Effect of proline rich domain of an RNA-binding protein Sam68 in cell growth process, death and B cell signal transduction.

BACKGROUND: Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line. METHODS: By using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions. RESULTS: A slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells. CONCLUSIONS: The proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide.

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes.

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit(+) cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT. Disclosure of potential conflicts of interest is found at the end of this article.

Treatment of Shwachman syndrome by Japanese herbal medicine (Juzen-taiho-to): stimulatory effects of its fatty acids on hemopoiesis in patients.

Juzen-taiho-to (a Japanese herbal medicine) has been traditionally administered to patients with anemia, neutropenia, or wasting syndrome. We previously attempted to isolate and purify the hemopoiesis-stimulatory components in Juzen-taiho-to extracts using an in vitro hemopoietic stem cell (HSC) assay method in which mouse HSCs can proliferate on a stromal cell line (MS-5). We have found that fatty acids (particularly oleic acid and linolenic acid) actively promote the proliferation of HSCs, and that the effect is mediated by stromal cells, rather than by any direct action on the HSCs. In the present study, we show, using human normal bone marrow cells (BMCs) and umbilical cord blood cells, that similar stimulatory effects are due to the presence of oleic acid and linolenic acid, which stimulate the proliferation of HSCs in stroma-based culture systems. Furthermore, a marked stimulatory effect was noted on BMCs from patients with Shwachman syndrome, which shows pancreatic and bone marrow dysfunctions. We also show the data on hemopoietic recovery after the administration of Juzen-taiho-to to a patient with Shwachman syndrome. These findings suggest that decreased fatty acid levels in the blood, caused by exocrine pancreatic insufficiency, induce bone marrow dysfunction in Shwachman syndrome.

Induction of tolerance in quadruple chimeric mice.

Human cord blood (CB) contains hematopoietic stem cells and progenitors. Because the major limitation to a widespread use of CB for transplantation lies in its limited volume, it is necessary to combine the CB from several donors. In this study, we show that lethally irradiated mice can be reconstituted with the injection of a mixture of T cell-depleted bone marrow cells (BMCs; total, 3 x 10(6)) obtained from three fully allogeneic mouse strains in two different mouse combinations. A higher survival rate was obtained in the triple injection group than in mice injected with BMCs (1 x10(6)) obtained from a single mouse strain. In the mixed chimeric mice, three kinds of donor-type and recipient-type cells were detected in all the hematopoietic organs 1 month after bone marrow transplantation (BMT). Mixed-lymphocyte reaction showed that the tolerance to both recipient-type and donor-type major histocompatibility complex determinants was induced in the chimeric mice. In the peripheral blood (PB) of these mice, only one type of cells from the three different donor strains became dominant in most chimeric mice and reached a stable level about 4 months after BMT. Polymerase chain reaction analyses, however, revealed that the skins from all the donors were accepted even when no cells with their phenotypes could be detected in the PB. These results suggest that both hemato-lymphoid reconstitution and stable tolerance to not only the recipient strain but also all the donor strains can be achieved in chimeric mice, indicating the possibility of mixed CB transplantation in humans.