Fusao Watanabe

Implantation of Mesenchymal Stem Cells Overexpressing Endothelial Nitric Oxide Synthase Improves Right Ventricular Impairments Caused by Pulmonary Hypertension

Background— Pulmonary hypertension (PH) is a life-threatening disease. Bone marrow cell transplantation is reported to reduce the development of PH by increasing vascular beds in pulmonary circulation. However, adenoviral overexpression of endothelial nitric oxide synthase (eNOS) in the lung is also known to reduce PH. Because mesenchymal stem cells (MSCs) are potential cell sources for neovascularization, the implantation of MSCs overexpressing eNOS (MSCs/eNOS) may further improve the surgical results. We evaluated the efficacy of MSCs/eNOS implantation in monocrotaline (MCT)-induced PH rats. Methods and Results— MSCs were isolated from rat bone marrow. PH was induced in rats by subcutaneous injection of MCT. One week after MCT administration, the rats received 3 different treatments: MSCs (MSC group), MSCs/eNOS (MSC/eNOS group), or nontreatment (PH group). As the negative control, rats received saline instead of MCT (control group). Right ventricular (RV) hypertrophy and the elevation of RV systolic pressure (RVSP) were evaluated 3 weeks after MCT administration. Moreover, the effects of MSCs/eNOS on survival were investigated in PH induced by MCT 3 weeks earlier. RVSP in both the MSC and MSC/eNOS groups was significantly lower than the PH group. RVSP in the MSC/eNOS group was significantly lower than the MSC group. The RV weight to body weight ratio was significantly lower in the MSC and MSC/eNOS groups than the PH group. The survival time of rats receiving MSCs/eNOS was significantly longer than the nontreatment rats. Conclusion— Intravenous implantation of MSCs/eNOS may offer ameliorating effects on PH-related RV impairment and survival time.

Correlation between cellular ATP level and bile excretion in the rat liver

The influence of the cellular level of adenosine triphosphate (ATP) in the liver on bile excretion was studied in rats. In ischemia, the cellular ATP level decreased rapidly--and, concomitantly, bile flow stopped within 5 min. Administration of L-ethionine i.p. to rats reduced the bile flow rate with decrease in the cellular ATP level. The correlation between the bile flow rate and the cellular ATP level was confirmed in a liver perfusion system. On anoxic perfusion, the ATP level and bile flow rate changed in the same manner as in ischemia. The recovery rates of both on reoxygenation decreased with increase in the anoxic perfusion period. During perfusion under oxygenated conditions, decrease in cellular ATP to various levels by infusion of various concentrations of potassium cyanide, an inhibitor of respiration, resulted in corresponding and concomitant suppression of bile excretion. Kinetic analysis of the bile flow rate revealed a Michaelis-Menten-type curve for the cellular ATP level. The apparent Kms for ATP of bile flow rate in L-ethionine-treated rat liver and liver perfused with potassium cyanide were 1.0 and 1.6 mM, and their Vmax values were 4.1 and 2.5 microliter/min/g liver, respectively. The concentrations of main bile components, such as phospholipids, cholesterol, and taurocholate increased, but their total outputs decreased with decrease in the ATP level, and returned to the normal range with recovery of the ATP level. Thus, it was shown experimentally that the extent of hepatic injury can be assessed simply by monitoring the bile flow rate, which reflects the cellular level of ATP.

Molecular cloning and sequence analysis of cDNA encoding human kidney D-amino acid oxidase

cDNA clones encoding D‐amino acid oxidase were isolated from a human kidney cDNA library by hybridization with cDNA for the pig enzyme. The cDNA insert of 2.0 kilobase pairs long provided coding information for a protein consisting of 347 amino acids. The molecular mass of the enzyme was calculated to be 39 410 Da. The amino acid sequence similarity between the pig and human enzymes is 84.4%, and among the active site residues proposed from chemical modification studies, methionine‐110 of the pig enzyme was replaced by threonine. Northern blot analysis confirmed the expression of an mRNA of 2.0 kilobases encoding the D‐amino acid oxidase in human kidney.

Novel Isoforms of Rat Brain Fructose 6‐Phosphate 2‐Kinase/Fructose 2,6‐Bisphosphatase Are Generated by Tissue‐Specific Alternative Splicing

Abstract: Fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate, which is the most potent activator of glycolysis. We have shown previously the occurrence of a partial cDNA (RB7) encoding the catalytic core domain of a novel brain‐type isozyme. To elucidate the full‐length sequence of RB7 cDNA, we have carried out cDNA cloning using the 3′‐ and 5′‐rapid amplification cDNA ends method and have isolated eight isoforms from rat brain. The cDNA sequences encoding the 5′‐untranslated region, the amino‐terminal domain, and the catalytic core domain were identical among all the isoforms. However, heterogeneity of the carboxyl‐terminus was found by sequence analysis. This heterogeneity was shown not to have resulted from transcription of multiple genes, as Southern blot and genomic sequence analysis revealed that the gene was a single copy in the rat genome. It is likely that these transcripts represent splice variants of the gene. High‐level expression of the gene was also observed by northern blot analysis in skeletal muscle. However, the pattern of alternative splicing was different from that of brain, and only four isoforms were detected in skeletal muscle.

Beneficial effect of fructose-1,6-bisphosphate on mitochondrial function during ischemia-reperfusion of rat liver

BACKGROUND/AIMS Several groups have reported that administration of fructose-1,6-bisphosphate (FBP) reduces ischemic injury. The aim of this study was to determine the protective effect of FBP on the impairment of mitochondrial oxidative phosphorylation by ischemia-reperfusion injury in the rat liver. METHODS The respiratory control ratio (RCR) and the adenine nucleotide content of mitochondria isolated from ischemic and reperfused livers with or without FBP treatment were measured. RESULTS In FBP-treated livers, the cellular adenosine triphosphate level was restored to more than 50% of normal after 120 minutes of reperfusion following 120 minutes of ischemia, whereas that of control livers only reached 15% of normal. The RCR and the adenine nucleotide content of mitochondria isolated from FBP-treated livers were significantly higher than those of mitochondria from control livers after ischemia and reperfusion. FBP strongly suppressed the formation of lipid peroxides during reperfusion. In vitamin E-deficient rats, the RCR decreased markedly during reperfusion, but FBP protected the mitochondria against reperfusion injury. CONCLUSIONS FBP has a protective effect against ischemia-reperfusion injury on the liver and especially preserves the oxidative phosphorylation capacity of hepatic mitochondria.

Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase.

We have reported the occurrence of eight splice variants of rat brain fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue‐specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue‐specific manner.

Effect of site-specific mutagenesis of tyrosine-55, methionine-110 and histidine-217 in porcine kidney D-amino acid oxidase on its catalytic function

To assess the contributions of Tyr‐55, Met‐110 and His‐217 in porcine kidney D‐amino acid oxidase (EC 1.4.3.3, DAO) to its catalytic function, we constructed three mutant cDNAs coding for the enzymes possessing Phe‐55, Leu‐110 and Leu‐217 by site‐specific mutagenesis. The mutant and wild type cDNAs could be expressed in vitro with similar efficiency. The three mutant enzymes thus synthesized showed catalytic activities comparable to that of the wild type oxidase. It is concluded that Tyr‐55, Met‐110 and His‐217 are not directly involved in the catalytic function.

Biosynthesis of porcine kidney D-amino acid oxidase

The biosynthesis of a porcine kidney peroxisomal enzyme, D-amino acid oxidase (EC 1.4.3.3., DAO), was investigated. Pig kidney mRNA as well as free and membrane-bound polysomes were used to investigate in vitro protein synthesis using a rabbit reticulocyte lysate. mRNA and free polysomes, but not membrane-bound polysomes, directed the synthesis of DAO. To examine the in vivo synthesis of the enzyme, a pig kidney cell line (LLC-PK1) was biosynthetically labelled. Both the in vitro and in vivo synthesized DAO had the same molecular weight, 38,000, as that of the purified enzyme. These results indicate strongly that DAO is synthesized on free ribosomes and transferred to the interior of peroxisomes without any proteolytic modification.

Synthetic vascular prosthesis impregnated with mesenchymal stem cells overexpressing endothelial nitric oxide synthase.

Background— Endothelial dysfunction is known to exaggerate coronary artery disease, sometimes leading to irreversible myocardial damage. In such cases, repetitive coronary revascularization including coronary artery bypass grafting is needed, which may cause a shortage of graft conduits. On the other hand, endothelial nitric oxide synthase (eNOS) is an attractive target of cardiovascular gene therapy. The vascular prostheses, of which the inner surfaces are covered with mesenchymal stem cells (MSCs) overexpressing eNOS, are expected to offer feasible effects of NO and angiogenic effects of MSCs on the native coronary arterial beds, as well as improvement of self-patency. Herein, we attempted to develop small caliber vascular prostheses generating the bioactive proteins. Also, we attempted to transduce eNOS cDNA into MSCs. Methods and Results— The MSCs were isolated from rat bone marrow and transduced with each adenovirus harboring rat eNOS cDNA and &bgr;-galactosidase (&bgr;-gal) (eNOS/MSCs and &bgr;-gal/MSCs). The &bgr;-gal/MSCs were impregnated into vascular prostheses, then the expressions of &bgr;-gal on the inner surfaces of them were evaluated by 5-bromo-4-chloro-3-indolyl &bgr;-d-galactoside staining. The NOS activity of eNOS/MSCs was assayed by monitoring the conversion of 3H-arginine to 3H-citrulline. The inner surfaces of the vascular prostheses were covered with MSCs expressing &bgr;-gal. The amount of the 3H-citrulline increased, and eNOS/MSCs were determined to generate enzymatic activity of eNOS. This activity was completely inhibited by NG-nitro-l-arginine methyl ester. Conclusions— The inner surface of expanded polytetrafluoroethylene vascular prostheses seeded with lacZ gene-transduced MSCs exhibited recombinant proteins. Development of eNOS/MSC-seeded vascular prostheses would promise much longer graft patency and vasculoprotective effects.

A new method of quantitating serum and urinary levels of 1,5-anhydroglucitol in insulin-dependent diabetes mellitus

A new method was developed for quantitating the serum and urinary levels of 1,5-anhydroglucitol (AG), a sensitive and informative marker of glycemic control. This method utilized a combination of ODS and pyranose oxidase-immobilized columns for HPLC, and monitored hydrogen peroxide production with an electrochemical detector. We applied this method to determine the serum and urinary AG levels in 15 patients with insulin-dependent diabetes mellitus (IDDM) as well as in control subjects. Baseline separation of AG from other sugars such as glucose and myoinositol was achieved. Quantitation of AG was achieved over the range from 0.2 ng to 0.3 micrograms based upon peak heights. The serum and urinary AG levels in the IDDM patients were 4.4 +/- 8.3 mg/l and 5.1 +/- 4.3 mg/day, respectively. We found that the urinary AG to serum AG ratio showed a linear correlation with the urinary glucose level in the IDDM patients (urinary glucose (y) vs. urinary AG to serum AG ratio (x): y = 9.071x-0.991; r = 0.968, P < 0.001). This method proved efficient and reliable for quantitating urinary AG. Since determination of both the AG and glucose levels in urine gives equivalent clinical information to the serum AG level, urinary monitoring could provide a valuable addition to the available methods for assessing the glycemic status of IDDM patients.