Biomedical subjects

Atsushi Muraguchi

Publications and source records attributed to Atsushi Muraguchi.

16 recordsLinked to original sources

PubMed2006

Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer.

BACKGROUND: Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. METHODS: Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated. RESULTS: Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction. CONCLUSION: The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae↗

PubMed2006

Amplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells.

The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell. In this method, the first-strand cDNA was directly synthesized from a single cell, and both the tailing reaction and second-strand cDNA synthesis were performed in the same tube without purifying the cDNA sample. Using this method, we were able to amplify the cDNA of the immunoglobulin (Ig) variable region gene from more than 50% of single B cells. The amplified cDNA fragment contained a full-length Ig variable region including a 5'-untranslated region, a leader sequence, and an initiation codon. This method may thus be applicable for a comprehensive analysis of the Ig variable genes of the lymphocyte repertoire in humans and animals, thereby contributing to the development of antibody-based therapeutics for infectious diseases.

B-Lymphocytes↗

PubMed2005

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Amino Acid Sequence↗

PubMed2005

Doxorubicin induces expression of multidrug resistance-associated protein 1 in human small cell lung cancer cell lines by the c-jun N-terminal kinase pathway.

Multidrug resistance (MDR) is a major impediment to successful chemotherapy for lung cancer. Overexpression of multidrug resistance-associated protein 1 (MRP1) appears to be involved in MDR development in lung cancer cells. A number of chemotherapeutic agents including doxorubicin (DOX) were reported to induce MRP1 expression in human lung cancer cells. In our study, we investigated the mechanism by which DOX induces MRP1 expression in human small cell lung cancer (SCLC) cell lines, GLC4 and NCI-H82. These cells expressed MRP1 protein at low levels and were sensitive to DOX. Doxorubicin at 50 nM induced a marked increase in MRP1 expression in 24 hr, and stimulated c-jun N-terminal kinase (JNK) activity. Treatment with a JNK inhibitor, SP600125, significantly inhibited MRP1 induction. Furthermore, transfection with JNK1 and JNK2 antisense oligonucleotides markedly inhibited DOX-induced MRP1 expression. Chromatin immunoprecipitation assays revealed an enhanced recruitment of phosphorylated c-jun to the MRP1 promoter containing the AP-1 site upon DOX stimulation, which was inhibited by pretreatment with SP600125. Surprisingly, GLC4 cells exposed to DOX for 24 hr maintained increased MRP1 expression and resistance to DOX for at least 3 weeks. Pretreatment with SP600125 before DOX stimulation blocked the appearance of the MDR phenotype as well as MRP1 induction in GLC4 cells. These findings suggest that JNK activation may play an essential role for the induction of MRP1 protein in human SCLC cells by chemotherapeutic agents and that combined treatment of a JNK inhibitor with anticancer drugs may prevent the development of MDR by the abrogation of MRP1 induction.

Antibiotics, Antineoplastic↗

PubMed2005

Characterization of the proximal enhancer element and transcriptional regulatory factors for murine recombination activating gene-2.

Recombination-activating gene (RAG)-1 and RAG-2 are essential for V(D)J recombination and are expressed specifically in lymphoid cells. We previously identified two putative enhancer elements, the proximal and distal enhancers, located at -2.6 and -8 kb, respectively, 5' upstream of mouse RAG-2, and characterized the distal enhancer element in detail. In this study, to characterize the proximal enhancer in vitro as well as in vivo, we first defined a 170-bp core enhancer element within the proximal enhancer (Ep) and determined its activity in various cells. Ep conferred enhancer activity only in B-lymphoid cell lines, but not in T- or non-lymphoid cell lines. Analysis of the transgenic mice carrying an EGFP reporter gene linked with Ep revealed that Ep activated the transcription of the reporter gene in bone marrow and spleen, but not in thymus or non-lymphoid tissues. Ep was active in both B220+IgM- and B220+IgM+ subpopulations in the bone marrow and in the B220+ subpopulation in the spleen. Using electrophoretic mobility shift assays and mutational assays, we found that Ikaros and CCAAT/enhancer binding protein cooperatively bind Ep and function as the transcription factors responsible for B cell-specific enhancer activity. These results demonstrate the role of Ep as a cis-regulatory enhancer element for RAG-2-specific expression in B-lymphoid lineages.

Animals↗

PubMed2005

A case study of the estimation of occupational internal dose using urinary excretion data obtained in a biomedical research facility.

This paper presents a fundamental investigation of the screening of intakes and the estimation of internal doses for workers at a biomedical research facility from their urinary excretion data. Urine samples from the workers were collected immediately before and several days after handling radioactive materials, and 5-mL portions were measured directly with a sodium iodide scintillation detector or a liquid scintillation detector. Intakes of radionuclides and effective doses were estimated from the data using the Internal Dose Easy Calculation code. Blank levels of radioactivity and detection limits for this method were determined from the radioactivity contained in urine samples collected from volunteers working outside the controlled area (control samples). Most of the radioactivity in the control samples was attributed to K. Under the present experimental conditions, the detection limits for the committed effective doses were 7.5 x 10(-4) mSv for 125I and 6.9 x 10(-2) mSv for 35S in the case of samples collected 1 d after handling. Accordingly, it can be determined whether workers were exposed more than the recording level (1 mSv of committed effective dose) or not. No workers in the surveyed facility were found to suffer internal exposure beyond the recording levels, and their intakes were estimated to be below the detection limits in the 2-y investigation period. In an ordinary facility of biomedical research, the amount of unsealed radioactivity handled at one time is within 10 MBq in many cases, and the air ventilation and filtering systems work adequately. Under such circumstances, the present results suggest that individual monitoring, such as urine analysis, is omissible in many cases as a routine monitoring of internal exposure for workers.

Adolescent↗

PubMed2004

Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein.

Recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the -41 to -17 nucleotides (nt) 5' -upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein-protein complex was responsible for its activity in immature B cells. In this study, we show that the -65/-42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter.

Animals↗

PubMed2004

RGA protein associates with a TRPV ion channel during biosynthesis and trafficking.

TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the protein-protein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function.

Animals↗

PubMed2004

Involvement of NF-kappaB in TGF-beta-mediated suppression of IL-4 signaling.

Control of immune response requires the coordinated integration of both stimulatory and inhibitory factors. Therefore, the cross-talk of different signaling pathways is critical in providing an integrated cellular response to multiple external signals. Both interleukin-4 (IL-4) and transforming growth factor (TGF-beta) are pleiotropic cytokines and play critical roles in controlling immune responses. For example, IL-4 mediates important pro-inflammatory functions in asthma including induction of the IgE isotype switch and expression of vascular cell adhesion molecules. Whereas, TGF-beta is secreted from B, T, and dendritic cells as well as macrophages, and negatively regulates their proliferation, differentiation, and activation by other cytokines. In this study, we examined the effect of TGF-beta on IL-4 signaling using B cells as well as embryonic kidney cells. TGF-beta inhibited IL-4-induced IgG1 production and gene expression of germline epsilon transcripts in B cells. In embryonic kidney cells, TGF-beta signals suppressed IL-4-induced transcription, when we monitored using germline epsilon promoter DNA. Furthermore, activation of NF-kappaB resulted in a resistance to TGF-beta-mediated suppression of IL-4 signaling. These results indicate that TGF-beta-mediated regulation of IL-4 signaling may act by targeting NF-kappaB signaling.

Animals↗

PubMed2003

Heat shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells.

DNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)-induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3-activated CAD in a cell-free system. Peptide-binding domain-deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)-binding domain-deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.

Caspases↗

PubMed2003

Proteomic approach to apoptotic thymus maturation.

Apoptosis is an essential process for selection of T lymphocytes specific for foreign antigen in the process of mammalian thymus maturation. Proteomics, a comprehensive study of proteins expressed in a cell, will facilitate the systematic analysis of protein molecules related to such a complicated biological system. Protein expression profiles including information about protein signatures, localization and their quantitative changes with extracellular stimulations are extremely useful to construct intracellular pathway models resulting in the apoptotic cell death.

Apoptosis↗

PubMed2003

Possible involvement of glial cell line-derived neurotrophic factor and its receptor, GFRalpha1, in survival and maturation of thymocytes.

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.

Animals↗

PubMed2002

Lymphoid enhancer-binding factor-1 binds and activates the recombination-activating gene-2 promoter together with c-Myb and Pax-5 in immature B cells.

The recombination-activating gene (RAG)-1 and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of Ag receptor genes. We studied the regulation of murine RAG-2 promoter and revealed that -41/-17 RAG-2 promoter region, which was indispensable for the RAG-2 promoter activity in B cell lines, contained binding sites for lymphoid enhancer-binding factor-1 (LEF-1), c-Myb, and Pax-5. We showed that these three transcription factors bound the promoter region in vitro and in vivo. Cotransfection assays using a human embryonic kidney cell line (293T) showed that LEF-1, c-Myb, and Pax-5 cooperatively activated the RAG-2 promoter, via their synergistic DNA binding. We also showed that LEF-1, c-Myb, and Pax-5 physically interact in the cells. Finally, we demonstrated that a dominant-negative LEF-1 protein, which lacks the binding site for beta-catenin, suppressed the RAG-2 promoter activity as well as the endogenous RAG-2 expression in a pre-B cell line (18.81). These results suggest that LEF-1/beta-catenin complex regulates the RAG-2 promoter activation in concert with c-Myb and Pax-5 in immature B cells. The link between LEF-1/beta-catenin and Wnt signaling in B lineage cells will be discussed.

5' Untranslated Regions↗

PubMed2002

Characterization of chromatin structure and enhancer elements for murine recombination activating gene-2.

Recombination-activating genes (RAGs) play a critical role in V(D)J recombination machinery and their expression is specifically regulated during lymphocyte ontogeny. To elucidate the molecular mechanisms regulating murine RAG-2 expression, we examined a chromatin structure of 25-kb DNA segment adjacent to murine RAG-2 by analyzing DNase I hypersensitive (HS) sites. In a RAG-2-expressing murine pre-B cell line, three lymphoid cell-specific HS sites (HS1, HS2, and HS3) were identified. Among these HS sites, one HS site (HS3) that locates in the RAG-2 promoter was associated only with RAG-2-expressing cell lines. Using the transient enhanced green fluorescence protein reporter gene assays, we identified two enhancer elements in the 5'-upstream region of RAG-2 that corresponded to HS1 and HS2. One of the enhancer elements (D3) exhibited enhancer activity only in the lymphoid cell lines. Analysis of the transgenic mice carrying the enhanced green fluorescence protein-reporter gene linked with D3 revealed that D3 activated the reporter gene-expression in the primary lymphoid tissues, but not in the secondary lymphoid tissues or nonlymphoid tissues. D3 was active in CD4(-)CD8(-), but not in CD4(+)CD8(+) or CD4(+)CD8(-) thymocytes in the thymus, and also active in B220(+)IgM(-), but not in B220(+)IgM(+), cells in the bone marrow. Finally, our data suggested that C/EBP may bind to the D3 enhancer and function as one of the transcription factor(s) responsible for the enhancer activity. These results show that the tissue- and stage-specific expression of murine RAG-2 is regulated by alteration of the chromatin structure as well as cis-regulatory enhancer elements.

3T3 Cells↗

PubMed2002

Cooperative binding of c-Myb and Pax-5 activates the RAG-2 promoter in immature B cells.

The recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in immature lymphoid cells undergoing the recombination of antigen receptor genes. The regulation of murine RAG-2 promoter was studied and it was revealed that the -41/-17 RAG-2 promoter region, which is conserved between humans and mice, was indispensable for the RAG-2 promoter activity in B-cell lines. The region contained 2 cis elements that bound c-Myb and Pax-5. Mutation in the c-Myb-binding site in the promoter reduced the promoter activity in B-cell lines. Cooperative activation of the RAG-2 promoter was seen by a combination of c-Myb and Pax-5 in a human embryonic kidney cell line (293T), via their synergistic DNA-binding. Deletion experiments showed that the C-terminus of c-Myb was responsible for their interaction. Furthermore, the dominant-negative c-Myb mutant suppressed the activation of the RAG-2 promoter in a pre-B-cell line as well as in 293T cells. These results suggest that cooperative binding of c-Myb and Pax-5 to the RAG-2 promoter is one of the mechanisms to direct the restricted expression of the RAG-2 in immature B cells.

Animals↗

PubMed2002

The regulation of DNAse activities in subcellular compartments of activated thymocytes.

Thymocytes expressing self-reactive T-cell receptors (TCR) are eliminated in the thymus through a TCR-mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase-activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co-localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase-3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase-3-dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony.

Animals↗