Sintering of Platinum-Containing Conjugated Polymers: Gas Adsorption and Catalysis of the Formed Pt-Carbon Composites.

Pt-containing meta- and para-linked poly(phenyleneethynylene)s were synthesized by the dehydrochlorination coupling polymerization of PtCl2(PBu3)2 with m- and p-diethynylbenzenes. The formed polymers were sintered at 900 °C to obtain Pt-graphene hybrids, whose structures were examined by Raman scattering spectroscopy, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) measurements. Shapes─facets, terraces, and steps─with average diameters of 2.0-3.4 µm were observed by field emission scanning electron microscopy (FE-SEM). The Pt-graphene hybrids moderately adsorbed CO2 and O2 and slightly adsorbed ethylene and methane. Epoxidation of stilbene was carried out using Pt-graphene hybrids as catalysts to obtain stilbene oxide.

Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation.

Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.

Natural dolapyrrolidone: Isolation and absolute stereochemistry of a substructure of bioactive peptides.

During the course of our chemical analysis of the hydrophilic fractions from marine cyanobacterium Moorena producens, we have isolated natural dolapyrrolidone (Dpy, 1), a natural pyrrolidone derived from phenylalanine, for the first time as a single compound. Compound 1, with an (S)-l absolute stereochemistry, was previously identified as a substructure that is common among several bioactive natural peptides. Surprisingly, the absolute stereochemistry of the isolated natural 1, determined through total synthesis, was (R)-d. This result was unambiguously determined by HPLC analysis using a chiral stationary column by comparing the retention times of the natural 1 and authentic samples of synthetic enantiomers. To verify the unexpected result, the absolute stereochemistry of the natural 1 was confirmed by X-ray crystallographic analysis of Pt-complex derivative using the synthetic enantiomer.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29H(sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29H(sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice.

A novel mutation in TAZ causes mitochondrial respiratory chain disorder without cardiomyopathy.

Tafazzin, encoded by the TAZ gene, is a mitochondrial membrane-associated protein that remodels cardiolipin (CL), an important mitochondrial phospholipid. TAZ mutations are associated with Barth syndrome (BTHS). BTHS is an X-linked multisystemic disorder affecting usually male patients. Through sequence analysis of TAZ, we found one novel mutation c.39_60del p.(Pro14Alafs*19) by whole-exome sequencing and a reported missense mutation c.280C>T p.(Arg94Cys) by Sanger sequencing in two male patients (Pt1 and Pt2). Patient with c.280C>T mutation had dilated cardiomyopathy, while another patient with c.39_60del mutation had no feature of cardiomyopathy. A reported m.1555A>G homoplasmic variant was also identified in the patient having mutation c.39_60del by whole mitochondrial DNA sequencing method. This variant was not considered to be the main cause of mitochondrial dysfunction based on a cytoplasmic hybrid (cybrid) assay. Tafazzin expression was absent in both patient-derived fibroblast cells. Complementation of TAZ expression in fibroblasts from the patient with the novel mutation c.39_60del restored mitochondrial respiratory complex assembly. High-performance liquid chromatography-tandem mass spectrometry-based metabolic analysis revealed the decline of CL and the accumulation of monolysocardiolipin, indicating the loss of tafazzin activity. Owing to phenotypic variability, it is difficult to diagnose BTHS based on clinical features only. We conclude that genetic analysis should be performed to avoid underdiagnosis of this potentially life-threatening inborn error of metabolism.

Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNA(Lys) gene.

We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNA(Lys) gene encoded by mtDNA for use in studies of its pathogenesis and transmission profiles. Because patients with mitochondrial diseases frequently carry mutations in the mitochondrial tRNA(Lys) and tRNA(Leu(UUR)) genes, we focused our efforts on identifying somatic mutations of these genes in mouse lung carcinoma P29 cells. Of the 43 clones of PCR products including the tRNA(Lys) or tRNA(Leu(UUR)) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNA(Lys) gene. P29 subclones with predominant amounts of G7731A mtDNA expressed respiration defects, thus suggesting the pathogenicity of this mutation. We then transferred G7731A mtDNA into mouse ES cells and obtained F0 chimeric mice. Mating these F0 mice with C57BL/6J (B6) male mice resulted in the generation of F1 mice with G7731A mtDNA, named "mito-mice-tRNA(Lys7731)." Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNA(Lys7731) with high proportions of G7731A mtDNA exclusively expressed respiration defects and disease-related phenotypes and therefore are potential models for mitochondrial diseases due to mutations in the mitochondrial tRNA(Lys) gene. Moreover, the proportion of mutated mtDNA varied markedly among the pups born to each dam, suggesting that selecting oocytes with high proportions of normal mtDNA from affected mothers with tRNA(Lys)-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children.

The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells.

Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for understanding the recurrence risks for mtDNA diseases.

A somatic T15091C mutation in the Cytb gene of mouse mitochondrial DNA dominantly induces respiration defects.

Our previous studies provided evidence that mammalian mitochondrial DNA (mtDNA) mutations that cause mitochondrial respiration defects behave in a recessive manner, because the induction of respiration defects could be prevented with the help of a small proportion (10%-20%) of mtDNA without the mutations. However, subsequent studies found the induction of respiration defects by the accelerated accumulation of a small proportion of mtDNA with various somatic mutations, indicating the presence of mtDNA mutations that behave in a dominant manner. Here, to provide the evidence for the presence of dominant mutations in mtDNA, we used mouse lung carcinoma P29 cells and examined whether some mtDNA molecules possess somatic mutations that dominantly induce respiration defects. Cloning and sequence analysis of 40-48 mtDNA molecules from P29 cells was carried out to screen for somatic mutations in protein-coding genes, because mutations in these genes could dominantly regulate respiration defects by formation of abnormal polypeptides. We found 108 missense mutations existing in one or more of 40-48 mtDNA molecules. Of these missense mutations, a T15091C mutation in the Cytb gene was expected to be pathogenic due to the presence of its orthologous mutation in mtDNA from a patient with cardiomyopathy. After isolation of many subclones from parental P29 cells, we obtained subclones with various proportions of T15091C mtDNA, and showed that the respiration defects were induced in a subclone with only 49% T15091C mtDNA. Because the induction of respiration defects could not be prevented with the help of the remaining 51% mtDNA without the T15091C mutation, the results indicate that the T15091C mutation in mtDNA dominantly induced the respiration defects.

G7731A mutation in mouse mitochondrial tRNALys regulates late-onset disorders in transmitochondrial mice.

We previously generated mito-mice-tRNA(Lys7731) as a model for primary prevention of mitochondrial diseases. These mice harbour a G7731A mtDNA mutation in the tRNA(Lys) gene, but express only muscle weakness and short body length by four months. Here, we examined the effects of their aging on metabolic and histologic features. Unlike young mito-mice-tRNA(Lys7731), aged mito-mice-tRNA(Lys7731) developed muscle atrophy, renal failures, and various metabolic abnormalities, such as lactic acidosis and anemia, characteristic of patients with mitochondrial diseases. These observations provide convincing evidence that the respiration defects induced by high G7731A mtDNA levels cause these late-onset disorders that are relevant to mitochondrial diseases.

Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene confers respiration defects.

We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNA(Leu(UUR)) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

Transmitochondrial mito-miceΔ and mtDNA mutator mice, but not aged mice, share the same spectrum of musculoskeletal disorders.

The spectra of phenotypes associated with aging and mitochondrial diseases sometimes appear to overlap with each other. We used aged mice and a mouse model of mitochondrial diseases (transmitochondrial mito-miceΔ with deleted mtDNA) to study whether premature aging phenotypes observed in mtDNA mutator mice are associated with aging or mitochondrial diseases. Here, we provide convincing evidence that all the mice examined had musculoskeletal disorders of osteoporosis and muscle atrophy, which correspond to phenotypes prevalently observed in the elderly. However, precise investigation of musculoskeletal disorders revealed that the spectra of osteoporosis and muscle atrophy phenotypes in mtDNA mutator mice were very close to those in mito-miceΔ, but different from those of aged mice. Therefore, mtDNA mutator mice and mito-miceΔ, but not aged mice, share the spectra of musculoskeletal disorders.

A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice.

The mitochondrial outer membrane protein mitoNEET is a binding protein of the insulin sensitizer pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) and is considered a novel target for the treatment of type II diabetes. Several small-molecule compounds have been identified as mitoNEET ligands using structure-based design or virtual docking studies. However, there are no reports about their therapeutic potential in animal models. Recently, we synthesized a novel small molecule, TT01001 [ethyl-4-(3-(3,5-dichlorophenyl)thioureido)piperidine-1-carboxylate], designed on the basis of pioglitazone structure. In this study, we assessed the pharmacological properties of TT01001 in both in vitro and in vivo studies. We found that TT01001 bound to mitoNEET without peroxisome proliferator-activated receptor-γ activation effect. In type II diabetes model db/db mice, TT01001 improved hyperglycemia, hyperlipidemia, and glucose intolerance, and its efficacy was equivalent to that of pioglitazone, without the pioglitazone-associated weight gain. Mitochondrial complex II + III activity of the skeletal muscle was significantly increased in db/db mice. We found that TT01001 significantly suppressed the elevated activity of the complex II + III. These results suggest that TT01001 improved type II diabetes without causing weight gain and ameliorated mitochondrial function of db/db mice. This is the first study that demonstrates the effects of a mitoNEET ligand on glucose metabolism and mitochondrial function in an animal disease model. These findings support targeting mitoNEET as a potential therapeutic approach for the treatment of type II diabetes.

Specific mitochondrial DNA mutation in mice regulates diabetes and lymphoma development.

It has been hypothesized that respiration defects caused by accumulation of pathogenic mitochondrial DNA (mtDNA) mutations and the resultant overproduction of reactive oxygen species (ROS) or lactates are responsible for aging and age-associated disorders, including diabetes and tumor development. However, there is no direct evidence to prove the involvement of mtDNA mutations in these processes, because it is difficult to exclude the possible involvement of nuclear DNA mutations. Our previous studies resolved this issue by using an mtDNA exchange technology and showed that a G13997A mtDNA mutation found in mouse tumor cells induces metastasis via ROS overproduction. Here, using transmitochondrial mice (mito-mice), which we had generated previously by introducing G13997A mtDNA from mouse tumor cells into mouse embryonic stem cells, we provide convincing evidence supporting part of the abovementioned hypothesis by showing that G13997A mtDNA regulates diabetes development, lymphoma formation, and metastasis--but not aging--in this model.

Lactic acidemia in the pathogenesis of mice carrying mitochondrial DNA with a deletion.

Lactic acidemia is one manifestation of the mitochondrial diseases caused by pathogenic mutant mitochondrial DNA (mtDNA). However, little is known about its chronic effects in the progression of mitochondrial disease phenotypes. To obtain experimental evidence on this point, we used trans-mitochondrial model mice (mito-mice) heteroplasmic for wild-type and deleted mtDNA (DeltamtDNA). Mito-mice carrying predominantly DeltamtDNA showed mitochondrial respiration defects and the resultant disease phenotypes, including lactic acidemia; they also showed a decrease in mitochondrial biogenesis regulated by the peroxisome proliferative activated receptor gamma, coactivator 1 alpha (PGC1alpha)-mediated pathway, such as the expression of mitochondrial transcription factor A and mtDNA-encoded gene products and the control of mtDNA content. When the accelerated lactate production of these mito-mice was pharmacologically inhibited by sodium dichloroacetate (DCA), the decrease in mitochondrial biogenesis improved, thus leading to the relaxation of mitochondrial respiration defects and extension of life span. These results showed that chronic overproduction of lactate caused by metabolic adaptation in mitochondrial diseases further deconditioned mitochondrial function. Mitochondrial respiration defects in mitochondrial diseases are therefore induced not only directly by the presence of mutant mtDNA, but also by the chronic lactic acidemia. Our in vivo study also suggested that inhibition of chronic lactic acidemia is a potential strategy for treating some mitochondrial diseases.

Regulation of metastasis; mitochondrial DNA mutations have appeared on stage.

It has been controversial whether mtDNA mutations are responsible for tumorigenesis and for the process to develop metastases. To clarify this issue, we established trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that possess high and low metastatic potential. The results revealed that the G13997A mutation in the ND6 gene of mtDNA from highly metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS). The transmitochondrial model mice possessing G13997A mtDNA showed symptoms of impaired glucose tolerability, suggesting that ROS generated mtDNA mutations can regulate not only metastatic potential, but also age-associated disorders such as diabetes. We also identified other mtDNA mutations that affect metastatic potential but the mechanisms are independent of ROS production. The mtDNA-mediated reversible control of metastasis and age-associated disorders are novel functions of mtDNA, and suggests that ROS scavengers may be therapeutically effective to suppress these phenotypes.

Comprehensive application of an mtDsRed2-Tg mouse strain for mitochondrial imaging.

Mitochondria are essential for many cellular functions such as oxidative phosphorylation and calcium homeostasis; consequently, mitochondrial dysfunction could cause many diseases, including neurological disorders. Recently, mitochondrial dynamics, such as fusion, fission, and transportation, have been visualized in living cells by using time-lapse imaging systems. The changes in mitochondrial morphology could be an indicator for estimating the activity of mitochondrial biological function. Here, we report a transgenic mouse strain, mtDsRed2-Tg, which expresses a red fluorescent protein, DsRed2, exclusively in mitochondria. Mitochondrial morphology could be clearly observed in various tissues of this strain under confocal microscope. Recently, many transgenic mouse strains in which enhanced green fluorescent protein (EGFP)-tagged proteins of interest are expressed have been established for physiological analysis in vivo. After mating these strains with mtDsRed2-Tg mice, red-colored mitochondria and green-colored proteins were detected simultaneously using fluorescent imaging systems, and the interactions between mitochondria and those proteins could be morphologically analyzed in cells and tissues of the F(1) hybrids. Thus, mtDsRed2-Tg mice can be a powerful tool for bioimaging studies on mitochondrial functions.

A 44-year experience of prosthetic heart valve implantation at Niigata University Hospital.

Based on a single hospital experience of heart valve implantation from 1965 to 2009, the superiority of prosthetic heart valves including Starr-Edwards caged ball valves, Omniscience aortic tilting disc valves, and St. Jude Medical bileaflet valves are reviewed. This review discusses the prominent antithrombogenicity of the Starr-Edwards model 1200 aortic prosthesis under selected conditions, the relatively rarely thrombosed (despite its decreased opening angle) Omniscience aortic valve, the long-term outcomes 10 as well as 30 years after St. Jude Medical valve replacement, and finally the latest results on the significance of patient-aortic prosthesis mismatch in relation to myocardial hypertrophy. The findings described here should be considered in further investigations of cardiac valve prostheses.

New evidence confirms that the mitochondrial bottleneck is generated without reduction of mitochondrial DNA content in early primordial germ cells of mice.

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells.

Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease.

Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte's number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.

Reduction of spinal cord ischemia/reperfusion injury with simvastatin in rats.

BACKGROUND: Surgery of the thoracic or thoracoabdominal aorta may cause spinal cord ischemia and subsequent paraplegia. However, conventional strategies for preventing paraplegia due to spinal cord ischemia provide insufficient protection and cause additional side effects. We hypothesized that simvastatin, a drug recently shown to be neuroprotective against brain ischemia/reperfusion, would be neuroprotective in a rat spinal cord ischemia/reperfusion model. METHODS: Rats were randomly assigned to simvastatin, vehicle, or sham-surgery (sham) groups (n = 6 per group). Simvastatin (10 mg/kg) or vehicle was administered subcutaneously once daily for 7 days before aortic balloon occlusion, and once at 24 hours after reperfusion. Spinal cord ischemia was induced by balloon inflation of a 2F Fogarty catheter in the thoracic aorta, and the proximal mean arterial blood pressure was maintained at 40 mm Hg for 12 minutes. The sham group received the same operation without inflation of the balloon. Ischemic injury was assessed by hindlimb motor function using the Motor Deficit Index score at 6 to 48 hours after ischemic reperfusion, and histological assessment of the spinal cord was performed 48 hours after reperfusion. RESULTS: The Motor Deficit Index scores at 24 and 48 hours after reperfusion were significantly improved in the simvastatin group compared with the vehicle group (P = 0.021 and P = 0.023, respectively). Furthermore, there were significantly more normal motor neurons in the simvastatin group than in the vehicle group (P = 0.037). The percentage area of white matter vacuolation was significantly smaller in the simvastatin group than in the vehicle group (P = 0.030). CONCLUSIONS: Simvastatin treatment can attenuate hindlimb motor dysfunction and histopathological changes in spinal cord ischemia/reperfusion injury in rats.