Biomedical subjects

K Shinmura

Publications and source records attributed to K Shinmura.

At least 19 recordsLinked to original sources

PubMed1998

Genetic polymorphisms and alternative splicing of the hOGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA.

The hOGG1 gene encodes a DNA glycosylase that excises 8-hydroxyguanine (oh8Gua) from damaged DNA. Structural analyses of the hOGG1 gene and its transcripts were performed in normal and lung cancer cells. Due to a genetic polymorphism at codon 326, hOGG1-Ser326 and hOGG1-Cys326 proteins were produced in human cells. Activity in the repair of oh8Gua was greater in hOGG1-Ser326 protein than in hOGG1-Cys326 protein in the complementation assay of an E. coli mutant defective in the repair of oh8Gua. Two isoforms of hOGG1 transcripts produced by alternative splicing encoded distinct hOGG1 proteins: one with and the other without a putative nuclear localization signal. Loss of heterozygosity at the hOGG1 locus was frequently (15/ 23, 62.2%) detected in lung cancer cells, and a cell line NCI-H526 had a mutation leading to the formation of the transcripts encoding a truncated hOGG1 protein. However, the oh8Gua levels in nuclear DNA were similar among lung cancer cells and leukocytes irrespective of the type of hOGG1 proteins expressed. These results suggest that the oh8Gua levels are maintained at a steady level, even though multiple hOGG1 proteins are produced due to genetic polymorphisms, mutations and alternative splicing of the hOGG1 gene.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Low concentrations of adenosine receptor blocker decrease protection by hypoxic preconditioning in ischemic rat hearts.

UNLABELLED: A role for adenosine in ischemic preconditioning and hypoxic preconditioning (HP) has been established in several species but is controversial in rats, due in part to the inconsistency of the data from the different experimental design. Our objective was to investigate the role of adenosine in the protection of the ischemic myocardium by HP in rats. METHODS: perfused hearts isolated from Sprague-Dawley rats were exposed to 5 min of hypoxic perfusion before 25 min of global ischemia followed by 20 min of reperfusion. The effects of adenosine receptor antagonist, 8-(p-sulfophenyl)-theophylline (8SPT) on HP-based changes in left-ventricular function, energy metabolites, and release of creatine kinase and lactate dehydrogenase were determined. To minimise non-specific effects of 8SPT, low concentrations of agent (0.5 or 1.0 micro mol/l) were used. RESULTS: 8SPT alone had no deleterious effects on normoxically perfused hearts or on ischemic/reperfused hearts. HP improved the recovery of LV function and creatine phosphate, and reduced the release of enzymes during reperfusion. 8SPT (1.0 micromol/l) ameliorated the beneficial effect of HP on cardiac function, but did not reverse the reduction in release of enzymes by HP completely. CONCLUSION: results suggest that the protective effect of HP on myocardial contractile function may be mediated by receptor(s) that can be inhibited by low concentrations of antagonist but may not have a primary role in the reduction of cellular damage by HP in rats.

Adenosine↗

PubMed1998

Genomic structure and chromosomal localization of the mouse Ogg1 gene that is involved in the repair of 8-hydroxyguanine in DNA damage.

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine: oh8Gua) is a damaged form of guanine induced by oxygen-free radicals and causes GC to TA transversions. Previously we isolated the hOGG1 gene, a human homolog of the yeast OGG1 gene, which encodes a DNA glycosylase and lyase to excise oh8Gua in DNA. In this study, we isolated a mouse homolog (Ogg1) of the OGG1 gene, characterized oh8Gua-specific DNA glycosylase/AP lyase activities of its product, and determined chromosomal localization and exon-intron organization of this gene. A predicted protein possessed five domains homologous to human and yeast OGG1 proteins. Helix-hairpin-helix and C2H2 zinc finger-like DNA-binding motifs found in human and yeast OGG1 proteins were also retained in mouse Ogg1 protein. The properties of a GST fusion protein were identical to human and yeast OGG1 proteins in glycosylase/lyase activities, their substrate specificities, and suppressive activities against the spontaneous mutagenesis of an Escherichia coli mutM mutY double mutant. The mouse Ogg1 gene was mapped to Chromosome (Chr) 6, and consisted of 7 exons approximately 6 kb long. Two DNA-binding motifs were encoded in exons 4 through 5. These data will facilitate the investigation of the OGG1 gene to elucidate the relationship between oxidative DNA damage and carcinogenesis.

Amino Acid Sequence↗

PubMed1998

RER phenotype and its associated mutations in familial gastric cancer.

To clarify the genetic background of gastric cancer, we collected 28 familial gastric cancers (FGCs) with reference to the Amsterdam criteria in hereditary non-polyposis colorectal cancer (HNPCC) and investigated the frequency of replication error (RER) at six microsatellite loci and frameshift mutations in its related genes in these tumors. RER was detected in seven (25%) of the 28 gastric cancers. Five (18%) cases showed RER at more than two loci. The apparent increased incidence of RER in FGC was not detected compared with that reported in sporadic gastric cancers previously. Among four cases with RER at more than three loci, frameshift mutations in the (A)8 track of the hMSH3 gene were detected in all the four cases and mutations in the (A)10 track of the transforming growth factor-beta type II receptor (TGF-beta RII) gene were detected in the three of them. Histologically, three of the four cases were of the intestinal type, and the other one was the diffuse type. No mutation was detected in the (C)8 and (GT)3 tracks of the hMSH6 and TGF-beta RII genes respectively. These results indicate that the acquisition of the RER phenotype equally influences the gastric carcinogenesis of both sporadic and familial cases, and that the majority of FGC is pathogenetically distinct from HNPCC.

Adult↗

PubMed1998

Mutational analyses of multiple target genes in histologically heterogeneous gastric cancer with microsatellite instability.

It has been recognized that gastric cancer often shows histological heterogeneity in a single tumor. Although microsatellite instability (MSI) has been reported in gastric cancer, the significance of genomic instability in gastric cancers with histological heterogeneity within a single tumor has never been addressed. We investigated MSI at 8 microsatellite loci in 40 normal/tumor DNA pairs from 20 gastric cancers with histological heterogeneity. Six of 20 patients (10 DNAs of 40 tumor DNAs) had severe MSI in more than 3 loci. Four of the MSI-positive cases had frameshift mutations in the poly(A)10 tract of the TGF beta RII gene. This mutation was found only in the MSI-positive component in the 2 cases (cases 4 and 5) in which only 1 component exhibited MSI. The other 4 cases demonstrated homozygous or heteroclonal mutations (1 and 2 base deletions) in the poly(A)8 tract of the hMSH3 gene; no mutation was detected in the poly(C)8 tract of the hMSH6 gene in any of the MSI-positive cases. The profile of alterations in multiple targets was different between the 2 components in most of the cases (5/6). These findings suggest that mismatch repair deficiency in MSI-positive tumors causes multiple gene inactivations through frameshift mutations in short repetitive sequences in a heterogeneous way within a histologically heterogeneous tumor.

Aged↗

PubMed1998

Infrequent mutations of the hOGG1 gene, that is involved in the excision of 8-hydroxyguanine in damaged DNA, in human gastric cancer.

DNA glycosylase, encoded by the hOGG1 gene, repairs 8-hydroxyguanine (oh8Gua), which is an oxidatively damaged mutagenic base. To clarify whether the DNA repair activity of hOGG1 protein is involved in gastric carcinogenesis, we examined 9 gastric cancer cell lines and 35 primary gastric cancers for mutations and genetic polymorphisms of the hOGG1 gene by polymerase chain reaction-single strand conformation polymorphism analysis. A G-to-A transition was detected in a gastric cancer cell line, MKN1. This nucleotide change caused the conversion of the amino acid from Arg to His at codon 154, which is located in a domain highly conserved among human, mouse, and yeast OGG1 proteins. No mutation was detected in primary gastric cancers. We compared the distribution of the polymorphic alleles associated with enzymatic activity (hOGG1-Ser326 vs. hOGG1-Cys326) between 35 gastric cancer patients and 42 healthy individuals. Although the frequency of the Cys326 allele, associated with low enzymatic activity, in gastric cancer patients was a little higher than that in healthy individuals, the difference did not reach statistical significance. These results suggest that low hOGG1 activity due to mutations and genetic polymorphisms is involved in the development of only a small subset of gastric cancers.

DNA Damage↗

PubMed1998

Myocardial uptake of iodine-125-labeled 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid is decreased in chronic diabetic rats with changes in subcellular distribution.

Iodine-123-labeled 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid (123I-BMIPP) is widely used to detect myocardial metabolic changes, but the preferred energy substrates in the myocardium would be expected to be altered in the presence of metabolic disorders such as diabetes mellitus (DM). We investigated the metabolism of branched-chain fatty acids in the myocardium of rats with DM. Streptozotocin-induced DM rats were examined 48 h (acute; AD) and 6 weeks (chronic; CD) after injection of streptozotocin. Hearts were excised 15 min or 60 min after injection of 0.185 MBq of 125I-BMIPP, followed by homogenization in an EDTA-Tris buffer. The homogenates were subjected to differential centrifugation to obtain the mitochondrial (MF) and cytoplasmic (CF) fractions. Myocardial 125I uptake tended to increase in the AD group, but the change was not significant. Myocardial 125I uptake at 15 min was significantly lower in the CD group than in the control group, even in the insulin-treated rats [control (CC), 4.4+/-0.4; not treated (CDN), 3.3+/-0.5; insulin-treated (CDI), 3.4+/-0.4 x 10(4) cpm/g, p<0.05 in each case]. The 125I count value corrected for the blood count (counts/min (cpm) per g of protein divided by blood cpm) in the MF decreased by 40% at 60 min in the CC group, but increased by 60% in the CDN group. The results of the present study suggest that the myocardial uptake of branched-chain fatty acids is decreased in rats with chronic diabetes, probably as a result of mitochondrial dysfunction.

Acute Disease↗

PubMed1998

Effect of E4031, a class III antiarrhythmic drug, on ischemia- and reperfusion-induced arrhythmias in isolated rat hearts.

UNLABELLED: The delayed outward rectifier K+ channel has a role in the increase in automaticity of myocytes under pathophysiological conditions. The purpose of the present study was to clarify the effect of blockade of outward rectifier K+ channels by a class III antiarrhythmic drug, E4031, on ischemia- and reperfusion-induced arrhythmias. Ion fluxes, energy metabolites and cardiac function were measured and the epicardial electrocardiograms of Langendorff-perfused rat hearts were recorded during initial perfusion, global or regional ischemia and reperfusion. 10(-7) M of E4031 administered during the initial perfusion did not prolong the QT interval, but slowed the heart rate ( CONTROL: 222, E4031: 183 bpm, p < 0.05), increased myocardial 45Ca2+ uptake ( CONTROL: 2.1, E4031: 2.9 mumol/g dwt, p < 0.05) and attenuated the loss of intracellular K+ during ischemia ( CONTROL: 238, E4031: 248 mumol/g dwt, p < 0.05). E4031 tended to reduce ischemia-induced ventricular tachyarrhythmias ( CONTROL: 60, E4031: 30%, n.s.), but reperfusion-induced ventricular tachyarrhythmias were sustained longer by the administration of E4031 (CONTROL: 255, E4031: 623 sec, p < 0.05). Prior exposure to E4031 decreased the depletion of high energy phosphates during ischemia, but suppressed their recovery during reperfusion. These results suggest that the attenuated loss of K+ from the ischemic myocardium and the decrease in heart rate by E4031 contributed to the reduction of ischemia-induced arrhythmias. However, the increase in myocardial Ca2+ uptake and altered energy metabolism may be responsible for the increase in reperfusion-induced arrhythmias.

Animals↗

PubMed1997

Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage.

We report the cloning of a human homolog of the yeast OGGC1 gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine). Since the deduced amino acid sequence (68 amino acids) of a human expressed sequence tag, N55394, matched a short stretch of yeast OGG1 protein with greater than 40% amino acid identity, a full length cDNA clone was isolated from a HeLa cell cDNA library with the N55394 clone as a probe. The cDNA clone encodes a predicted protein of 345 amino acids which is homologous to yeast OGG1 protein throughout the entire polypeptide sequence and shares 38% amino acid identity with yeast OGG1 protein. Moreover, we found that both a human homolog and yeast OGG1 protein possess two distinct DNA binding motifs, a helix-hairpin-helix (HhH) motif and a C2H2 zinc finger like motif, and a domain homologous to human and E. coli MutY proteins. Expression of a human homolog suppressed spontaneous mutagenesis of an E. coli (mutM mutY) mutant as in the case of yeast OGG1 protein. The gene was ubiquitously expressed in a variety of human organs and mapped to chromosome 3p26.2. These results strongly suggest that the gene isolated here is a human counterpart of the yeast OGGI gene and is involved in the repair of oxidative DNA damage in human cells.

Amino Acid Sequence↗

PubMed1997

Changes in ischemic tolerance and effects of ischemic preconditioning in middle-aged rat hearts.

BACKGROUND: Although both clinical and animal studies have shown that ischemic tolerance is reduced in the senescent myocardium, it has not been clarified when myocardium becomes more vulnerable to ischemia. Preconditioning protects the hearts of young adult animals of various species, but its effects are not identical in human studies. We investigated whether ischemic tolerance and the effect of preconditioning decreased in isolated hearts of middle-aged rats. METHODS AND RESULTS: The hearts of young adult rats (12 weeks old: group Y, n = 44) and middle-aged rats (50 weeks old: group M, n = 44) were subjected to global ischemia for 15, 20, or 25 minutes followed by reperfusion. Hearts were also subjected to preconditioning and then to 20 (group Y, n = 22) or 15 (group M, n = 22) minutes of ischemia followed by reperfusion. Left ventricular developed pressure (LVDP) was decreased by 40% to 60%, and the level of ATP was decreased by 60% to 70% in group M compared with group Y. Preconditioning increased LVDP (% LVDP, 40.5% to 72.4%) and levels of high-energy phosphates (ATP, 11.8 to 14.1; creatine phosphate, 17.0 to 23.1 mumol/g dry wt) and reduced left ventricular end-diastolic pressure (LVEDP, 32.8 to 10.3 mm Hg), creatine kinase release (257 to 132 U/g dry wt), and ryanodine-sensitive sarcoplasmic reticulum Ca2+ release after ischemia in group Y. Preconditioning exerted opposite effects in group M (% LVDP, 45.9% to 15.8%; LVEDP, 21.0 to 28.5 mm Hg; ATP, 14.1 to 8.5 mumol/g dry wt; and CK release, 176 to 332 U/g dry wt). Preconditioning was associated with increases in the incidence of reperfusion-induced ventricular fibrillation (0% to 62.5%) and the rate of sarcoplasmic reticulum Ca2+ release in group M. CONCLUSIONS: These results indicate that hearts became more vulnerable to ischemia with age and that the beneficial effects of preconditioning were reversed in middle-aged rat hearts.

Adenosine Triphosphate↗

PubMed1997

Decrease in ischemic tolerance with aging in isolated perfused Fischer 344 rat hearts: relation to increases in intracellular Na+ after ischemia.

While the ischemic tolerance of the myocardium has been reported to decrease with senescence, it is not known when and how this occurs. Our objectives were to determine whether the tolerance to myocardial ischemia in rats decreased before the onset of senescence and whether an increase in myocardial ionic imbalance was associated with an enhanced myocardial injury with aging. Hearts were isolated from Fischer 344 rats categorized as young (12 weeks old), mature adult (24 weeks), middle-aged (50 weeks) or senescent (100 weeks). Hearts were perfused isovolumically by the Langendorff procedure and subjected to 25 min of global ischemia followed by 30 min of reperfusion. In the 50- and 100-week-old rats, the recovery of ventricular function and high-energy phosphate levels was lower and there was increased incidence of ventricular fibrillation after 25 min of global ischemia followed by reperfusion. The release of creatine kinase and lactate dehydrogenase during reperfusion was greater in the 50-and 100-week-old rats than in the 12- and 24-week-old rats, indicating the irreversible myocardial damage due to ischemia-reperfusion increased by middle-age. Intracellular levels of Na+ and K+ before ischemia were higher in the 50- or 100-week-old rats than in the 12-week-old rats. The increase in intracellular Na+ at end of ischemia was greater in the older (50-week-old, 215% of the pre-ischemic value; 100-week-old, 232% of the pre-ischemic value) than in the younger rats (12-week-old, 158% of the pre-ischemic value). Results indicated that the rat heart becomes more vulnerable to ischemia in middle-age. This decrease in ischemic tolerance may be caused by an acceleration of myocardial ionic imbalance with aging.

Aging↗

PubMed1997

8-hydroxyguanine (7,8-dihydro-8-oxoguanine) DNA glycosylase and AP lyase activities of hOGG1 protein and their substrate specificity.

Recently we cloned a structural human homolog (hOGG1) of the yeast OGG1 (yOGG1) gene that is involved in the excision repair of 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine; oh8Gua), hOGG1 protein shares 38% amino acid identity with yOGG1 protein. In this paper, we define the substrate specificity of oh8Gua DNA glycosylase and AP lyase activities of the hOGG1 protein. The oh8Gua released from oh8Gua containing DNA was measured by analysis with HPLC coupled with electrochemical detector (ECD) and cleavage sites in the DNA were identified by cleavage assay using gel electrophoresis. GST-hOGG1 protein possessed the oh8Gua DNA glycosylase/AP lyase activity and weak delta-elimination activity, oh8Gua opposite the C in duplex oligonucleotide was most efficiently released by GST-hOGG1 protein and oh8Gua opposite the T was also released, while oh8Gua opposite the G or A was very slowly done. The rank order of DNA cleavage efficiency was the same as that of oh8Gua glycosylase activity. Glycosylase/AP lyase activities and their substrate specificities of the GST-hOGG1 protein was similar to GST-yOGG1 protein but different from MutM protein. These results indicate that the dominant function of hOGG1 protein is a oh8Gua glycosylase reaction by specifically recognizing oh8Gua and pyrimidine opposite the oh8Gua and delta-elimination reaction in the same manner as yOGG1 protein. Thus, the hOGG1 gene is a functional human homolog of the yOGG1 gene on oh8Gua excision repair in spite of the low structural identity at amino acid level between hOGG1 and yOGG1 proteins.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Effects of alpha 1-adrenoreceptor subtype blockade on ischemia-reperfusion injury.

To clarify the roles of subclasses of alpha 1-adrenoreceptors in ischemic-reperfused myocardium, we compared the effect of the nonselective alpha 1-blocker bunazosin with that of the alpha 1A-blocker WB4101 and the alpha 1B-blocker chlorethylclonidine (CEC) in isolated rat hearts. After 30 min of preperfusion, Langendorff-perfused hearts were subjected to 25 min of global ischemia followed by 30 min of reperfusion. Hearts were randomly divided into 4 groups, with one of the following substances being added to the perfusate: buffer alone (control), 10(-6) mol/L bunazosin, 10(-7) mol/L WB4101, or 10(-7) mol/L CEC. Bunazosin had a negative inotropic effect and preserved the postischemic ATP content, reduced the postischemic increase in intracellular Na+ content and then enhanced postreperfusion recovery of creatine phosphate. Bunazosin also reduced myocardial 45Ca2+ uptake during reperfusion (control 5.2 vs bunazosin 2.5 mumol/g dry weight of tissue (dwt), p < 0.01). However, the recovery of left ventricular developed pressure (DP) was not improved when bunazosin was added to the perfusate during reperfusion. WB4101 had neither a negative inotropic nor an energy-sparing effect, but it improved the recovery of DP (control 43% vs WB4101 56% of preischemic value, p < 0.05) with no reduction in myocardial 45Ca2+ uptake. CEC had a negative inotropic and energy-sparing effect and then reduced myocardial 45Ca2+ uptake (CEC 3.1 mumol/g dwt, p < 0.05), but it did not improve the recovery of DP. These results suggest that the preischemic administration of an alpha 1B-adrenoreceptor subtype blocker protected ischemic-reperfused myocardium via reduction of Ca2+ overload, whereas the selective blockade of the alpha 1A-adrenoreceptor subtype reduced myocardial damage via mechanism(s) other than Ca2+ metabolism.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1997

[Clinical characteristics and cardiac events in elderly patients with apical hypertrophic cardiomyopathy].

Apical hypertrophic cardiomyopathy (apical HCM) has been believed to be a special type of hypertrophic cardiomyopathy. It usually occurs in middle-aged or elderly men and the prognosis is thought to be good. However, recent reports suggest that approximately 10% of middle-aged patients with apical HCM have cardiac events and poor outcomes. We studied electrocardiograms and echocardiograms, the occurrence of cardiac events, and clinical characteristics in elderly patients with apical HCM (13 men and 7 women, 61 to 95 years old, mean age 74 +/- 7). Three of 20 patients (15%) had cardiac events (sudden death, 1; heart failure, 1; chest pain, 1), 2 had cerebral infarction with atrial fibrillation, and 2 died of non-cardiac causes. There were no differences in age, sex, medication, complications, or in initial values of electrocardiographic or echocardiographic variables between patients who had and did not have cardiac events. However, left ventricular end-systolic dimension (LVEDs) and left atrial diameter at the time of the last evaluation were larger in patients who had cardiac events than in those who did not (26 vs. 34 mm, p = 0.019, 33 vs. 38 mm, p = 0.1325, respectively). These results suggest that the prognosis for patients with apical HCM is not necessarily good, and that enlargement of the LVEDs might be used to predict cardiac events in elderly patients with apical HCM.

Aged↗

PubMed1997

[Microsatellite instability in gastric cancer with varied structure].

BACKGROUND: The intratumoral histological heterogeneity of cancer has been investigated by many pathologists. Although microsatellite alteration has been reported in gastric cancer, the significance of genomic instability in these histologically heterogeneous cases has not been elucidated. METHODS: Microsatellite alteration detected by polymerase chain reaction (PCR) in 13 primary advanced gastric cancers with varied structure was examined at 8 microsatellite loci. RESULTS: We were able to detect a greater prevalence of replication errors (RER) (6/13, 46.2%) at the primary site of gastric cancer than previously reported and loss of heterozygosity (LOH) at 17p (4/13, 30.8%) was demonstrated at the primary sites. All the same time, we also examined metastatic tumors in the regional lymph nodes in 12 of these cases. The frequency of RER (8/12, 66.7%) in metastatic lesions was higher than that in primary tumors. Detectability of RER was more frequent in the poorly differentiated portions than the well-differentiated portions of lymph node metastases. CONCLUSIONS: These findings suggest that gastric cancer with varied structure acquired frequent microsatellite instability during progression and metastasis, and we reasoned that the mutator phenotype detected by microsatellite alterations may represent heterogeneous tumor clones in gastric cancer and lymph node metastases.

Adenocarcinoma↗

PubMed1997

Stage-dependent evaluation of microsatellite instability in gastric carcinoma with familial clustering.

Familial clustering of gastric cancer is probably caused by multifactorial processes, both environmental and genetic. In this report, the incidence of microsatellite instability (MSI) in 31 cases of gastric cancer in Japanese (33 lesions) with familial clustering (two or more gastric cancers within second-degree relatives) was compared to MSI in Japanese cases without a family of any cancer in age ( +/- 10 years)-, stage-, and histological subtype-matched case-control study. Although the difference noted was not significant, we noted a strong trend for MSI at any of up to seven loci of CA repeats to occur more frequently in the patients with a family history of gastric than in the control patients in early cancer (intramucosal and submucosal), whereas the prevalence of MSI was similar in both groups in more advanced cases, in which the tumor invaded beyond the proper muscle layer of the gastric wall. Because the contribution of a family history of gastric cancer to MSI apparently differs in early and advanced gastric cancer, interpretation of MSI in familial gastric cancer cases published previously require reevaluation in terms of stage and proper controls. An acquisition of CA repeat alterations in the early stage rather than in the late stage of gastric carcinogenesis may have in common etiological factors, at least in some cases, with the familial clustering of gastric cancer.

Adult↗

PubMed1996

Localization of Menkes gene expression in the mouse brain; its association with neurological manifestations in Menkes model mice.

Menkes gene (Mc1 or MNK, encoding putative copper-transporting ATPase) expression was investigated and compared in normal and macular mutant mouse brain. Northern blot analysis showed a distinct 8.3-kb transcript and no obvious difference in size or extent in normal mice and macular mutants on postnatal days 0, 4, 7, 10 or 13. In situ hybridization revealed that certain specific populations of cells in the brain express Menkes mRNA, and that their localization in normal and mutant mice did not differ and was conserved on days 4, 10 and 13. The most intense hybridization signals were observed in the hippocampal CA1 region and dentate gyrus, the olfactory bulb nuclei, the cerebellar granular cell layer, the choroid plexus and the ependyma, with less intense signals in the hippocampal CA3 region and cerebellar Purkinje cells. In addition, necrotic neuronal cell death was predominantly observed in the CA3 region and the Purkinje cells of macular mice after postnatal day 10. The finding that the regions that had lower expression level of Menkes mRNA corresponded to those showing neuronal necrosis suggests that the Menkes gene may be responsible for the neuronal degeneration in some specific portions of the brain and clinical manifestations in this mutant.

Adenosine Triphosphatases↗

PubMed1996

Effect of methylisobutyl amiloride on [Na+]i, reperfusion arrhythmias, and function in ischemic rat hearts.

With 2 microM methylisobutyl amiloride (MIA), an inhibitor of Na+/H+ exchange, we tested the hypothesis that ion imbalance due to H+/Na+/Ca2+ exchange exacerbates reperfusion injury and arrhythmias. Isolated rat hearts were subjected to 25-min global ischemia and 30-min reperfusion. In the MIA-treated group, MIA was added throughout the perfusion protocol. Left ventricular pressure (LVP), arrhythmias, myocardial Na+ and K+ content, 45Ca2+ uptake, and the levels of energy metabolites were analyzed. The recovery of LV developed pressure (LVDP) and +dP/dt and -dP/dt were improved in the MIA group (53 vs. 80, 71 vs. 86, 77 vs. 94%: each p < 0.05). MIA inhibited the increase in Na+ content and the decrease in K+ content that occurred at the end of the ischemic phase and reduced 45Ca2+ uptake after reperfusion (28.6 vs. 17.1, 248 vs. 296, 2.79 vs. 1.36 microM/g dry weight of tissue; each p < 0.05). The incidence of ventricular tachycardia (VT) or ventricular fibrillation (VF) was lower in the MIA group [VT 11 of 20 (55%) vs. 4 of 20 (20%), p < 0.05; VF 13 of 20, (65%) vs. 6 of 20 (30%), 0.05 < p < 0.1], although the incidence of VF just escaped statistical significance. ATP level was higher in the MIA group after the ischemic phase and reperfusion (5.3 vs. 9.9, 12.3 vs. 14.7 microM/g dry weight of tissue; each p < 0.05). Our results suggest that MIA reduced reperfusion arrhythmias and improved functional recovery in isolated rat hearts subjected to global ischemia apparently by preserving high-energy phosphates during ischemia and by inhibiting Na+/H+ exchange, with attenuated cellular imbalance between Na+ and Ca2+.

Adenosine Triphosphate↗