Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases

The Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 are critical signaling regulators in mammalian cells. The deletion of both Rac1 and Rac2 murine alleles leads to a massive egress of hematopoietic stem/progenitor cells (HSC/Ps) into the blood from the marrow, whereas Rac1-/- but not Rac2-/- HSC/Ps fail to engraft in the bone marrow of irradiated recipient mice. In contrast, Rac2, but not Rac1, regulates superoxide production and directed migration in neutrophils, and in each cell type, the two GTPases play distinct roles in actin organization, cell survival, and proliferation. Thus, Rac1 and Rac2 regulate unique aspects of hematopoietic development and function.     

p21WAF1/CIP1蛋白在小鼠卵泡与黄体中的表达

【目的】研究p21WAF1/CIP1蛋白在成年小鼠卵泡发育与黄体形成及退化过程中表达的变化规律。【方法】取20只成年雌性小鼠的卵巢,制作石蜡切片,经免疫组织化学SP法染色后,观察p21WAF1/CIP1蛋白在原始卵泡、初级卵泡、次级卵泡、三级卵泡、成熟卵泡、闭锁卵泡、早期黄体、中期黄体以及晚期黄体中的定位及表达。【结果】卵泡发育和黄体形成及退化过程中均有p21WAF1/CIP1蛋白的表达;p21WAF1/CIP1蛋白在此过程中呈先升高后降低的趋势,峰值出现在三级卵泡时期。【结论】p21WAF1/CIP1蛋白在卵母细胞中发挥一定作用,可能参与优势卵泡的选择,并对卵泡发育及黄体形成和退化有影响。     

Control of the reversibility of cellular quiescence by the transcriptional repressor HES1

The mechanisms by which quiescent cells, including adult stem cells, preserve their ability to resume proliferation after weeks or even years of cell cycle arrest are not known. We report that reversibility is not a passive property of nondividing cells, because enforced cell cycle arrest for a period as brief as 4 days initiates spontaneous, premature, and irreversible senescence. Increased expression of the gene encoding the basic helix-loop-helix protein HES1 was required for quiescence to be reversible, because HES1 prevented both premature senescence and inappropriate differentiation in quiescent fibroblasts. In some human tumors, the HES1 pathway was activated, which allowed these cells to evade differentiation and irreversible cell cycle arrest. We conclude that HES1 safeguards against irreversible cell cycle exit both during normal cellular quiescence and pathologically in the setting of tumorigenesis.     

Tissue factor gene localized to human chromosome 1 (1pter----1p21)

Tissue factor (tissue thromboplastin, coagulation factor III), a protein component of cell membranes, is an essential cofactor for factor VII-dependent initiation of blood coagulation. Since no tissue factor-deficient condition has been described, it is one of only a few proteins of the coagulation system for which the pattern of inheritance has not been ascertained. Because of the species-specificity of tissue factor activity and the availability of a very sensitive chromogenic assay, it was possible in the present study to use somatic cell hybrids to assign the chromosomal location of the tissue factor structural gene (F3) to human chromosome 1 (1pter----1p21).     

Inhibition of myosin light chain kinase by p21-activated kinase

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.     

The oncogenic activation of human p21ras by a novel mechanism

Single amino acid changes were introduced into normal (non-oncogenic) and activated forms of the human H-ras protein at a position (residue 116) proposed on structural grounds to represent a contact site with guanine nucleotides. Substitutions at this site could significantly reduce the ability of both forms to bind and hydrolyze guanosine 5'-triphosphate; these substitutions, however, did not necessarily diminish the transforming capacity of activated derivatives. One substitution that severely impairs these functions activated the transforming potential of the otherwise normal polypeptide.     

Nanos maintains germline stem cell self-renewal by preventing differentiation

Despite much progress in understanding how extrinsic signaling regulates stem cell self-renewal, little is known about how cell-autonomous gene regulation controls this process. In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically to produce daughter GSCs and cystoblasts, the latter of which develop into germline cysts. Here, we show that removing the translational repressor Nanos from either GSCs or their precursors, the primordial germ cells (PGCs), causes both cell types to differentiate into germline cysts. Thus, Nanos is essential for both establishing and maintaining GSCs by preventing their precocious entry into oogenesis. These functions are likely achieved by repressing the translation of differentiation factors in PGCs and GSCs.     

Est1p as a cell cycle-regulated activator of telomere-bound telomerase

In Saccharomyces cerevisiae, the telomerase components Est2p, TLC1 RNA, Est1p, and Est3p are thought to form a complex that acts late during chromosome replication (S phase) upon recruitment by Cdc13p, a telomeric DNA binding protein. Consistent with this model, we show that Est1p, Est2p, and Cdc13p are telomere-associated at this time. However, Est2p, but not Est1p, also binds telomeres before late S phase. The cdc13-2 allele has been proposed to be defective in recruitment, yet Est1p and Est2p telomere association persists in cdc13-2 cells. These findings suggest a model in which Est1p binds telomeres late in S phase and interacts with Cdc13p to convert inactive, telomere-bound Est2p to an active form.     

Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation

The mitogen-activated protein (MAP) kinase cascade is inactivated at the level of MAP kinase by members of the MAP kinase phosphatase (MKP) family, including MKP-1. MKP-1 was a labile protein in CCL39 hamster fibroblasts; its degradation was attenuated by inhibitors of the ubiquitin-directed proteasome complex. MKP-1 was a target in vivo and in vitro for p42(MAPK) or p44(MAPK), which phosphorylates MKP-1 on two carboxyl-terminal serine residues, Serine 359 and Serine 364. This phosphorylation did not modify MKP-1's intrinsic ability to dephosphorylate p44(MAPK) but led to stabilization of the protein. These results illustrate the importance of regulated protein degradation in the control of mitogenic signaling.     

利用CRISPR/Cas9技术构建永生化脐带间充质干细胞系及MYC-AS1转基因研究

人脐带间充质干细胞(hMSCs)是一种具有多向分化潜能的多能干细胞,在组织修复和某些疾病治疗的临床应用上发挥着重要作用.利用CRISPR/Cas9技术在腺相关病毒AAVS1位点插入具有磷酸甘油酸激酶PGK启动子和SV40-polyA的SV40 LT基因,使hMSCs达到永生化.并利用慢病毒构建具有抑癌作用的反义长链非编...     

Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.     

Modulation of hematopoietic stem cell homing and engraftment by CD26

Hematopoietic stem cell homing and engraftment are crucial to transplantation efficiency, and clinical engraftment is severely compromised when donor-cell numbers are limiting. The peptidase CD26 (DPPIV/dipeptidylpeptidase IV) removes dipeptides from the amino terminus of proteins. We present evidence that endogenous CD26 expression on donor cells negatively regulates homing and engraftment. By inhibition or deletion of CD26, it was possible to increase greatly the efficiency of transplantation. These results suggest that hematopoietic stem cell engraftment is not absolute, as previously suggested, and indicate that improvement of bone marrow transplant efficiency may be possible in the clinic.     

Suppression of human colorectal carcinoma cell growth by wild-type p53

Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.     

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.     

MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha

Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.     

Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling

The adult Drosophila midgut contains multipotent intestinal stem cells (ISCs) scattered along its basement membrane that have been shown by lineage analysis to generate both enterocytes and enteroendocrine cells. ISCs containing high levels of cytoplasmic Delta-rich vesicles activate the canonical Notch pathway and down-regulate Delta within their daughters, a process that programs these daughters to become enterocytes. ISCs that express little vesiculate Delta, or are genetically impaired in Notch signaling, specify their daughters to become enteroendocrine cells. Thus, ISCs control daughter cell fate by modulating Notch signaling over time. Our studies suggest that ISCs actively coordinate cell production with local tissue requirements by this mechanism.     

Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome

Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. Here, we show that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation.     

Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR

Differentiation of hepatic stellate cells (HSCs) to extracellular matrix- and growth factor-producing cells supports liver regeneration through promotion of hepatocyte proliferation. We show that the neurotrophin receptor p75NTR, a tumor necrosis factor receptor superfamily member expressed in HSCs after fibrotic and cirrhotic liver injury in humans, is a regulator of liver repair. In mice, depletion of p75NTR exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. p75NTR-/- HSCs failed to differentiate to myofibroblasts and did not support hepatocyte proliferation. Moreover, inhibition of p75NTR signaling to the small guanosine triphosphatase Rho resulted in impaired HSC differentiation. Our results identify signaling from p75NTR to Rho as a mechanism for the regulation of HSC differentiation to regeneration-promoting cells that support hepatocyte proliferation in the diseased liver.     

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.