Masaharu Isobe

Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity.

The cellular origin, in vivo function and fate of donor bone marrow‐derived cells residing in the recipient intestinal epithelial cells, pericryptal myofibroblasts or endothelial cells remain obscure. Although ‘immunoprivileged’ mesenchymal stem cells (MSCs) are prime candidates for cell‐ and gene‐based therapy, their precise role in colitis remains largely undetermined. Using a dextran sulphate sodium (DSS) colitis with busulphan (BU)‐induced hypoplastic marrow model, we examined the therapeutic effects of MSC transplantation, focusing on the role of MSCs as both cell providers and immunomodulators. Donor‐derived MSCs were detected by eGFP immunofluorescence and fluorescence in situ hybridization for Y‐chromosome (Y‐FISH) analysis. Western blot analysis of apical‐most tight junction proteins was performed with antibodies against claudin‐2, ‐7, ‐8, ‐12, ‐13, ‐15 and ZO‐1. Cytokine and cell cycle profiles were analysed by semi‐quantitative RT‐PCR and flow cytometry. Susceptibility to DSS colitis was significantly increased by co‐existing BU‐induced bone marrow hypoplasia and this increase was significantly reduced by enhancing epithelial engraftment of MSCs, an effect depending on restoring epithelial barrier integrity rather than inhibiting host immune responses. We provide evidence that implicates MSCs in maintaining epithelial barrier function by reassembling apical‐most tight junction proteins, claudins. The therapeutic efficacy of extrinsic MSCs depends on enhancing epithelial engraftment in damaged crypts by busulphan conditioning. Such a role for the MSC‐derived intestinal cells in colitis therapy merits further examination and may offer a promising new treatment for inflammatory bowel disease (IBD). Copyright

Molecular cloning and chromosomal mapping of a human protein-tyrosine phosphatase LC-PTP†

We isolated cDNA clones encoding a protein-tyrosine phosphatase (PTP) from a human T cell PEER cDNA library. The predicted open reading frame encodes a approximately 40-kDa protein composed of 360 amino acids and has no apparent hydrophobic segments, suggesting that it is a nontransmembrane PTP, which was designated as LC-PTP (leukocyte PTP). Northern blot analysis revealed that the LC-PTP mRNA was preferentially expressed in a variety of hematopoietic cells and the transcriptional sizes were approximately 4.0 kilobases and approximately 2.9 kilobases. Fluorescent in situ hybridization revealed that the human LC-PTP gene is located on the chromosome region 1q32.1, which is known to be a site associated with chromosomal deletion in malignant lymphomas, where candidate tumor suppressor genes might be present.

Loss of Heterozygosity of 14q32 in Colorectal Carcinoma

Previous allelotyping studies on colorectal carcinoma suggest that loss of heterozygosity (LOH) on chromosome 14q may be a common genetic alteration in this tumor type. The purpose of this study was to determine precise frequency of LOH at 14q32 region in colorectal carcinomas and to define a minimal region of LOH. LOH at 14q32 in 66 primary colorectal carcinomas were analyzed by polymerase chain reaction-based deletion mapping using six highly polymorphic microsatellites. The average rate of informative data was 68.2% in all cases of colorectal carcinoma analyzed. The average rate of LOH at 14q32 was 44.1%, with the highest frequency of 50.0% at D14S267 locus in the informative cases. The minimal region of LOH was defined by markers D14S65 and D14S250 at 14q32. The present data provide useful information for the isolation of tumor suppressor genes from this region.

Identification of the TCL6 genes within the breakpoint cluster region on chromosome 14q32 in T-cell leukemia.

A region on chromosome 14q32.1 is often involved in chromosomal translocations and inversions with one of the T-cell receptor loci in T-cell lymphoproliferative diseases. The breakpoints of the different rearrangements segregate into two clusters; a cluster due to inversion on the centromeric side and a cluster due to simple balanced translocations on the telomeric side. If the target gene activated by these different types of chromosomal rearrangements is the same, the gene must be localized between the two clusters of breakpoints in a region of around 160 kb. Within this breakpoint cluster region, we isolated two genes; namely, TCL1 and TML1/TCL1b genes. In the course of characterizing the TML1 gene, we further identified a third novel gene, which we named TCL6 (T-cell leukemia/lymphoma 6), from a region 7 kb upstream of the TML1 locus. The TCL6 gene expressed at least 11 isoforms through very complex alternative-splicing, including splicing with the TML1 gene. Those isoforms encode at least five open reading frames (ORFs) with no homology to known sequences. The localization of the proteins corresponding to these ORF was determined by fusing green fluorescence protein at the carboxyl terminal of each ORF. ORF141 and ORF72 were observed in the cytoplasmic region, while ORF105, ORF119, and ORF163 were predominantly localized in the nuclear region. Since the TCL6 gene was expressed in T-cell leukemia carrying a t(14;14)(q11;q32.1) chromosome translocation and was not expressed in normal T-cells (just like the TML1 and TCL1 genes), it is also a candidate gene potentially involved in leukemogenesis.

Cryopreservation of competent intact yeast cells for efficient electroporation

We have developed a simple method for cryopreserving Schizosaccharomyces pombe and Saccharomyces cerevisiae competent intact cells that permits high transformation efficiency and long‐term storage for electroporation. Transformation efficiency is significantly decreased if intact cells are frozen in common permeating cryoprotectants such as glycerol or dimethyl sulphoxide. On the other hand, we found that a high transformation efficiency could be maintained if the cells were frozen in a non‐permeating cryoprotectant such as sorbitol. The optimum concentration of sorbitol was found in a hypertonic solution of around 2 M. It was also very important to use S. pombe cells grown in minimal medium and S. cerevisiae cells grown in nutrient medium in the exponential growth phase. A slow freezing rate of 10°C/min and a rapid thawing rate of 200°C/min resulted in the highest transformation efficiency. We also found it necessary to wash the thawed cells with 1.0 M of non‐electrolyte sorbitol, since the intracellular electrolytes had leaked as a result of cryoinjury. The frozen competent cells stored at −80°C could be used for more than 9 months without any loss of transformation efficiency. This cryopreservation method for electroporation is simple and useful for routine transformations of intact cells. Frozen competent cells offer the advantages of long‐term storage with high efficiency and freedom from the preparation of fresh competent cells for each transformation. Copyright

Rapid isolation of viral integration site reveals frequent integration of HTLV-1 into expressed loci

AbstractAlthough there is tight association of the human T-cell leukemia virus type-1 (HTLV-1) with adult T-cell leukemia/lymphoma (ATLL), it has remained unresolved whether the HTLV-1 integration into the host genome has any role in the development of this disease. We isolated a total of 58 HTLV-1 integration sites using newly developed, adaptor-ligated PCR from 33 ATLL patients and five ATLL cell lines. We compared our data as well as the previously reported ones with the complete human genomic sequence for the location of its placement, structure, and expression of genes nearby the integration site. The chromosomal target for integration was selected at random, but the integration favorably occurred within the transcription units; more than 59.5% of total integration was observed within the transcriptional unit. All inserted genes by HTLV-1 integration were expressed in normal T cells. Upregulation of genes due to viral integration was found in two out of nine ATLL cases; about 4.4- and 102-fold elevated ankyrin-1 (ANK-1) and gephyrin (GPHN) gene expressions were observed, respectively. These data suggest that the preferential integration of HTLV-1 into an expressed locus occasionally causes deregulation of corresponding gene, which may lead to leukemogenesis of a fraction of ATLL.

Chromosomal imbalances in adult T-cell leukemia revealed by comparative genomic hybridization: gains at 14q32 and 2p16-22 in cell lines

AbstractComparative genomic hybridization was used to identify chromosomal imbalances in eight cell lines and 12 blood samples from patients with adult T-cell leukemia/lymphoma (ATL). The chromosomes most often over-represented in the cell lines were 2p (6 cases), 7q (4 cases), and 14q (4 cases), with minimal common regions at 2p16-22, 7q21-36, and 14q32, respectively. Distinct imbalances were detected in only 7 of the clinical samples. Chromosomes 14q32 and 2p16-22 harbor TCL1 and a transcription factor, HTLF (human T-cell leukemia virus enhancer factor), respectively. FISH analysis revealed that TCL1 did not juxtapose to TCRA, and we detected no expression of TCL1 in any of the ATL cell lines despite the 14q32 amplifications. Moreover, expression of HTLF was not elevated in the ATL cell lines bearing multiplication of 2p. These results suggest that chromosomal regions 2p16-22 and 14q32 harbor genes other than HTLF and TCL1 that are involved in cellular immortalization or in the pathogenesis of ATL.

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

BackgroundAlthough a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging.ResultsWe propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals.ConclusionsOur technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

Involvement of Growth-Associated Protein-43 with Irreversible Neurite Outgrowth by Dibutyryl Cyclic AMP and Phorbol Ester in NG108–15 Cells

Simultaneous treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and dibutyryl cyclic AMP (diBu‐cAMP) for 72 h induced neurites in NG108–15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu‐cAMP alone disappeared after the withdrawal of the drug. The expression of growth‐associated protein‐43 (GAP‐43) mRNA was also observed by a combined application of TPA and diBu‐cAMP. The increased level of GAP‐43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP‐43 cDNA, neurites induced by treatment with diBu‐cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP‐43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in the accumulation of GAP‐43.

Allelic imbalance of 14q32 in esophageal carcinoma

It has been demonstrated that the accumulation of alterations in several oncogenes and tumor suppressor genes plays a role in the initiation and progression of esophageal carcinoma. However, to our knowledge, very few studies have described the molecular genetic changes of chromosome arm 14q in esophageal carcinoma. In this study, we examined 35 primary esophageal carcinomas for allelic imbalance on 14q32. Analysis of four polymorphic microsatellite markers identified 13 (37%) tumors exhibiting allelic imbalance on 14q32 in at least one locus. In particular, the allelic imbalance of the D14S267 marker that has been reported in various tumors as having a high frequency of deletion was observed in 10 of 26 informative cases (38.5%). The commonly deleted regions were delineated by markers D14S65 and D14S250 on 14q32. In regard to the macroscopic features of tumor, the 14q allelic imbalance rate of protruding type tumors was higher than that of the ulcerative type. These data suggest that potential suppressor loci on 14q32 are related to the development and progression of esophageal carcinoma.