Biomedical subjects
Publications and source records attributed to Toshio Tanaka.
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OBJECTIVE: To evaluate the incidence and effect of cytomegalovirus (CMV) reactivation in patients with inflammatory connective tissue diseases (CTD) undergoing immunosuppressive therapy. METHODS: A total of 18 consecutive CMV seropositive patients undergoing immunosuppressive therapy for inflammatory CTD were enrolled. CMV reactivation was determined by detection of CMV-DNA in peripheral blood leukocytes (PBL) or plasma using quantitative real-time polymerase chain reaction. RESULTS: CMV reactivation was detected in PBL in 7 of 17 evaluable patients (41%), and in plasma in 5 of 17 patients (29%). Patients with detectable CMV-DNA in plasma were exclusively positive for CMV-DNA in PBL. Conclusion. Patients with inflammatory CTD under immunosuppressive therapy are at high risk for CMV reactivation. The clinical significance of such an event and indications for antiviral therapy should be examined further.
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A UMP-derivative, uridine 5'-hexadecylphosphate (UMPC16), exhibited a fungicidal action against various yeast strains including the fission yeast Schizosaccharomyces pombe in combination with Ba2+ ion. UMPC16 accelerated reactive oxygen species (ROS) generation in medium with Ba2+ ion in a dose- and time-dependent manner. Additional supplementation of Ca2+ ion into medium could suppress such a combined fungicidal action due to oxidative stress induction.
BACKGROUND: Activation of mast cells and basophils through allergen stimulation releases chemical mediators and synthesizes cytokines. Among these cytokines, IL-4, IL-13, and IL-5 have major roles in allergic inflammation. OBJECTIVE: We sought to determine the potency of flavonoids (astragalin, fisetin, kaempferol, myricetin, quercetin, and rutin) for the inhibition of cytokine expression and synthesis by human basophils. METHODS: The inhibitory effect of flavonoids on cytokine expression by stimulated KU812 cells, a human basophilic cell line, and freshly purified peripheral blood basophils was measured by means of semiquantitative RT-PCR and ELISA assays. The effects of flavonoids on transcriptional activation of the nuclear factor of activated T cells were assessed by means of electrophoretic mobility shift assays. RESULTS: Fisetin suppressed the induction of IL-4, IL-13, and IL-5 mRNA expression by A23187-stimulated KU812 cells and basophils in response to cross-linkage of the IgE receptor. Fisetin reduced IL-4, IL-13, and IL-5 synthesis (inhibitory concentration of 50% [IC(50)] = 19.4, 17.7, and 17.4 micromol/L, respectively) but not IL-6 and IL-8 production by KU812 cells. In addition, fisetin inhibited IL-4 and IL-13 synthesis by anti-IgE antibody-stimulated human basophils (IC(50) = 5.1 and 6.2 micromol/L, respectively) and IL-4 synthesis by allergen-stimulated basophils from allergic patients (IC(50) = 4.8 micromol/L). Among the flavonoids examined, kaempferol and quercetin showed substantial inhibitory activities in cytokine expression but less so than those of fisetin. Fisetin inhibited nuclear localization of nuclear factor of activated T cells c2 by A23187-stimulated KU812 cells. CONCLUSION: These results provide evidence of a novel activity of the flavonoid fisetin that suppresses the expression of T(H)2-type cytokines (IL-4, IL-13, and IL-5) by basophils.
The serum LDL-cholesterol (LDL-C)-lowering effects of two types of canned beverage containing mixed green vegetables and fruits, with or without broccoli and cabbage (B&C), were examined in a randomized double-blind study design. Seventy-seven adults subjects with mild to moderate hypercholesterolemia participated in this study after giving their informed consent. The subjects were randomly divided into two groups. One group(test group) was allocated a test sample, containing B&C as the main materials. Another group(control group) was allocated a placebo sample made from the same materials but without B&C. The subjects were administered 2 cans of the assigned sample (160 g contents/can) per day for 12 weeks. Forty-nine out of 77 subjects, whose LDL-C levels were greater than or equal to 140 mg/dl and less than 180 mg/dl, were analyzed for the effectiveness. Serum LDL-C levels in the test group were significantly(p < 0.05) reduced at 3, 6 and 9 weeks after administration in comparison to the baseline levels (155.7 mg/dl in average). The average LDL-C value at 9 weeks was 142.5 mg/dl and the reduction rate was 8.5%. But serum LDL-C levels in the control group were not significantly reduced. Significant differences(p < 0.05) between the groups were observed in the LDL-C levels at 6 and 9 weeks and also in the total cholesterol levels at 9 week. Thus daily intake of the beverage tested containing B&C are useful for lowering serum LDL-C levels in hypercholesterolemic subjects.
Pharmacogenomics is defined to identify the therapeutic gene clusters which are involved in determining the responsiveness and to distinguish responders and non-responders to a given drug. Genome sequencing, transcriptome, proteome and metabolome analysis are of particular significance in pharmacogenomics. Sequencing is used to locate polymorphisms, and monitoring of functional gene expression can provide clue about the genomic response to disease and treatment. The transcriptome analysis can be done by methods of random cDNA sequencing, mRNA display(fluorescent differential display et al.) and differential hybridization(cDNA microarray, oligomicrochip et al.). We used transcriptome/proteome/metabolome analysis to identify therapeutic target genes by studying change of gene expression in animal models and human model cells of various diseases and found novel drug target candidates through this pharmacogenomic strategy. The present study describes combined transcriptome and metabolome analysis for therapeutic target validation in hypoxia-induced vascular remodeling. The pharmacogenomic analysis and pharmainformatics has the potential for strategy to define novel therapeutic gene clusters in various diseases and holds the promise that drugs might be tailor-maid for individuals and adapted to each person's own genetic makeup. These techniques provided an excellent strategy for screening and validation of targets.
We have characterized the gene for human phosphodiesterase 8B, PDE8B, and cloned the full-length cDNA for human PDE8B (PDE8B1) and two splice variants (PDE8B2 and PDE8B3). The PDE8B gene is mapped to the long arm of chromosome 5 (5q13) and is composed of 22 exons spanning over approximately 200kb. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. PDE8B1 encodes an 885 amino acid enzyme, containing an N-terminal REC domain, a PAS domain, and a C-terminal catalytic domain. PDE8B2 and PDE8B3 both have deletion in the PAS domain and encode 838 and 788 amino acid proteins, respectively. RT-PCR analysis revealed that while PDE8B1 is the most abundant variant in thyroid gland, PDE8B3, but not PDE8B1, is the most abundant form in brain. These findings suggest that selective usage of exons produces three different PDE8B variants that exhibit a tissue-specific expression pattern.
Analysis of patients with atopic dermatitis (AD) for differential expression of genes, as compared to normal individuals, will be useful for understanding the molecular pathogenesis of AD. We found that the expression of the gene ETEA in human peripheral blood CD3-positive cells from patients with atopic dermatitis was significantly higher than in normal individuals. Eosinophils from AD patients expressed ETEA at a significantly higher level than the healthy controls. The overall sequence of the 445 aa deduced polypeptide from the cloned ETEA cDNA showed homology to human Fas-associated factor 1 (FAF1), which is involved in Fas-mediated apoptosis. However, the interaction of ETEA with the Fas death domain was weaker than that of FAF1, as studied in yeast two-hybrid experiments. The ETEA-EGFP fusion protein was expressed in cytoplasm. During the course of activation-induced cell death of primary T cells, transcription levels of ETEA and FAF1 were upregulated with similar kinetics. The enhanced expression of ETEA may play a role in the regulating the resistance to apoptosis that is observed in T cells and eosinophils of AD patients.
Recombinant human CRP (rhCRP) was secreted into culture supernatant of Escherichia coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell. Highly purified 5 g rhCRP was produced from 180 L culture supernatant by affinity chromatography. The purified rhCRP was indistinguishable from the native one with respect to Ca(2+)-dependent binding ability to phosphorylcholine, electrophoretic behavior, N-terminal amino acid analysis, and immunochemical properties. The molecular weight of rhCRP monomer was determined to be 23059.7 Da by TOF/MS analysis. These results indicate that rhCRP has the same protein structure as native one and that rhCRP has the potential as a reference material and/or calibrator of high-sensitivity CRP assay to predict the risk of cardiovascular disease.
The effects of a canned mixed green vegetable and fruit beverage, containing broccoli (Brassica oleracea L. var. botrytis L.) and cabbage (Brassica oleracea L. var. capitata L.) as main materials, on serum lipid levels in hypercholesterolemic patients were investigated. Thirty-one adult subjects were administered two cans of the beverage (160 g/can) per day for 3 weeks. Their serum total cholesterol (TC) levels significantly decreased from 6.7 +/- 0.8 to 6.1 +/- 0.6 mmol/L, and, more strikingly, the level of low-density lipoprotein cholesterol (LDL-C) significantly decreased from 4.4 +/- 0.8 to 3.8 +/- 0.7 mmol/L. At 9 weeks after the cessation of the administration, these levels had returned to the preadministration levels. Furthermore, 14 other subjects who were administered one can of the sample for 12 weeks also showed a significant reduction in the levels of serum TC and LDL-C. Thus, daily consumption of this mixed green vegetable and fruit beverage may be useful in lowering serum TC and LDL-C levels in hypercholesterolemic patients.
1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, initially induced morphological changes similar to those of typical apoptosis in human leukemia HL-60 cells but FPy-treated cells were characterized by the absolute absence of final apoptotic events such as fragmentation into apoptotic bodies. FPy-induced cell death was considered to be apoptotic on the basis of the induction of DNA fragmentation and the protection against these events by the coaddition of a pan-caspase inhibitor. The increase in the cytoplasmic cytochrome c level supported the possibility that FPy-treated cells should have the ability to complete the entire apoptotic process ending in cell fragmentation and apoptotic body formation. At concentrations too low to induce apoptosis, FPy could suppress the induction of apoptotic body formation in HL-60 cells by typical inducers of apoptosis such as actinomycin D or anisomycin. FPy exhibited a cytochalasin-like effect on spatial arrangement of actin filament independent of its apoptosis-inducing activity.
We have found that an HIV-1 accessory gene product Vpr enhanced HIV-1 reproduction in U1 cells chiefly by the induction of TNF, a proinflammatory cytokine, which was also known to be an activator of HIV-1 reproduction. We have generated the functional HIV-1 accessory gene product Vpr in bacterial cells. Vpr was generated in an Escherichia coli system (rVpr), purified with antibodies (Ab) to the 16 C-terminal amino acids of Vpr. The purified rVpr of 15 kDa was examined for its ability to upregulate HIV-1 reproduction in U1 cells, which is a reported function of the authentic Vpr. rVpr upregulated HIV-1 reproduction in U1 cells in a dose-dependent manner and induced the secretion of TNF. The upregulation of HIV-1 by rVpr was completely inhibited not only by anti-Vpr antibodies but also by anti-TNF antibody. These findings suggested that Vpr caused an HIV-1 reproduction in U1 cells through the induction of TNF.
BACKGROUND: Analysis of genes that are differentially expressed in patients with atopic dermatitis (AD) and normal individuals will provide important information on the underlying molecular pathogenetic mechanisms of AD. METHODS: Transcript of freshly isolated peripheral blood T cells from 59 individuals were analyzed with a fluorescent differential display (FDD) method. Ninety-two differentially expressed genes were identified in this manner. Additionally, real-time quantitative RT-PCR was employed to investigate the expression of the FDD-selected genes and also genes related to T cell function. RESULTS: A number of genes, including CC chemokine receptor 4, T cell-specific tyrosine kinase (Emt/Itk), integrin beta1, integrin alpha6, IQGAP1 and MAR/SAR DNA-binding protein (SATB1), were shown to be more highly expressed in patients with moderate and/or severe AD than in controls or patients with mild AD. Because the products of these upregulated genes influence chemotaxis, adhesion, migration and Th2 polarization, it is suggested that in more severe AD, circulating T cells may function differently in this regard. Several other genes, the role of which in T cell function is currently unknown, were also found to be differentially expressed in AD. These included the heat shock protein 40 and vasopressin-activated calcium-mobilizing receptor 1. CONCLUSION: The upregulated genes identified in this work may serve as useful markers for moderate to severe AD as opposed to normal or mild AD and also as markers indicating progression to more severe AD. Further functional characterization will provide a better understanding of the pathophysiology of circulating T cells in AD.
The Human Genome Project provides insights so profound that it has the ability to change everything we know about medicine and how medicines are developed. Pharmacogenomics is defined as studies to identify the genes that are involved in determining the responsiveness to a given drug and to distinguish responders and non-responders to a given drug. Genome sequencing, transcriptome, and proteome analysis are of particular significance in pharmacogenomics. Transcriptome analysis can be done by methods of random cDNA sequencing, mRNA display and, differential hybridization (i.e., cDNA microarray and associated methods). Our results suggest that the pharmacogenomic transcriptome analysis and pharmainformatics have potential as strategies for defining novel drug targets in various diseases. Pharmacogenomics enhances the development, commercialization, and clinical use of conventional pharmaceutical products for common diseases, and it will eventually become a powerful tool for Evidence-Based Medicine. It is also important to predict interindividual pharmacokinetic differences by genetic polymorphisms of transporters or pharmacokinetic changes by transporter-mediated drug interactions during drug development. Pharmacogenomics and pharmainformatics enable us to move quickly and efficiently from targets to appropriate medicines.
The polyol macrolide niphimycin (NM) exhibited fungicidal activity against Saccharomyces cerevisiae cells accompanying the leakage of cytoplasmic components including nucleotide-like materials in addition to K+ at 10 microM or above. Such a dynamic change in the plasma membrane was observed upon treatment of cells with H2O2 but not with the polyene macrolide antibiotic amphotericin B (AmB). The NM-induced cell death could be prevented by the exogenous addition of phosphatidylcholine (PC) whereas such a protective effect was only weakly observed with ergosterol, the molecular target of AmB. NM-treated cells were further characterized with a dramatic loss of glutathione even at a dose of 5 microM or less, representing NM-triggered metabolic conversion of the antioxidant molecule. NM-treatment indeed accelerated the cellular production of reactive oxygen species (ROS) such as H2O2 detectable with a specific fluorescent probe in a dose-dependent manner. These results suggested a synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of NM against S. cerevisiae.
A long-chain acyl-CoA synthetase (Faap) inhibitor, adenosine 5'-hexadecylphosphate (AMPC16), caused lethal plasma membrane damage to Saccharomyces cerevisiae cells as reflected by the leakage of cytoplasmic K+ into medium in which Mg2+ was supplemented at 10 mM. AMPC16 did not interfere with the de novo synthesis of phospholipids using acetyl-CoA as a starting material, but the AMP analog accelerated the liberation of long- chain fatty acids from phospholipids with the aid of exogenous Mg2+, suggesting that the role of this divalent cation is to maximize the rate of the acyl group turnover. This Mg2+-dependent fungicidal effect of AMPC16 was similarly observed with a mutant lacking any of the phospholipase B (Plbp) isozymes, whereas it was mostly suppressed in a phospholipase C (Plc1p) deletion mutant in which the liberation of fatty acids from phospholipids was completely prevented. These results suggest that Plc1p is normally functional for phospholipid reconstitution in exponentially growing cells and enhancement of its activity by exogenous Mg2+ could be a cause of the irreversible deacylation of plasma membrane phospholipids when their reacylation is blocked at the step of activation of long-chain fatty acids by one of the Faap isozymes.
A 51-year-old man with recurrent episodes of angioedema was diagnosed as having autoimmune acquired angioedema, based on adult onset, lack of apparent family history, decreased activity of C1 esterase inhibitor (C1 INH) and CH50, decreased levels of serum C4 and Clq and the presence of autoantibodies to C1 INH. The danazol treatment relieved the symptoms of angioedema and increased the C1 INH activity and concentration with the normalization of CH50, C1q and C4 levels. To our knowledge, this is the first case of autoimmune acquired angioedema in Japan.