Biomedical subjects

Tadashi Furusawa

Publications and source records attributed to Tadashi Furusawa.

10 recordsLinked to original sources

PubMed2007

Relation between human decay-accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti-pig cytotoxicity: value of highly expressed hDAF for xenotransplantation.

BACKGROUND: Although the successful production of alpha1,3-galactosyltransferase-knockout (GT-KO) pigs has increased expectations of clinical xenotransplantation, additional modifications of genetically engineered pigs are still being explored, because even GT-KO pigs are incapable of inhibiting the host's immunological response completely. One of the potential candidates is a complement-regulatory protein, such as human decay-accelerating factor (hDAF). However, there are few reports on how high the expression level of hDAF in pig cells would be required for suppression of complement activation. The purpose of this study was to examine the relationship between the level of hDAF expression and its inhibitory effect on human serum cytotoxicity. METHODS: An expression (pCAGGS) vector containing the hDAF gene was transfected into pig fibroblasts using an electroporation system (Gene Pulser II). Forty-eight to fifty-two hours after transfection, the cells were stained with FITC-labeled anti-hDAF antibody and then applied to the cell sorter. hDAF-transfected cells with various expression levels were collected by gating on fluorescence intensity. The level of hDAF expression was determined relative to that in human control endothelial cells. Collected cells expressing x1, x5, x10, x15 and x30 hDAF were incubated into 96-well plates for 16 h, and the cells were subjected to 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: hDAF expression levels in transfected cells at the time of MTT assay (16 h after sorting) were comparable to those immediately after sorting. hDAF expression in pig cells five times higher than in human endothelial cells was effective in inhibiting complement-dependent cytotoxicity of most human sera. However, 15- to 30-fold expression of hDAF was required for effective inhibition of human sera with the highest cytotoxic capacity. CONCLUSIONS: A much higher level of hDAF expression in pig cells than previously considered necessary might be required to provide additional benefit in inhibiting antibody-mediated rejection. Genetically engineered pigs that express very high levels of hDAF would be beneficial for xenotransplantation.

Animals↗

PubMed2006

Gene expression profiling of mouse embryonic stem cell subpopulations.

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.

Animals↗

PubMed2006

Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity with anti-Myc antibody.

The Cre-loxP system has been recognized as a tool for conditional gene targeting in mice. However, most anti-Cre antibodies fail to react with Cre expressed in vivo. In an attempt to directly detect Cre by antibodies in vivo, we constructed the tagged-NCre (NCreMH) gene by connecting the human Myc and His tag sequences to the 3' end of the NCre gene carrying a nuclear localizing signal (NLS) sequence. The production of NCre protein and the recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17 carrying the loxP-flanked lacZ gene into NIH3T3 cells. This activity was also confirmed in vivo after gene transfer of pCMV-NCreMH and pCRTEIL-6 carrying loxP-flanked HcRed1 and EGFP cDNAs, into oviductal epithelium by electroporation. Immunohistochemical staining using anti-Myc antibody demonstrated that the area positive for enhanced green fluorescent protein (EGFP) fluorescence was immunostained with the antibody. These findings indicate that NCreMH is useful as an alternative to NCre for gene targeting.

Animals↗

PubMed2006

Anti-bovine CD34 monoclonal antibody reveals polymorphisms within coding region of the CD34 gene.

OBJECTIVE: Monoclonal antibodies (mAbs) against CD34 are widely used for purification of CD34+ hematopoietic as well as nonhematopoietic stem/progenitor cells. We produced mAbs against bovine CD34 (boCD34) to facilitate the study of hematopoiesis in cattle. METHODS: MAbs were produced by immunizing BALB/c mice with BALB/3T3 cells transfected with boCD34 cDNA. Staining of bone marrow mononuclear cells (BMMNCs) from 10 newborn Holstein calves with the mAbs was examined by flow cytometry. The nucleotide sequence of the coding region for boCD34 in each calf was determined after amplification of the cDNA by reverse-transcription polymerase chain reaction (RT-PCR). BoCD34 fusion proteins, each representing one of the boCD34 alleles found to exist in the calves, were expressed in HeLa cells by DNA transfection, and the staining of these proteins with the mAbs was assessed. RESULTS: One mAb, N21, stained relatively high percentages of BMMNCs from 4 calves but failed to stain those from the other calves. RT-PCR analysis revealed single-nucleotide polymorphisms within the coding region, 3 of which led to amino-acid substitutions. A CD34 mutation experiment indicated that mAb N21 bound to a boCD34 allele with tryptophan at amino acid 167 but not to that with arginine. CONCLUSION: By using mAb N21 as an allelic cell marker, it would be feasible to detect and isolate boCD34+ cell species derived from N21+ donors in N21- recipients following allogeneic in utero transplantation; this would make cattle potentially useful as large animal models with a unique experimental advantage.

Amino Acid Sequence↗

PubMed2005

Differences in gene expression patterns between somatic cell nuclear transfer embryos constructed with either rabbit granulosa cells or their derivatives.

Successful production of offspring by somatic cell nuclear transfer (SCNT) is affected by the nature of the donor cells used. The purpose of this study was to determine whether characteristic changes induced in donor cells by culture conditions influenced gene expression patterns in the resultant SCNT embryos. Rabbit granulosa cells (rGC) were cultured under different conditions, either with or without hCG, and the two derivative cell types obtained (named respectively cGC+ and cGC-) were used as donor cells for SCNT. There were characteristic differences between fresh rGC and the two derivative cell types: p450scc expression and progesterone secretion were both higher in cGC+ than in cGC-; expression of bmp4 and fgfr2 was decreased in cGC+ and cGC- compared with rGC; and cGC+ and cGC- cell types gained collagenIV expression. Use of fresh rGC, or cGC+ and cGC- derivative cells, did not alter either the developmental potencies of SCNT oocytes or cell numbers at the blastocyst stage. The expression patterns of four genes (bmp4, fgfr2, gata4, oct3/4) in SCNT embryos and in fertilized embryos were analyzed by quantitative RT-PCR. We found that oct3/4 was expressed in all embryos. The expression patterns of the other three genes showed considerable variation between the different types of embryo: bmp4 was found in most fertilized embryos but only some of rGC and none of cGC+ and cGC- derived SCNT embryos; fgfr2 was present in fertilized embryos but was present in some rGC and cGC- NT embryos and in all cGC+ NT embryos; gata4 was not expressed in fertilized embryos but was present in a few rGC and cGC+ NT embryos and in most cGC- NT embryos. Our results suggest that the gene expression patterns in SCNT embryos derived from granulosa donor cells are affected by characteristic changes to the cells during in vitro culture.

Animals↗

PubMed2005

Subcellular localization and dynamics of MysPDZ (Myo18A) in live mammalian cells.

MysPDZ is an unconventional myosin belonging to the class XVIII myosin containing a KE (lysine and glutamine)-rich domain and a PDZ domain, which codistributes with actin fibers partially without any canonical actin binding sequence in its myosin head domain. Recently, we reported the identification of a novel isoform of MysPDZ lacking these domains and exhibiting subcellular localization and expression profile different from the original form of MysPDZ. In order to delineate domains directing the subcellular localization of MysPDZ, we performed co-immunoprecipitation experiments and image analyses using mutants of MysPDZ fused with enhanced yellow fluorescent protein. Co-immunoprecipitation analyses showed that MysPDZ can self-associate through its C-terminus coiled-coil domain and the KE-rich domain mediates the interaction with actin. We observed by image analyses that the codistribution with actin fibers and the localization in inner surface of cell membrane of MysPDZ are controlled by the KE-rich domain and the PDZ domain, respectively. Time lapse video microscopy showed that MysPDZ in the cytoplasm moves randomly and rapidly within short range and is allocated to a subcellular compartment without ATP hydrolysis by MysPDZ. This suggests that MysPDZ is a protein which is unlike most unconventional myosins. Our study uncovers a novel role of the KE-rich and PDZ domains in directing subcellular localization and also contributes to a better understanding of functional differences in MysPDZ isoforms.

Actins↗

PubMed2004

Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo.

We examined the expression of cell-surface markers on subpopulations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripotency. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1+ cells expressed a high level of PECAM- 1, and reversible repopulation was observed between PECAM- 1+SSEA-1+ and PECAM-1+SSEA-1- cells. The ES cells carrying the lacZ gene were sorted into three subpopulations: PECAM- 1-SSEA-1-, PECAM-1+SSEA-1-, and PECAM-1+SSEA-1+. Quantitative reverse transcription-polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1- cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1+ SSEA-1+/- cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM- 1- cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0-7.0 days post coitum, many PECAM-1+SSEA- 1+ cells were found in the epiblast, but few beta-gal+ cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.

Animals↗

PubMed2003

Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo.

To generate severe combined immunodeficient (SCID) livestocks for xenotransplantation, we have attempted to generate a SCID phenotype without gene knockout. Based on the reported mouse RAG1 mutants, we constructed the corresponding rabbit RAG1 mutants by mutagenesis of three residues within the catalytic domain: D602A, D710A, and E964A. As expected, these mutants each exhibited no catalytic activity on artificial substrates and inhibited recombination by the wild type RAG1. Moreover, replacement of the N-terminus of RAG1 with enhanced green fluorescent protein (EGFP) greatly increased protein stability, and the triple mutant RAG1 showed a twofold increase in its ability to inhibit wild type activity in vitro. We generated mice transgenic for the latter mutant to assess its effect on V(D)J recombination in vivo. Serum IgM levels in four out of seven transgenic mice were reduced to approximately 30-50% of control levels in four out of seven transgenic mice. Our results suggest that immunodeficient animals for regenerative medicine could be generated without gene knockout.

3T3 Cells↗

PubMed2003

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Abnormalities, Multiple↗

PubMed2003

Genome structure and differential expression of two isoforms of a novel PDZ-containing myosin (MysPDZ) (Myo18A).

We previously cloned a gene for a novel myosin (called MysPDZ) containing a PDZ-domain from bone marrow stromal cells. This new myosin is found in humans and classified as one of the class XVIII myosins (Myo18A). Here, we report the hematopoietic cell-specific splicing isoform (MysPDZbeta) in addition to the previously reported isoform (MysPDZalpha). Combined with mouse genome sequence data, the overall genome structure and generation of the two spliced isoforms are deduced. The MysPDZbeta protein lacks a PDZ-domain in the N-terminal region. Studies of the subcellular localization of the two spliced isoforms indicated that MysPDZalpha containing the PDZ domain co-localizes with the ER-Golgi complex, while MysPDZbeta, which lacks the PDZ domain, localizes diffusely in the cytoplasm. These results suggest that the isoforms differ in their subcellular localization and may have different functions in membrane ruffling and membrane traffic pathways. The PDZ-containing spliced isoform (MysPDZalpha) is not expressed in bone marrow hematopoietic cells, whereas MysPDZbeta lacking the PDZ is specifically expressed in most hematopoietic cells. It is noted that neither isoform is expressed in red blood cells. Interestingly, MysPDZalpha was detected in mature but not in immature macrophages, and its level increased after the induction of differentiation of M1 cells, suggesting a functional role of PDZ-containing myosin in macrophages.

Animals↗