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Biomedical subjects

H Nagai

Publications and source records attributed to H Nagai.

At least 73 records · Page 4Linked to original sources

High-throughput PCR in silicon based microchamber array.

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

Hot Temperature↗

High-dose tacrolimus and lengthy survival of the combined rat pancreas/spleen graft in a high-responder combination.

Pancreas grafts do not survive for lengthy periods, especially in a high-responder rat combination. Recent data indicated that a combined spleen/pancreas graft protects against acute graft rejection and induces donor-specific tolerance. In this study, we performed a combination spleen/pancreas transplantation using high-dose tacrolimus in a high-responder rat combination of DA (RT1a) to LEW (RT1) and induced permanent survival in the few recipient rats. In these recipients, there was no difference in the mixed lymphocyte reaction (MLR) of the recipients when compared with that of the naive LEW splenic cells, but MLR inhibition by the serum from the recipients was significantly decreased. We also performed immunoblotting and detected a protein that has an affinity for the anti-DA class antibody. This protein may be an anti-idiotypic antibody and contribute to donor- and tissue-specific tolerance.

Animals↗

Disruption of antigen-induced airway inflammation and airway hyper-responsiveness in low affinity neurotrophin receptor p75 gene deficient mice.

1. Recently, much attention has been paid to the relationship between the nervous and immune systems. The present study was conducted to clarify the role of neurotrophin low affinity receptor (p75N) in allergic airway inflammation and hyper-responsiveness (AHR) in mice by employing p75N gene deficient mice. 2. Mice were immunized twice by intraperitoneal injections of ovalbumin (OA) at intervals of 12 days. OA was inhaled 10 days after the secondary immunization and repeated three times at 4 days interval. Twenty-four hours after the last inhalation, airway responsiveness to acetylcholine was measured and bronchoalveolar lavage fluid (BALF) was obtained for examining the number of inflammatory cells and the level of cytokines. Serum immunoglobulin was measured as a marker of systemic immune response before the final inhalation. 3. In wild-type mice, repeated antigen provocation resulted in airway eosinophilia, AHR and elevations in serum IgE and interleukin (IL)-4 and -5 in BALF. In p75N gene deficient mice, none of the above parameters was observed after antigen provocation. The antigen-induced production of interferon (IFN)-gamma and nerve growth factor (NGF) were not altered by depletion of p75N gene. 4. The present findings suggest that p75 gene deficiency disrupt an allergic airway inflammation and AHR in mice by interfering type 2 helper T (Th2) cell responses.

Acetylcholine↗

Two novel single-nucleotide polymorphisms of the Caspase-9 (CASP9) gene in the Japanese population.

We identified two single-nucleotide polymorphisms (SNPs) in the human Caspase-9 (CASP9) gene (1p36.3), which encodes an apoptosis-related cysteine protease, by screening all exons and exon-intron boundaries. These SNPs were present in coding regions. One of coding SNPs reflected an amino-acid substitution; an A to G transition at codon 221 in exon 5 would encode arginine instead of glutamine. As the gene is implicated in apoptotic cascade, the polymorphic sites will serve as useful markers for genetic studying of disorders affecting immune response and cancer susceptibility in humans.

Caspase 9↗

Hypermethylation of p16INK4A gene promoter during the progression of plasma cell dyscrasia.

Recent studies have indicated a close relationship between inactivation of tumor suppressor genes (TSGs) and disease progression. The genes encoding the cyclin-dependent kinase inhibitors p16INK4A and p15INK4B are potent TSGs, and correlations between their inactivation and disease progression have also been reported in various malignancies. In this study, we analyzed the methylation status of p16INK4A and p15INK4B gene promoters in plasma cell dyscrasias (PCDs) by methylation-specific PCR (MSP). In analyses using DNAs extracted from bone marrow mononuclear cells (BM-MNCs), patients with multiple myeloma (MM) showed frequent hypermethylation of the p16INK4A gene (15/37, 41%), whereas p15INK4B gene methylation was not so frequent (5/37, 14%). Many patients whose BM-MNC showed dense methylation of the p16INK4A gene had extramedullary plasmacytoma (extra-PC), and all available extra-PC samples showed alterations of the p16INK4A gene (4; dense methylation, 1; homozygous deletion). In contrast to MM, hypermethylation of the p16INK4A gene was significantly infrequent in indolent PCDs (2/22, 9%, P= 0.0055). The infrequency in indolent PCDs was also confirmed by analyses using DNAs extracted from BM smears (1/29, 3%). It is possible that hypermethylation of the p16INK4A gene promoter contributes to progression to aggressive MM from indolent PCD, especially to extra-PC development.

Biomarkers↗

Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreB.

BACKGROUND: The initiation arrest at a modified lambdaPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown. RESULTS: The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the lambdaPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6-9-fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not. CONCLUSIONS: All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.

Base Sequence↗

Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability.

BACKGROUND & AIMS: Methylation of the hMLH1 promoter region has been suggested to cause microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC). We studied the methylation profile in a wide region of the hMLH1 promoter and compared with the hMLH1 protein expression and MSI status in 88 cases of sporadic CRC. METHODS: Na-bisulfite treatment and polymerase chain reaction single-strand conformation polymorphism analysis was performed using 5 sets of polymerase chain reaction primers spanning the promoter region of the hMLH1 to examine methylation status. Results were compared with immunostaining using anti-hMLH1 monoclonal antibody and MSI status of the tumor samples. RESULTS: Methylation status was classified as full or partial methylation. Full methylation indicates the methylation of all CpG sites in the examined regions. Methylation of the hMLH1 promoter was observed in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), among which 89% (14 of 16) had full methylation with reduced hMLH1 protein expression. All cases showing full methylation were proximal colon tumors with MSI-H. In cases with partial methylation, only the upstream region of the hMLH1 promoter was methylated. Partial methylation was also shown in 33.3% (6 of 18) of the normal mucosa of MSI-H cases. Frequencies of methylation were significantly correlated with female gender (P = 0.0009) and aging (P = 0.007). CONCLUSIONS: Full methylation of the hMLH1 promoter region and subsequent gene inactivation may play a crucial role in the carcinogenesis of MSI-H CRCs in the proximal colon. Methylation upstream of the hMLH1 promoter appears to be an early event in the carcinogenesis of MSI-H tumors.

Adaptor Proteins, Signal Transducing↗

Clinical evaluation of Amplicor Mycobacterium detection system for the diagnosis of pulmonary mycobacterial infection using sputum.

SETTING: The Amplicor Mycobacterium detection kit was evaluated for the diagnosis of active pulmonary mycobacterial infection using sputum. OBJECTIVE: To assess the clinical usefulness of the Amplicor Mycobacterium kit for the diagnosis of pulmonary tuberculosis and non-tuberculous mycobacterial infection in the country of medium prevalence. DESIGN: All the patients were diagnosed with bacterial, histopathological, and clinical 'gold standard'. The sensitivity and specificity for diagnosing clinically active pulmonary tuberculosis and Mycobacterium avium and Mycobacterium intracellulare infections were evaluated comparing Amplicor results and clinical diagnosis. RESULTS: A total of 1088 sputum specimens were collected from 780 in and out patients. Mycobacteria were recovered from 339 specimens by culture. The sensitivity and specificity of conventional culture method for the diagnosis of pulmonary tuberculosis were 60.2% and 99.8% respectively based on the number of patients. The figures for Amplicor were 61.8% and 97.4% respectively. There was no statistical significant difference between these methods. In rapidity, the Amplicor was significantly superior to the microscopy method in sensitivity. CONCLUSION: Patients with Amplicor positive and conventional negative result had mostly mycobacteria related diseases. The Amplicor positive result indicated mostly active mycobacterial infection and was clinically useful for rapid diagnosis.

Chi-Square Distribution↗

Endoscopic partial resection of a duodenal duplication cyst.

A case of symptomatic duodenal duplication cyst is reported. The patient underwent endoscopic partial resection of the cystic wall using the O-ring ligation kit. After resection, the abdominal pain disappeared. Endoscopic partial resection is useful for diagnosis and treatment of duodenal duplication cyst.

Adult↗

Secreted type of modified interleukin-18 gene transduced into mouse renal cell carcinoma cells induces systemic tumor immunity.

PURPOSE: Interleukin (IL)-18 is a novel cytokine that has been identified as a strong interferon-gamma inducer. IL-18 has bioactivity similar to that of IL-12 and demonstrates antitumor effects. Since IL-18 does not have a signal sequence, we constructed the gene, consisting of the signal sequences of interferon-beta and mature IL-18 complementary (c) DNA. The modified gene was subsequently transduced into a mouse renal cell carcinoma cell line to induce IL-18 secretion from tumor cells to establish whether this IL-18 secreting tumor cell line may induce systemic tumor immunity. MATERIALS AND METHODS: Modified IL-18 cDNA consisting of the signal sequences of interferon-beta and mature type of IL-18 cDNA was constructed by the overlap extension method. The modified and original IL-18 cDNA was transduced into the RenCa mouse renal cell carcinoma cell line. Expression of IL-18 messenger RNA and concentration of IL-18 in the culture supernatant were determined. Direct antitumor and tumor vaccine effects were investigated in syngeneic Balb/c mice. To determine the mechanism of the antitumor effect immunodeficient mice were challenged with IL-18 secreting RenCa cells. RESULTS: Although the modified and original IL-18 genes transduced RenCa showed almost the same level of IL-18 messenger RNA expression, only RenCa cells transduced with modified IL-18 gene secreted IL-18 into the culture supernatant and were completely rejected when transplanted into syngeneic mice. T lymphocytes were involved in this antitumor effect. Moreover, the modified IL-18 transduced RenCa induced tumor vaccine effect against parental RenCa cells injected at a distant site. CONCLUSIONS: Immune gene therapy using modified IL-18 cDNA appears to be effective and IL-18 secreting cancer cells may be a candidate for tumor vaccine therapy.

Animals↗

Fusion of a sequence from HEI10 (14q11) to the HMGIC gene at 12q15 in a uterine leiomyoma.

Uterine leiomyoma, a benign smooth-muscle tumor of the myometrium, is the most commonly encountered neoplasm in women of reproductive age. Band q15 of chromosome 12 is often rearranged in benign mesenchymal tumors such as uterine leiomyomas, and the HMGIC gene, encoding a protein of the high-mobility-group (HMG), is present in that region. Using 3' rapid amplification of cDNA ends (3'RACE) experiments, we isolated an ectopic sequence that was fused to HMGIC in a uterine leiomyoma. Cloning of the fusion cDNA identified a gene termed rising dbl quote, left (low)homo sapiens enhancer of invasion 10" (HEI10) as the fusion partner. Radiation hybrid mapping revealed that the normal location of HEI10 is at 14q11. In the fusion transcript the first two exons of the HMGIC gene, which encode DNA-binding domains, were fused to the 3' portion of the HEI10 gene. This rearrangement implicates HMGIC in the tumorigenesis of uterine leiomyoma, and suggests that its fusion HMGIC product may play a role in mesenchymal differentiation.

Amino Acid Sequence↗

Down-regulation in multiple human cancers of a novel gene, DMHC, from 17q25.1 that encodes an integral membrane protein.

Frequent observations of allelic loss in chromosomal band 17q25.1 in a variety of human cancers have suggested that one or more tumor suppressor genes are present in that region. Moreover, a genetic locus for hereditary focal non-epidermolytic palmoplantar keratoderma, a condition associated with cancer of the esophagus (TOC; Tylosis with Oesophageal Cancer), lies in the same region. We screened cell lines derived from a variety of human cancers by reverse transcription-polymerase chain reaction (RT-PCR) to detect alterations in expression of genes within the region in question, by examining expressed sequence tags located there. These experiments identified an 1834-bp full-length cDNA encoding a novel, 441-amino acid integral membrane protein with seven putative transmembrane domains. This gene showed loss or extreme decrease of expression in 6 of 10 uterine cancer-cell lines, 2 of 11 hepatic cell carcinoma-cell lines, 2 of 7 lung cancer-cell lines, 1 of 6 gastric cancer-cell lines, and 1 of 10 breast cancer-cell lines. (We named it DMHC ("down-regulated in multiple human cancers").) Our results suggest that loss of expression of DMHC at 17q25.1 may play an important role in development of variety of human cancers.

Amino Acid Sequence↗

[Clinical characterization of blaIMP positive gram-negative rods isolated cases].

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.

Bacterial Proteins↗

Inhibition of the motility and growth of B16F10 mouse melanoma cells by dominant negative mutants of Dok-1.

Dok-1 (p62(Dok)) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.

Animals↗

The effect of leukotriene D(4) inhalation on the antigen-induced airway hyperresponsiveness and inflammation in 5-lipoxygenase gene-deficient mice.

BACKGROUND: The role of 5-lipoxygenase (5-LO) products in the asthmatic bronchoconstriction is evident. However, the role of 5-LO products in airway hyperresponsiveness (AHR) and airway inflammation is still under discussion. The aim of the present study is to investigate the role of leukotriene D(4) (LTD(4)) in AHR and allergic airway eosinophilia in mice. METHODS: The effect of LTD(4) inhalation on antigen-induced AHR and airway eosinophilia was investigated in 5-LO gene-deficient mice. RESULTS: After three inhalations of LTD(4), airway responsiveness to acetylcholine was not altered in normal or allergic wild-type and 5-LO knockout (KO) mice. In contrast, the number of eosinophils in 5-LO KO allergic mice increased to the level of wild-type allergic mice after the inhalation of LTD(4). These observations were confirmed by a histopathological study of the lungs. No change in the cytokine levels in bronchoalveolar lavage fluid and serum immunoglobulin levels was shown after LTD(4) inhalation. CONCLUSION: These findings suggest that LTD(4) plays a role in eosinophilic airway inflammation but not in AHR in mice.

Administration, Inhalation↗

Prednisolone inhibits an IgE-mediated late-phase allergic cutaneous reactionby interfering with the activation of mast cells in mice.

Epicutaneous antigen challenge in passively sensitized mice with IgE produces a biphasic cutaneous response which peaks 1 h (immediate-phase reaction) and 24 h (late-phase reaction; LPR) after the antigen challenge. In this model, anaphylactic degranulation and interleukin 6 (IL-6) expression between 4 and 8 h are observed in resident mast cells as the preceding stage of LPR. Prednisolone at a dose of 3 mg kg(-1) clearly inhibited the LPR when administered 2 h before and 4 h after antigen challenge. Slight or no inhibition of LPR was observed by prednisolone administered 6-12 h after challenge. Histologically, prednisolone treatment 2 h before antigen challenge completely inhibited edema and inflammatory cell infiltration, while treatment at 6 h did not at all. In order to investigate the relationship between inhibition of LPR by prednisolone and mast cell activation, the effects of prednisolone on degranulation of mast cells and IL-6 expression in mast cells were investigated. 8 h after antigen challenge, prednisolone clearly inhibited the increase in the number of anaphylactic degranulated and IL-6-positive mast cells by administration 2 h before challenge, but did not affect it by administration 6 h after challenge. These data indicate that the inhibitory mechanism of prednisolone on LPR, at least, involves the inhibition of mast cell activation before LPR.

Animals↗