Masaki Kanazawa

Reduction in Myocardial Ischemia/Reperfusion Injury in Group X Secretory Phospholipase A2-Deficient Mice

Background— Group X secretory phospholipase A2 (sPLA2-X) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA2. sPLA2-X is expressed in neutrophils, but its pathogenic role remains unclear. Methods and Results— We generated mice that lack sPLA2-X and studied their response to myocardial ischemia/reperfusion. The sPLA2-X−/− mice had a significant reduction in myocardial infarct size and a decrease in myocardial myeloperoxidase activity compared with sPLA2-X+/+ mice. Myocardial infarct size was also significantly reduced in lethally irradiated sPLA2-X+/+ mice reconstituted with sPLA2-X−/− bone marrow compared with sPLA2-X+/+ bone marrow. The extent of myocardial ischemia/reperfusion injury was comparable between sPLA2-X−/− and sPLA2-X+/+ mice in Langendorff experiments using isolated hearts and blood-free perfusion buffer, supporting a potential role of sPLA2-X in blood in myocardial ischemia/reperfusion injury. In the infarcted myocardium of sPLA2-X+/+ mice, sPLA2-X was released from neutrophils but not myocardial tissues and platelets and was undetectable in the peripheral serum. The sPLA2-X−/− mice had lower accumulation of neutrophils in ischemic myocardium, and the isolated sPLA2-X−/− neutrophils had lower release of arachidonic acid and attenuated cytotoxic activities including respiratory burst compared with sPLA2-X+/+ neutrophils. The attenuated functions of sPLA2-X−/− neutrophils were reversible by the exogenous addition of sPLA2-X protein. Furthermore, administration of a sPLA2 inhibitor reduced myocardial infarct size and suppressed the cytotoxic activity of sPLA2-X+/+ neutrophils. Conclusions— Myocardial ischemia/reperfusion injury was attenuated in sPLA2-X−/− mice partly through the suppression of neutrophil cytotoxic activities.

Visualization of Mitochondrial Protein Import in Cultured Mammalian Cells with Green Fluorescent Protein and Effects of Overexpression of the Human Import Receptor Tom20

The presequence of the ornithine transcarbamylase precursor (pOTC) was fused to green fluorescent protein (GFP), yielding pOTC-GFP and pOTCN-GFP containing the presequence plus 4 and 58 residues of mature ornithine transcarbamylase, respectively. When GFP cDNA was transfected into COS-7 cells, the cytosol and nucleus were fluorescent. On the other hand, pOTC-GFP cDNA gave strong fluorescence of a unique mitochondrial pattern. After fractionation of cells expressing pOTC-GFP with digitonin, fluorescence was recovered mostly in the particulate fraction. Immunoblot analysis showed that processed GFP was present in the particulate fraction, whereas pOTC-GFP was recovered in both the soluble and particulate fractions. pOTC-GFP and pOTCN-GFP synthesized in vitro were imported efficiently into the isolated mitochondria. Single and triple amino acid mutations in the presequence resulted in impaired mitochondrial import and in a loss of mitochondrial fluorescence. Perinuclear aggregation of fluorescent mitochondria was observed when the human mitochondrial import receptor Tom20 (hTom20) was coexpressed with pOTC-GFP. Overexpression of hTom20 (not ΔhTom20, which lacks the anchor sequence) resulted in stimulated mitochondrial import of pOTC-GFP in COS-7 cells. When pOTC-GFP cDNA was microinjected into nuclei of human fibroblast cells, mitochondrial fluorescence was detected as early as 2-3 h after injection. These results show that GFP fusion protein can be used to visualize mitochondrial structures and to monitor mitochondrial protein import in a single cell in real time.

Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106–125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitroimport of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.

Magnetic resonance imaging in late-onset ornithine transcarbamylase deficiency

We examined brain magnetic resonance imaging (MRI) in a cohort of seven patients with ornithine transcarbamylase deficiency (OTCD), and correlated MRI findings with clinical manifestations. Seven patients with OTCD, aged 3-27 years, all with a missense mutation, were involved in the study. We classified the OTCD patients clinically into four stages. MR study was performed with a 1.5-T superconducting magnet during asymptomatic periods. MRI revealed white matter lesions in two patients with an advanced clinical stage, i.e. T1 and T2 prolongated round lesions in the deep white matter and posterolateral angle of the lateral ventricle in one patient; small foci of T2 and T1 prolongation in the subcortical white matter in another. Parenchymal lesions, and cerebral and cerebellar atrophy were not found in the other five patients. MRI might be normal in the early stage of the disease, and progress in proportion to the clinical stage of OTCD. OTCD should be considered as a differential diagnosis of small foci in the white matter in children.

Evaluation of endogenous nitric oxide synthesis in congenital urea cycle enzyme defects

Nitric oxide (NO) is synthesized from arginine and O(2) by nitric oxide synthase (NOS). Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by the 2 enzymes acting in the urea cycle: argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Although the complete urea cycle is expressed only in the liver, ASS and ASL are expressed in other organs including the kidney and vascular endothelium. To examine possible alterations of the NO pathway in urea cycle defects, we measured plasma concentrations of arginine and citrulline and serum concentrations of nitrite/nitrate (NOx(-), stable NO metabolites) and asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor) in patients with congenital urea cycle disorders of 3 types: ornithine transcarbamylase (OTC) deficiency, ASS deficiency, and ASL deficiency. All were receiving oral arginine replacement at the time of this study. The same parameters were also measured in healthy subjects, who participated as controls. The OTC-deficient patients had significantly high NOx(-) and nonsignificantly high ADMA concentrations. Their NOx(-) was significantly positively correlated with arginine. The ASS-deficient patients had significantly low NOx(-) and significantly high ADMA concentrations. The ASL-deficient patients had normal NOx(-) and nonsignificantly high ADMA concentrations. In ASS-deficient and ASL-deficient patients, the NOx(-) was significantly inversely correlated with citrulline. These results suggest that NO synthesis is enhanced in OTC-deficient patients while receiving arginine but that NO synthesis remains low in ASS-deficient patients despite receiving arginine. They also suggest that endogenous NO synthesis is negatively affected by citrulline and ADMA in ASS-deficient and ASL-deficient patients. Although the molecular mechanisms remain poorly understood, we infer that the NO pathway might play a role in the pathophysiology related to congenital urea cycle disorders.

Effects of arginine treatment on nutrition, growth and urea cycle function in seven Japanese boys with late-onset ornithine transcarbamylase deficiency

BackgroundThe aim of this study was to investigate the effects of arginine on nutrition, growth and urea cycle function in boys with late-onset ornithine transcarbamylase deficiency (OTCD). Seven Japanese boys with late-onset OTCD enrolled in this study resumed arginine treatment after the cessation of this therapy for a few years. Clinical presentations such as vomiting and unconsciousness, plasma amino acids and urinary orotate excretion were followed chronologically to evaluate urea cycle function and protein synthesis with and without this therapy. In addition to height and body weight, blood levels of proteins, lipids, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein -3 (IGFBP-3) were monitored.ResultsThe frequency of hyperammonemic attacks and urinary orotate excretion decreased significantly following the resumption of arginine treatment. Despite showing no marked change in body weight, height increased gradually. Extremely low plasma arginine increased to normal levels, while plasma glutamine and alanine levels decreased considerably. Except for a slight increase in high-density lipoprotein cholesterol level, blood levels of markers for nutrition did not change. In contrast, low serum IGF-I and IGFBP-3 levels increased to age-matched control levels, and normal urinary GH secretion became greater than the level observed in the controls.ConclusionArginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.

Expression analysis of two mutations in carnitine palmitoyltransferase IA deficiency

AbstractCarnitine palmitoyltransferase I (CPT I) is one of the carnitine cycle enzymes that plays a role in the transportation of long-fatty acids into the mitochondria for β-oxidation. Hepatic carnitine palmitoyltransferase I (CPT IA) is one of the isozymes of CPT I, and its deficiency results in an autosomal recessive mitochondrial fatty acid oxidation disorder. To date, 19 patients with CPT IA deficiency and 9 CPT IA mutations have been reported. Recently, six novel mutations in the CPT IA gene were reported in Japanese patients with CPT I deficiencies who were clinically diagnosed as having a Reye-like syndrome. One of these mutations was a missense mutation, 1079A>G (E360G). The other was a splicing mutation, 2027-2028+2delAAGT, which caused aberrant splicing transcripts, whereas 1876-2028del, 2027-2028insGTCTCTTCC ACTTCTTCC, and 2026-2028del were three aberrant transcripts that kept reading in-frame. In this report, an expression assay using SV40 transformed fibroblasts was performed to investigate the consequences of these two mutations on enzyme activity and protein levels. Molecular analysis in this study revealed that the two mutations 1079A>G and 2028+2delAAGT were the disease-causing mutations.

Identification of three novel mutations in the MNK gene in three unrelated Japanese patients with classical Menkes disease

AbstractMenkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations to the Menkes (MNK) gene. We identified three novel mutations of the MNK gene in three unrelated Japanese patients with classical Menkes disease by analyzing reverse-transcriptase polymerase chain reaction products and genomic DNA of the MNK gene. Firstly, an insertional mutation was found, 1173 ins A, which led to a premature termination and resulted in a very immature Menkes protein. Secondly, we found a point mutation, T2763G, resulting in a leucine-to-arginine conversion, which we predicted would cause a change in the secondary structure of the Menkes protein. Finally, we identified a splicing mutation, 2317+5G>C, which resulted in the skipping of both exons 8 and 9 or exon 9 only, and led to a truncation of the protein. Each of these mutations is hypothesized to destroy copper-ATPase-mediated copper transport. We propose that each of these mutations in the MNK gene plays a causative role in the disease.

Enzyme-linked immunosorbent assay of carbamoylphosphate synthetase I: plasma enzyme in rat experimental hepatitis and its clearance.

Carbamoylphosphate synthetase I (CPS I), a urea cycle enzyme, is located almost exclusively in the mitochondria of hepatocytes. The enzyme is unique in that it constitutes about 2-6% of total liver protein and is composed of a large subunit of 160 kD. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for measurement of the enzyme in plasma using an antibody against the rat enzyme. In galactosamine-induced rat acute hepatitis, plasma concentration of CPS I that was 1-2 micrograms/ml blood before the treatment, increased up to 125 micrograms/ml blood in 24 h after the treatment and decreased to a near control level in 72 h. Plasma concentration of ornithine carbamoyl-transferase (OCT), another urea cycle enzyme, reached a maximum in 24 h and then decreased a little more rapidly than that of CPS I. On the other hand, alanine aminotransferase activity reached a maximum in 36 h and decreased to a normal level in 96 h. In immunoblot analysis, the native CPS I polypeptide of 160 kD and its fragments of 140 and 125 kD were detected 24-48 h after the treatment. When purified rat CPS I and bovine OCT were injected intravenously into rats, the enzymes disappeared from blood roughly exponentially with apparent half-lives of about 67 and 18 min, respectively. Development of an ELISA for human CPS I and determination of the serum enzyme in various liver diseases remain to be performed.

Mutations of carnitine palmitoyltransferase II (CPT II) in Japanese patients with CPT II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving β‐oxidation of long‐chain fatty acids. CPT II deficiency is a wide‐spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult‐onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non‐consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction‐amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult‐onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult‐onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult‐onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.