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ABSTRACT: Although tachycardia is commonly present in patients with congestive heart failure, its role in the development of congestive heart failure remains unclear. We studied the effect of rapid electrical stimulation of contraction on beta-adrenergic receptor (beta-AR) signal pathway in cultured cardiomyocytes of neonatal rats.
Contraction of cardiomyocytes was induced by electrical stimulation at 50 V with twice the threshold pulse width. beta-ARs were identified by [(3)H]CGP-12177 and [(3)H]dihydroalprenolol. Electrical stimulation reduced cell-surface but not total beta-AR density; the effect was dependent on pacing frequency (a reduction of 11%, 28%, and 18% in cells paced at 2.5, 3. 0, and 3.3 Hz, respectively). This reduction was apparent at 3 hours, in contrast to reduced beta-AR density after exposure to isoproterenol (ISP) for 1 hour. The fraction and inhibition constant of beta-AR binding agonist with high affinity were not affected by rapid electrical stimulation. In cardiomyocytes paced at 3.0 Hz for 24 hours, the response to ISP decreased compared with unpaced cells, 142% versus 204% of baseline with 1 micromol/L ISP, whereas the responses to forskolin or acetylcholine were not different. Treatment of cardiomyocytes with 2,3-butanedione monoxime (10 mmol/L) or taxol (10 micromol/L) inhibited the rapid pacing-induced reduction in beta-AR density.
Our results suggest that contractile activity is involved in regulation of cardiac function by modulating the beta-AR system independently of hemodynamic and neurohormonal factors. This may help to elucidate the role of mechanical stress in the development of heart failure.
Circulation 07/2000; 101(22):2625-30. · 14.74 Impact Factor
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ABSTRACT: The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L-type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats.
Journal of Molecular and Cellular Cardiology 05/2000; 32(4):655-64. · 5.17 Impact Factor
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ABSTRACT: The aim of this study was to elucidate the role of bradykinin in mediating captopril-induced upregulation of beta-adrenergic receptor (beta-AR). The density of beta-AR on the surface of cardiac myocytes was measured by binding assay using [(3)H]CGP-12177. Treatment of cultured neonatal rat cardiomyocytes with captopril resulted in a time-dependent elevation of bradykinin concentration in the culture medium. The increased bradykinin concentration was significant at 2, 3 and 6 h, but not at 12 h after exposure to captopril. This time-dependent effect of captopril on enhancement of bradykinin levels paralleled that of beta-AR upregulation. Exogenously applied bradykinin increased beta-AR density by 22, 30 and 35% at 0.01, 0.1 and 1 microm concentrations, respectively. Myocytes treated with 1 microm bradykinin responded to isoproterenol (ISP) in a dose-dependent manner, as demonstrated by acceleration of spontaneous beating frequency. These beating acceleration effects of bradykinin were abolished by Hoe 140. Stimulation of bradykinin B2 receptor by exogenously added bradykinin for 6 h was sufficient to produce beta-AR up-regulation to a level similar to that seen after 24 h. Our results indicate that bradykinin potentiation by ACE inhibitors regulates, at least in part, captopril-induced beta-AR up-regulation.
Journal of Molecular and Cellular Cardiology 02/2000; 32(1):153-9. · 5.17 Impact Factor
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K Hamaguchi,
A Kimura,
N Seki,
T Higuchi, S Yasunaga,
M Takahashi,
T Sasazuki,
Y Kusuda,
T Okeda,
K Itoh,
T Sakata
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ABSTRACT: Polymorphisms in the 5'-flanking region of the tumor necrosis factor (TNF)-alpha gene were examined to study the genetic background of type 1 diabetes in Japanese. Five different biallelic polymorphisms were examined in 136 type 1 diabetic patients and 300 control subjects. The frequencies of individuals carrying TNF-alpha-857T allele (designated as TNFP-D allele) or -863A/-1,031C allele (designated as TNFP-B allele) were significantly increased in the patients as compared with the controls. Since these TNF-alpha alleles are in linkage disequilibria with certain DRB1 and HLA-B alleles, two-locus analyses were carried out. The TNFP-D allele did not increase the risk in either the presence or absence of the DRB1*0405 or HLA-B54 allele, while the DRB1*0405 and HLA-B54 alleles per se could confer susceptibility in both the TNFP-D allele-positive and -negative populations. Moreover, an odds ratio was remarkably elevated in the population carrying both DRB1*0405 and HLA-B54. Similarly, the TNFP-B allele did not show significant association with the disease in either the HLA-B61-positive or -negative population, while the HLA-B61 allele could significantly increase the risk in the TNFP-B allele-positive population. These data suggest that the associations of TNFP-D and -B alleles may be secondary to their linkage disequilibria with the susceptible HLA class I and class II alleles. Because HLA-B and DRB1 genes were independently associated, both of these genes may be contributed primarily to the pathogenesis of type 1 diabetes in Japanese.
Tissue Antigens 02/2000; 55(1):10-6. · 2.59 Impact Factor
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ABSTRACT: With a view to establishing an accurate evaluation of the genetic predisposition to insulin-dependent type I diabetes (IDDM), we have built a model based on the characteristics of the relevant pockets of HLA-DR and -DQ molecules. Three independent populations were investigated. Group I and group II were Caucasoids, while group III was Japanese, including a total of 1,166 IDDM patients and 2,391 healthy controls. We formulate the hypothesis that suceptibility to IDDM is not only explained by the absence of Aspartate 57 (negative charge) from pocket 9 of DQB1 (P9DQ), but also by the presence of an electric charge (+/- vs. neutral), generated by residues 70, 71 and 74 in pockets 4 of DRB1 (P4DR) and DQB1 (P4DQ) molecules. The respective weight of each pocket, was evaluated in a multivariate analysis based on the logistic regression method. The 4 components (2 loci and 2 pockets) were systematically analysed in the computer model. It was clearly shown that the structural characteristics of pockets P9DQ-P4DR and, to a lesser degree that of P4DQ, account for IDDM predisposition. On applying the model to the whole international series, it appears that the highest risk concerns individuals with P9DQ non-Asp 57 and both the charged P4 of DRB1 and P4 of DQB1, conferring a 80% prediction of susceptibility. Conversely, P9DQ Asp and neutral P4DR and P4DQ give the lowest risk with a predictive value of 5%. This model of risk susceptibility prediction fits remarkably well with the observed distribution in a worldwide study. It allows a better evaluation of the respective role of HLA-DR and -DQ molecules as a major component of susceptibility to IDDM.
Tissue Antigens 11/1999; 54(4):341-8. · 2.59 Impact Factor
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ABSTRACT: The pathogenesis of histiocytic necrotizing lymphadenitis (HNL), which was reported first by Kikuchi et al. and Fujimoto et al. in 1972, is as yet unknown. HNL is frequently reported in Asian countries including Japan, however it is rare in Europe and North America. To elucidate whether the human leukocyte antigen (HLA) alleles and haplotypes are associated with HNL, we performed DNA typing of HLA class II genes (HLA-DR, -DQ, and -DP) in 86 patients with HNL and 525 unrelated healthy Japanese controls with polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). In this study, we found DPA1*01 and DPB1*0202 allele frequencies in HLA class II genes are significantly higher in HNL patients than in normal controls. It is known that the frequency of DPB1*0202 alleles is extremely low or absent in Caucasians (e.g., French 0.4%, Italian 0.8%) and Negroid (e.g., South African 0%, Hottentot 0%), but relatively frequent in Asians (e.g., Korean 9.9%, Japanese 4.5%). Previous reports have said the incidence of HNL is frequent in Asians but rare in other races. In light of this background, HLA class II genes of HNL and the incidence of HNL in Asian countries, including Japan, might have a positive relationship to DPA1*01 and DPB1*0202 allele.
Tissue Antigens 10/1999; 54(3):246-53. · 2.59 Impact Factor
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ABSTRACT: This study was undertaken to investigate the possible association of Fas gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE) in Japanese.
Screening for structural defects of the Fas gene was performed by using reverse transcriptase-polymerase chain reaction (RT-PCR)/single-strand conformation polymorphism (SSCP) analysis in 57 patients with SLE, followed by direct sequencing for the aberrantly migrating bands. The frequency of Fas polymorphism was determined by sequence-specific oligonucleotide probe (SSOP) hybridization in 82 SLE patients and 132 ethnically matched healthy individuals.
We found a novel polymorphism at nucleotide 297 (T297C), which was linked to Fas polymorphism at nucleotide 416 (A416G). The 297C/416G genotype was present in four of the 132 (3.0%) healthy controls, none of whom was homozygous for the genotype. The allele frequency for 297C/416G in the controls was 1.5%. In contrast, 10 of the 82 (12.2%) SLE patients carried the 297C/416G allele, including one patient homozygous for the genotype. The allele frequency in SLE patients was 6.7%. The 297C/416G allele was significantly frequent in SLE patients (P = 0.01, chi2) with a relative risk of 5.00.
As the polymorphism 297C/416G is silent at the amino acid level, it may affect the expression of Fas itself or be linked to a neighbouring genetic abnormality that is responsible for the pathogenesis of SLE.
Rheumatology 07/1999; 38(6):516-20. · 4.06 Impact Factor
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ABSTRACT: Objectives. This study was undertaken to investigate the possible association of Fas gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE) in Japanese. Methods. Screening for structural defects of the Fas gene was performed by using reverse transcriptase-polymerase chain reaction (RT-PCR)/single-strand conformation polymorphism (SSCP) analysis in 57 patients with SLE, followed by direct sequencing for the aberrantly migrating bands. The frequency of Fas polymorphism was determined by sequence-specific oligonucleotide probe (SSOP) hybridization in 82 SLE patients and 132 ethnically matched healthy individuals. Results. We found a novel polymorphism at nucleotide 297 ( T297C ), which was linked to Fas polymorphism at nucleotide 416 (A416G). The 297C/416G genotype was present in four of the 132 (3.0%) healthy controls, none of whom was homozygous for the genotype. The allele frequency for 297C/416G in the controls was 1.5%. In contrast, 10 of the 82 (12.2%) SLE patients carried the 297C/416G allele, including one patient homozygous for the genotype. The allele frequency in SLE patients was 6.7%. The 297C/416G allele was significantly frequent in SLE patients (P= 0.01, x2) with a relative risk of 5.00. Conclusion. As the polymorphism 297C/416G is silent at the amino acid level, it may aVect the expression of Fas itself or be linked to a neighbouring genetic abnormality that is responsible for the pathogenesis of SLE. Systemic lupus erythematosus (SLE ) is an inflammatory and its ligand ( FasL) have been reported in mouse disorder aVecting multiple organ systems; it is character- models of human SLE: MRL-lpr, MRL-lprcg and C3H- ized by the presence of antibodies reactive against vari- gld mice (3-5). In patients with SLE, increased levels ous self-antigens. The breakdown of self-tolerance in of the soluble form of Fas have been reported which SLE is caused by a defect in the elimination of auto- may interfere with the apoptosis of autoreactive lympho- reactive T or B cells (1).
Rheumatology. 01/1999; 38(6):516-520.
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ABSTRACT: Although bradykinin is thought to contribute to the effects of ACE inhibitors on the cardiovascular system, its precise role remains to be elucidated. Evidence suggests that bradykinin might be important in the upregulation of beta-adrenergic receptors (beta-ARs) induced by ACE inhibitors, and the role of bradykinin in this effect has now been investigated with cultured neonatal rat cardiac myocytes.
The density of beta-ARs on the myocyte surface was determined with a binding assay with [3H]CGP-12177. Incubation of cultured myocytes for 24 hours with the ACE inhibitor captopril (1 micromol/L) increased beta-AR density by 35% and enhanced the response of cells to isoproterenol but not to forskolin. Neither an angiotensin-II type 1 (AT1) receptor antagonist, CV-11974, nor angiotensin-I affected beta-AR density. However, the bradykinin B2 receptor antagonist Hoe 140 abolished the effect of captopril on beta-AR upregulation in a dose-dependent manner. The protein kinase C inhibitor staurosporine (20 nmol/L) but neither indomethacin nor L-NAME also inhibited captopril-induced upregulation of beta-ARs. Exogenous bradykinin increased the spontaneous beating frequency of cultured myocytes and Hoe 140 abolished this effect. Bradykinin level in the medium increased 1.4-fold by the treatment of cultured myocytes with captopril for 24 hours.
The results suggest that captopril enhances beta-AR responsiveness by inducing beta-AR upregulation and that the latter effect is mediated by activation of bradykinin B2 receptors and protein kinase C. These observations also offer insight into the different roles of ACE inhibitors and AT1 receptor antagonists in the treatment of heart failure.
Circulation 07/1998; 97(22):2268-73. · 14.74 Impact Factor
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Arthritis & Rheumatism 05/1998; 41(4):757-8. · 7.87 Impact Factor
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ABSTRACT: X-linked adrenal hypoplasia congenita (AHC) is characterized by primary adrenal insufficiency and is frequently associated with hypogonadotropic hypogonadism (HHG). Mutations of the DAX1 gene have been reported in patients with AHC and HHG. We found a novel DAX1 mutation in our patient. Sequence analysis of the patient's DAX1 demonstrated a 1-bp (G) deletion at codon 49 in exon 1. The mutation shifts the reading frame, resulting in completely different amino acid sequences from codon 49 to the premature stop codon at 84. The G was present at this position in the sequences of the father and 2 younger brothers. Direct sequence and single-strand conformation polymorphism analyses of polymerase chain reaction fragments revealed that the mutation at codon 49 was heterozygously present in the mother's DAX1 gene. The codon 84 is located in the first half of the DNA binding domain, and the mutation site is closer to the N-terminus than those in previously reported cases. The onset of adrenal insufficiency in the neonatal period as seen in our patient has also been reported in other patients with different DAX1 mutations, especially in a patient with DAX1 protein lacking 11 amino acids at the C-terminus. Therefore, it is less likely that position of termination codons correlate to clinical manifestations.
American Journal of Medical Genetics 02/1998; 76(1):62-6.
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ABSTRACT: We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.
Biochimica et Biophysica Acta 01/1998; 1389(3):178-86. · 4.66 Impact Factor
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ABSTRACT: Gastric cancer is multifactorial disease and several reports have described genetic factors involved in pathogenesis of gastric cancer. Recently, it was reported that HLA class II gene DQB1*0301 was strongly associated with gastric cancer in Caucasian population (20). We performed DNA typing of HLA class I (HLA-A) and HLA class II genes (HLA-DR, DQ and DP) to elucidate the HLA alleles or HLA haplotypes associated with gastric cancer in Japanese population using polymerase chain reaction-sequence-specific oligonucleotide probe analysis in 88 unrelated patients with gastric cancer and 525 unrelated healthy controls. We observed slight difference in frequencies of some HLA alleles and haplotypes between gastric cancer patients and controls; however, after Bonferroni correction, statistical significance was not confirmed. It is possible that environmental factors such as diet cover the contribution of genetic factors to the disease in Japanese population, which has a higher frequency of gastric cancer than do Caucasian populations, most likely due to more exposure to environmental risk factors.
Tissue Antigens 10/1997; 50(3):277-82. · 2.59 Impact Factor
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ABSTRACT: In patients with congestive heart failure (CHF), beta-receptor up-regulation is regarded as one of the mechanisms leading to improved function and prognosis. To clarify whether beta-receptor up-regulation is involved in the mechanisms underlying the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors, we investigated the actions of ACE inhibitors and an angiotensin II type 1 receptor (AT1) antagonist on beta-receptors of neonatal rat cultured cardiac myocytes. Angiotensin II (A-II) increased the spontaneous beating frequency of the cells, and the effect was completely antagonized by the AT1 antagonist CV-11974. Under control conditions, beta-receptor density (Bmax) and affinity (Kd) were measured by radiobinding assay with the hydrophilic ligand [3H]CGP-12177, and were 103 +/- 11 fmol/mg protein and 3.4 +/- 0.4 nmol/L, respectively. Captopril increased the beta-receptor density of myocytes and augmented the response to isoproterenol. Bmax was increased by 34% after 24 h treatment with 10(-6) mol/L captopril. CV-3480, and ACE inhibitor that contains no sulfhydryl group, but neither A-II nor the AT1 antagonist, also up-regulated beta-receptors. The results suggest that beta-receptor up-regulation contributes at least partly to the beneficial cardiac effects of ACE inhibitors in patients with CHF. ACE inhibitors and AT1 antagonists seem to play different roles in clinical practice.
Japanese Circulation Journal 03/1997; 61(2):170-9.
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 05/1996; 41(5):553-5.
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H Nishizaka,
T Horiuchi,
Z B Zhu,
Y Fukumori,
K Nagasawa,
K Hayashi,
R Krumdieck,
C G Cobbs,
M Higuchi, S Yasunaga,
Y Niho,
J E Volanakis
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ABSTRACT: Deficiency of the sixth component of complement (C6D) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular bases of C6D in two unrelated subjects, one African American (case 1) and the other Japanese (case 2). Screening all 17 exons of the C6 gene and their boundaries by exon-specific PCR/single strand conformation polymorphism demonstrated aberrant single stranded DNA fragments in exon 12 of case 1 and exon 2 of case 2. Nucleotide sequencing of the amplified DNA fragments revealed a homozygous single-base deletion (G1936) in exon 12 case 1 and a heterozygous single base deletion (C291/C292/C293/C294) in exon 2 of case 2. Both mutations resulted in frame shifts and premature termination of the C6 polypeptide. Sequence-specific oligonucleotide probe hybridization and direct sequencing of exon 12 amplified from genomic DNA further supported the homozygosity of the mutation in case 1. Case 2 is apparently compound heterozygote, but the putative mutation in the other allele of the C6 gene remains unknown. Both case 1 and case 2 were homozygous for the C6A allotype. These data indicate that at least three distinct mutational events can cause C6D, single nucleotide deletions in exons 2 and 12, and a mutation yet unidentified. Thus, similar to other complement protein deficiencies, the pathogenesis of C6D appears to be heterogeneous.
The Journal of Immunology 04/1996; 156(6):2309-15. · 5.79 Impact Factor
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ABSTRACT: Previous studies have indicated that certain alleles of HLA-DR and -DQ genes were strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM), and the role of DQ molecule in IDDM has been suggested. To further clarify the association of DQ alleles with IDDM, we determined the nucleotide sequences of full-length cDNA from 13 DQA1 alleles and 14 DQB1 alleles. The sequencing analysis revealed sequence polymorphisms outside the hypervariable region of DQ genes. We then analyzed the DQA1 and DQB1 polymorphisms along with that of DRB genes in 86 B-lymphoblastoid cell lines (B-LCLs) from various ethnic groups and in healthy unrelated Japanese and Norwegian individuals. The allelic and haplotypic distributions in each population revealed the characteristic haplotypic formation in the HLA class II region. HLA genes in 139 Japanese and 100 Norwegian IDDM patients were analyzed. DQB1*0301 was negatively associated with IDDM in both ethnic groups, irrespective of associated DRB1 and DQA1 alleles. In DQB1*0302 positive populations, which represented a positive association with IDDM in both ethnic groups, DRB1*0401, *0404, *0802 haplotypes increased in the patients, whereas DRB1*0406 haplotype decreased. Considering about the hierarchy in DRB1 alleles with IDDM susceptibility (DRB1*0401>*0404>*0403 in Norwegian and DRB1*0802>*0403>*0406 in Japanese), the genetic predisposition to IDDM is suggested to be defined by the combination of DR-associated susceptibility and DQ-associated susceptibility and by the DQ-associated resistance which is a dominant genetic trait.
Tissue Antigens 02/1996; 47(1):37-48. · 2.59 Impact Factor
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S Yasunaga
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ABSTRACT: The present study was conducted to examine the hypothesis that children's feelings of uncertainty would mediate between pre-experience and rule acquisition in observational learning. After received a pretest, subjects were requested to estimate their response uncertainty concerning a task used in the pretest. Then they observed a model responding to a series of pictures each of which depicting one of four directions. The model's responses were governed by a rule consisted of two dimensions: the progression and the direction. After observing the demonstration, subjects received two post-tests which differed in the order of stimulus presentation and in terms of the reinforcement. Subjects enhancing their feelings of uncertainty in the pretest phase acquired a rule which contained a dimension of the progression, while subjects who did not enhance them acquired a rule used by the model. These findings were interpreted as generally supporting the hypothesis of response uncertainty as a mediator of rule learning.
Shinrigaku kenkyu: The Japanese journal of psychology 09/1987; 58(3):137-43.
