, 1983a, 1985a, b, 1987a, b). To date, we have identified 36 mutations in the ASS gene located on chromosome 9q34 and have clarified the pathogenesis of most CTLN1 patients at the molecular level (Kobayashi et al., 1987, 1990, 1991, 1994, 1995a; Kakinoki et al., 1997).

Pathogenesis and pathophysiology of citrin (a mitochondrial aspartate glutamate carrier) deficiency

Adult-onset type II citrullinemia (CTLN2), characterized by a liver-specific deficiency of urea cycle enzyme, argininosuccinate synthetase, is caused by mutations in SLC25A13 that encodes a calcium binding mitochondrial solute carrier protein, citrin. Citrin deficiency causes not only CTLN2 but also neonatal intrahepatic cholestasis caused by citrin deficiency at neonatal period. Moreover citrin and its isoform aralar were found to be aspartate glutamate carrier. From the viewpoint of the metabolic functions of citrin as aspartate glutamate carrier in urea synthesis and NADH shuttle, symptoms of CTLN2 and neonatal intrahepatic cholestasis caused by citrin deficiency are analyzed.

Brain oxymetry in the operating room: current status and future directions with particular regard to cytochrome oxidase

Near-infrared spectroscopy (NIRS) is a cerebral monitoring method that noninvasively and continuously measures cerebral hemoglobin oxygenation and the redox state of cytochrome oxidase using highly tissue-permeable near-infrared light. This technique now has wide clinical application, and its usefulness in the measurement of cerebral hemoglobin oxygenation has been confirmed under global cerebral injury and/or hypoxemic hypoxia; however, regional cerebral infarction located far from the monitoring site may not be detected by NIRS. Furthermore, the specificity and accuracy of the measurement of the redox state of cytochrome oxidase remain controversial. We apply NIRS to both animal and clinical investigations. Based on these results, we discuss the significance of the measurement of cerebral hemoglobin oxygenation and cytochrome oxidase in vivo and in clinical medicine. Using our algorithm, cytochrome oxidase signals are unaffected by hemoglobin signals, even when hematocrit values change from 35 to 5% under cardiopulmonary bypass in a dog model. In the clinical study, cytochrome oxidase during surgery is likely to be a good (though not perfect) predictor of postoperative cerebral outcome. NIRS appears to be a promising technology, but additional investigations are required to establish its clinical efficacy and justify its routine use during operative and perioperative periods.