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Biomedical subjects

N Hamasaki

Publications and source records attributed to N Hamasaki.

At least 19 recordsLinked to original sources
PubMed2000

Binding of human mitochondrial transcription factor A, an HMG box protein, to a four-way DNA junction.

Mitochondrial transcription factor A (mtTFA), the only known transcription factor in mitochondria, is also implicated in maintenance of mitochondrial genome although little is elucidated about its molecular basis. mtTFA is a member of HMG box proteins family. Some HMG proteins bind with high affinity to four-way DNA junctions that mimic a Holliday structure, a putative intermediate in DNA recombination. To explore possible involvement of a Holliday-like structure in the maintenance of mitochondrial genome, we examine the binding of recombinant human mtTFA to a synthetic four-way DNA junction. The human mtTFA binds to the four-way DNA junction with an approximately 10-fold higher affinity than to the corresponding linear duplex DNA and with essentially the same affinity as to a 40-mer DNA containing the human mitochondrial light strand promoter sequence. The mtTFA binds to the four-way as a monomer. Both of the two HMG box domains of human mtTFA are required for the high affinity binding to the four-way junction.

Base Sequence↗
PubMed2000

Accumulation of adenine DNA glycosylase-sensitive sites in human mitochondrial DNA.

The mitochondrial respiratory chain inevitably produces reactive oxygen species as byproducts of aerobic ATP synthesis. Mitochondrial DNA (mtDNA), which is located close to the respiratory chain, is reported to contain much more 8-oxoguanine (8-oxoG), an oxidatively modified guanine base, than nuclear DNA. Despite such a high amount of 8-oxoG in mtDNA (1-2 8-oxoG/10(4) G), mtDNA is barely cleaved by an 8-oxoG DNA glycosylase or MutM, which specifically excises 8-oxoG from a C:8-oxoG pair. We find here that about half of human mtDNA molecules are cleaved by another 8-oxoG-recognizing enzyme, an adenine DNA glycosylase or MutY, which excises adenine from an A:8-oxoG pair. The cleavage sites are mapped to adenines. The calculated number of MutY-sensitive sites in mtDNA is approximately 1.4/10(4) G. This value roughly corresponds with the electrochemically measured amount of 8-oxoG in mtDNA (2.2/10(4) G), raising the possibility that 8-oxoG mainly accumulates as an A:8-oxoG pair.

Adenine↗
PubMed2000

Genetic variants of IL-13 signalling and human asthma and atopy.

Asthma and atopy show epidemiological association and are biologically linked by T-helper type 2 (T(h)2) cytokine-driven inflammatory mechanisms. IL-4 operates through the IL-4 receptor (IL-4R, a heterodimer of IL-4Ralpha and either gammac or IL-13Ralpha1) and IL-13 operates through IL-13R (a heterodimer of IL-4Ralpha and IL-13Ralpha1) to promote IgE synthesis and IgE-based mucosal inflammation which typify atopy. Recent animal model data suggest that IL-13 is a central cytokine in promoting asthma, through the stimulation of bronchial epithelial mucus secretion and smooth muscle hyper-reactivity. We investigated the role of common genetic variants of IL-13 and IL-13Ralpha1 in human asthma, considering IgE levels. A novel variant of human IL-13, Gln110Arg, on chromosome 5q31, associated with asthma rather than IgE levels in case-control populations from Britain and Japan [peak odds ratio (OR) = 2.31, 95% CI 1.33-4.00]; the variant also predicted asthma and higher serum IL-13 levels in a general, Japanese paediatric population. Immunohistochemistry demonstrated that both subunits of IL-13R are prominently expressed in bronchial epithelium and smooth muscle from asthmatic subjects. Detailed molecular modelling analyses indicate that residue 110 of IL-13, the site of the charge-modifying variants Arg and Gln, is important in the internal constitution of the ligand and crucial in ligand-receptor interaction. A non-coding variant of IL-13Ralpha1, A1398G, on chromosome Xq13, associated primarily with high IgE levels (OR = 3. 38 in males, 1.10 in females) rather than asthma. Thus, certain variants of IL-13 signalling are likely to be important promoters of human asthma; detailed functional analysis of their actions is needed.

Adult↗
PubMed2000

Ten-years clinical follow-up following successful percutaneous transvenous mitral commissurotomy: single-center experience.

The purpose of this study is to report the long-term follow-up outcome of patients undergoing percutaneous transvenous mitral commissurotomy (PTMC). The follow-up of 68 of 82 (83%) consecutive patients undergoing successful PTMC (mitral valve area of more than 1.5 cm(2) without major complications) in 1987 using the Inoue balloon was analyzed. The mean age at the time of PTMC was 52 +/- 11 years and 81% were female patients. The mean follow-up interval was 98 +/- 37 months (6 to 123). Actuarial survival rate was 98%, 97%, and 86% at 1, 5, and 10 years, respectively; the event-free (death, mitral valve replacement, and repeat PTMC) survival rate was 90%, 85%, and 66% at 1, 5, and 10 years, respectively. According to the echocardiographic findings, patients could be divided into three groups: pliable valve, semipliable valve, and rigid valve. Multivariable analysis identified echocardiographic subgrouping as the major significant predictor of any event: the event-free survival rate being 70% in group 1, 66% in group 2, and 20% in group 3 (P < 0.05). Echocardiographic follow-up was available in 49 of 68 patients (72%); the mitral valve area changed from 1.4 +/- 0.5 before to 2.1 +/- 0.4 immediately post-PTMC, and 1.8 +/- 0.4 cm(2) 10 years after the procedure. The long-term follow-up outcome following successful PTMC was favorable and seems to support it as a viable alternative to surgical commissurotomy in selected patients. Patients with rigid valves should be selected very carefully.

Adult↗
PubMed2000

Clinical and angiographic follow-up after single long GFX coronary stent implantation.

In order to identify predictors of late restenosis after GFX stent implantation, procedural and 6-month clinical and angiographic follow-up data of prospectively entered 141 consecutive lesions treated with a single long (24 or 30 mm) GFX stent were compared to 66 consecutive lesions requiring a single short (12 or 18 mm) stent. The initial clinical success rate of 97% and thrombosis rate of 1.4% with long stents were similar to 97% and 0% with short stents (P = NS). Their respective binary restenosis rates were 34.7% and 23.3% for long and short stents as a whole (P = NS), but being 10.0% for 12 mm, 26.0% for 18 mm, 31.3% for 24 mm, and 39.2% for 30 mm. When proximal and distal reference diameters at baseline were compared between the lesions with and without restenosis, proximal reference diameters were not statistically different (3.02+/-0.42 mm vs. 3.18+/-0.62 mm) and the restenosis group had significantly smaller distal reference diameters (2.15+/-0.48 mm vs. 2.55+/-0.53 mm, P<0.0001). The treatment of long lesions with single long-stent implantation can be accomplished with high success and low complication rates. Single long-stent implantation may be effective, if the distal reference size of the long narrowing is big enough to accept the stent.

Aged↗
PubMed2000

The D-loop structure of human mtDNA is destabilized directly by 1-methyl-4-phenylpyridinium ion (MPP+), a parkinsonism-causing toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism via its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+), which inhibits complex I of the mitochondrial respiratory chain. Its parkinsonism-causing mechanisms attract a great deal of interest as a model of the disease. Recently, we reported that MPP+ strongly decreases the amount of mtDNA independent of the inhibition of complex I. Maintenance of a proper amount of mtDNA is essential for the normal function of mitochondria as exemplified in many mitochondrial diseases. The most characteristic feature in vertebral mtDNA replication is that H-strand synthesis proceeds displacing the parental H-strand as a long single strand. It forms the D-loop, a triplex replication intermediate composed of the parental L-strand, nascent H-strand and displaced H-strand. Here we show that MPP+ does not inhibit DNA synthesis by DNA polymerase gamma, but rather releases the nascent H-strands from mtDNA both in organello and in vitro. This indicates that MPP+ directly destabilizes the D-loop structure, thereby inhibiting replication. This study raises a new mechanism, i.e. destabilization of replication intermediates, for depletion of mtDNA.

1-Methyl-4-phenylpyridinium↗
PubMed2000

Highly selective antibacterial activity of novel alkyl quinolone alkaloids from a Chinese herbal medicine, Gosyuyu (Wu-Chu-Yu), against Helicobacter pylori in vitro.

To elucidate the antibacterial activity of Gosyuyu, the crude extract from the fruit of Evodia rutaecarpa, a Chinese herbal medicine, has been fractionated chromatographically, and each fraction was assayed for antibacterial activity against Helicobacterpylori (H. pylori) in vitro. As the result, a single spot having marked antibacterial activity against H. pylori was obtained and the chemical structure was analyzed. The isolated compound was revealed to be a novel alkyl quinolone alkaloid based on the solubility, IR spectra, NMR analysis and mass spectrometric data after purification by TLC of silica. We compared the antimicrobial activity of this compound with that of other antimicrobial agents and examined susceptibility of various intestinal pathogens. As the result, the new quinolone compounds obtained from Gosyuyu extracts were found to be a mixture of two quinolone alkaloids, 1-methyl-2-[(Z)-8-tridecenyl]-4-(1H)-quinolone and 1-methyl-2-[(Z)-7-tridecenyl]-4-(1H)-quinolone (MW: 339), reported previously. The minimum inhibitory concentration (MIC) of these compounds against reference strains and clinically isolated H. pylori strains were less than 0.05 microg/ml, which was similar to the MIC of amoxicillin and clarithromycin that are used worldwide for the eradication of H. pylori, clinically. Furthermore, it was noted that the antimicrobial activity of these compounds was highly selective against H. pylori and almost non-active against other intestinal pathogens. The above results showed that these alkyl methyl quinolone (AM quinolones) alkaloids were useful for the eradication of H. pylori without affecting other intestinal flora.

Alkaloids↗
PubMed2000

In vivo administration of mycobacterial cord factor (Trehalose 6, 6'-dimycolate) can induce lung and liver granulomas and thymic atrophy in rabbits.

Trehalose 6,6'-dimycolate (TDM) is a cell surface molecule of Mycobacterium tuberculosis. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6, 6'-tetraacyl trehalose 2'-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.

Animals↗
PubMed2000

Childhood atopic asthma: positive association with a polymorphism of IL-4 receptor alpha gene but not with that of IL-4 promoter or Fc epsilon receptor I beta gene.

We examined the relative contributions of three representative candidate genes for atopy (Fc epsilon receptor I beta, IL-4, and IL-4 receptor alpha) to the development of atopic asthma. Four polymorphisms of the three candidate genes including Ile50Val and Gln551Arg of IL-4 receptor alpha, -590C/T of IL-4 promoter and Glu237Gly of Fc epsilon receptor I beta were studied in 100 patients with atopic asthma and 100 nonatopic controls in the northern Kyushu area in Japan. Among the four polymorphisms of the three candidate genes, the Ile50 allele of the IL-4 receptor alpha chain gene demonstrated an association with atopic asthma subjects (p = 0.044), especially in patients with onset at 2 years of age or earlier (p = 0.034) and in patients with moderate to severe atopic asthma (p = 0. 031). Gln551Arg of IL-4 receptor alpha, -590C/T of IL-4 promoter and Glu237Gly of Fc epsilon receptor I beta showed no association with atopic asthma. A slight linkage disequilibrium between Ile50Val and Gln551Arg polymorphisms of the IL-4 receptor alpha chain gene was observed in both patients and nonatopic controls. The identification of additional atopy genes in areas with a certain genetic background is essential for genetic diagnosis and to establish new therapeutic modalities for atopic asthma.

Adolescent↗
PubMed1999

Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site.

The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.

Aphidicolin↗
PubMed1999

Thrombectomy with AngioJet catheter in native coronary arteries for patients with acute or recent myocardial infarction.

The AngioJet thrombectomy catheter removes thrombi by rheolytic fragmentation and suction. The purpose of this study was to identify the efficacy and safety of this new device. Myocardial infarction (MI) is associated with intracoronary thrombus. Intracoronary thrombus has been identified as a risk factor of unfavorable outcome after percutaneous transluminal coronary angioplasty. To what extent the AngioJet is applicable or effective for acute or recent MI in native coronary artery is not clear. Thrombectomy with the AngioJet was attempted in 31 patients with 31 native coronary arteries selected from 304 patients with acute or recent MI. Follow-up angiography was performed at 3 to 6 months. Procedure success was achieved in 29 patients (94%). Adjunctive balloon angioplasty was performed after AngioJet thrombectomy in 30 patients (97%), and in only 1 patient (3%) AngioJet thrombectomy was the sole procedure. Subsequent stenting after balloon angioplasty was attempted successfully in 12 patients (40%) without thrombotic complications. Thrombolysis In Myocardial Infarction trial flow grading increased from 0.70 +/- 0.97 before to 2.61 +/- 0.88 after AngioJet thrombectomy (p <0.0001), to 2.84 +/- 0.64 after adjunctive procedures (p = 0.070). At follow-up angiography restenosis rate was 21% but Thrombolysis In Myocardial Infarction flow 3 was present in all patients. The restenosis rate of stented patients was 8%. There were no major events during in-hospital and follow-up. The AngioJet can be used safely and successfully to remove thrombus from the native coronary artery of patients with MI. Thrombus removal makes subsequent stenting safe and uncomplicated. The restenosis rate was considered to be acceptable.

Angioplasty, Balloon, Coronary↗
PubMed1999

R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase.

Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells. The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase. A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA. Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication. This mitochondrial R-loop is resolved by RecG in a dose-dependent manner. Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG. A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop. This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase. The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG.

Bacterial Proteins↗
PubMed1999

Antagonistic effects of an alternative splice variant of human IL-4, IL-4delta2, on IL-4 activities in human monocytes and B cells.

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.

Alternative Splicing↗
PubMed1999

Cutting edge: dominant effect of Ile50Val variant of the human IL-4 receptor alpha-chain in IgE synthesis.

Two variants of the IL-4R alpha-chain (IL-4Ralpha) gene have been recently identified in association with different atopic disorders. To clarify the etiological relationship between the two variants, we analyzed responsiveness to IL-4 of transfectants with four kinds of IL-4Ralpha carrying either Val or Ile at 50 and either Gln or Arg at 551. The substitution of Ile for Val augmented STAT6 activation, proliferation, and transcription activity of the Iepsilon promoter by IL-4, whereas that of Arg for Gln did not change these IL-4 signals. Arg551 was not associated with atopic asthma in the Japanese population. CD23 expression and IgE synthesis by IL-4 were augmented in Ile50-bearing PBMC, compared with those bearing Val50. Taken together, substitution of Arg551 does not enhance the IL-4 signal for generation of germline epsilon transcript, whereas the substitution of Ile50 contributes to enhancement of IgE synthesis.

Amino Acid Substitution↗
PubMed1999

Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.

Animals↗
PubMed1999

Association of the interleukin-4 receptor alpha chain with p47phox, an activator of the phagocyte NADPH oxidase in B cells.

Interleukin (IL)-4 plays an important role in IgE synthesis in B cells and in Th2 differentiation in T cells. IL-4 conducts its biological activities through binding to the IL-4 receptor (IL-4R) on the surface of target cells. IL-4R are thought to be composed of the IL-4R alpha chain (IL-4R alpha) and either the IL-2R gamma chain or the IL-13R alpha chain. We have previously shown that the membrane-proximal portion in the cytoplasmic domain of the human IL-4R alpha (hIL-4R alpha) is critical for proliferation, generation of germline epsilon transcript, and activation of STAT6, based on analyses of truncated hIL-4R alphas. In this study, we found that p47phox, an activator of the phagocyte NADPH oxidase, binds to this portion by the two-hybrid system. Furthermore, we observed the association of p47phox with the hIL-4R alpha in B cells derived from a normal donor. These results suggest that p47phox is involved in the signal transduction of IL-4 in B cells. However, activation of STAT6, CD23 expression, and IgE synthesis induced by IL-4 were not affected in p47phox-deficient patients, which raises the possibility that p47phox may be important in other signaling activities as well in B cells.

Amino Acid Sequence↗