Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming

Upon maturation, dendritic cells (DCs) acquire the unique ability to activate na?ve T cells. We used time-lapse video microscopy and two-photon imaging of intact lymph nodes to show that after establishing initial contact between their dendrites and na?ve T lymphocytes, mature DCs migrate toward the contacted lymphocytes. Subsequently, the DCs tightly entrap the T cells within a complex net of membrane extensions. The Rho family guanosine triphosphatases Rac1 and Rac2 but not Rho itself control the formation of dendrites in mature DCs, their polarized short-range migration toward T cells, and T cell priming.     

Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation

T helper 1 (TH1) cells mediate cellular immunity, whereas TH2 cells potentiate antiparasite and humoral immunity. We used a complementary DNA subtraction method, representational display analysis, to show that the small guanosine triphosphatase Rac2 is expressed selectively in murine TH1 cells. Rac induces the interferon-gamma (IFN-gamma) promoter through cooperative activation of the nuclear factor kappa B and p38 mitogen-activated protein kinase pathways. Tetracycline-regulated transgenic mice expressing constitutively active Rac2 in T cells exhibited enhanced IFN-gamma production. Dominant-negative Rac inhibited IFN-gamma production in murine T cells. Moreover, T cells from Rac2-/- mice showed decreased IFN-gamma production under TH1 conditions in vitro. Thus, Rac2 activates TH1-specific signaling and IFN-gamma gene expression.     

Little evidence for developmental plasticity of adult hematopoietic stem cells

To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.     

Stem cell depletion through epidermal deletion of Rac1

Mammalian epidermis is maintained by self-renewal of stem cells, but the underlying mechanisms are unknown. Deletion of Rac1, a Rho guanosine triphosphatase, in adult mouse epidermis stimulated stem cells to divide and undergo terminal differentiation, leading to failure to maintain the interfollicular epidermis, hair follicles, and sebaceous glands. Rac1 exerts its effects in the epidermis by negatively regulating c-Myc through p21-activated kinase 2 (PAK2) phosphorylation. We conclude that a pleiotropic regulator of cell adhesion and the cytoskeleton plays a critical role in controlling exit from the stem cell niche and propose that Rac and Myc represent a global stem cell regulatory axis.     

Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling

The Rac1 guanosine triphosphatase (GTPase) has been implicated in multiple cellular functions, including actin dynamics, proliferation, apoptosis, adhesion, and migration resulting from signaling by multiple receptors, including the B cell antigen receptor (BCR). We used conditional gene targeting to generate mice with specific Rac1 deficiency in the B cell lineage. In the absence of both Rac1 and the highly related Rac2, B cell development was almost completely blocked. Both GTPases were required to transduce BCR signals leading to proliferation, survival and up-regulation of BAFF-R, a receptor for BAFF, a key survival molecule required for B cell development and maintenance.     

Integrins regulate Rac targeting by internalization of membrane domains

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.     

Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis

The immune system develops in waves during ontogeny; it is initially populated by cells generated from fetal hematopoietic stem cells (HSCs) and later by cells derived from adult HSCs. Remarkably, the genetic programs that control these two distinct stem cell fates remain poorly understood. We report that Lin28b is specifically expressed in mouse and human fetal liver and thymus, but not in adult bone marrow or thymus. We demonstrate that ectopic expression of Lin28 reprograms hematopoietic stem/progenitor cells (HSPCs) from adult bone marrow, which endows them with the ability to mediate multilineage reconstitution that resembles fetal lymphopoiesis, including increased development of B-1a, marginal zone B, gamma/delta (γδ) T cells, and natural killer T (NKT) cells.     

Hematopoietic stem cell quiescence maintained by p21cip1/waf1

Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.     

Localized Rac activation dynamics visualized in living cells

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.     

稻瘟菌Rac1蛋白的原核表达与纯化

Ras相关的C3肉毒素底物1(Rac1)是Rho族蛋白主要成员,在稻瘟菌(Magnaporthe oryzae)的侵染致病过程中发挥重要作用.本研究目的是采用结构生物学的方法进一步探究Rac1结构与功能以及与其他蛋白互作的机制,进一步阐释其致病机制.试验以稻瘟菌的cDNA为模板,根据Ras相关的C3肉毒素底物1基因(MoRac1)序列设计特异性引物进行PCR扩增,克隆了MoRac1基因并构建原核表达载体pHAT2-Rac1,异丙基硫代半乳糖苷(IPTG)诱导表达.SDS-PAGE检测与Western blot分析表明,pHAT2-Rac1在BL21(DE3)中表达,大小为25kD.通过亲和层析、离子交换与分子筛对重组蛋白进行纯化,获得了高纯度的目的蛋白.该蛋白的表达与纯化对其结构功能的研究奠定了基础.     

哈茨木霉Rac基因的克隆及特征分析

Rac基因是普遍存在于真核生物细胞中参与环境应急反应的重要基因。本文采用RT-PCR方法从哈茨木霉中成功克隆了哈茨木霉的Rac基因的eDNA,全长718bp,推测编码204个氨基酸,蛋白分子量为22.4kD。通过数据库检索等生物信息学方法分析发现哈茨木霉Rac与羊茅香柱菌相似性高达82％。与柄篮状菌相似性也达到75％。哈茨木霉Rac基因含有GTP／GDP结合和Effeetor区。Rac基因的cDNA序列及推测的氨基酸序列在GenBank上登录（登录号分别FJ595933和ACM24223）。     

Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.     

Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment

Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell-based therapeutics.     

土荆芥腐解液对蚕豆幼苗光合特性和保卫细胞活性的影响

[目的]探讨土荆芥腐解液对蚕豆幼苗光合特性和保卫细胞活性的影响,为揭示土荆芥的入侵机制提供理论依据.[方法]采用表皮条分析法和盆栽试验,分析土荆芥腐解液作用下蚕豆幼苗株高、地上部鲜重和干重、叶面积、实际光化学效率(ΦPsⅡ)和最大光化学效率(Fv/Fm)等指标的变化情况,并检测保卫细胞的活性及细胞核异常率.[结果]土荆芥腐解液处理蚕豆幼苗的叶面积变化不明显,但地上部生物量和株高显著下降(P<0.05,下同),且具有时间和浓度依赖性;土荆芥腐解液处理蚕豆叶片的叶绿素荧光参数Fv/Fm和ΦPsⅡ均发生不同程度变化,但与对照(CK)均无显著差异(P>0.05).在土荆芥腐解液的作用下,蚕豆叶片的保卫细胞活性降低,细胞核出现错位、固缩、拉长或降解等畸变特征;随腐解液浓度的增加,保卫细胞死亡率及细胞核畸变率显著升高.[结论]土荆芥腐解液对蚕豆幼苗的光反应影响不明显,但其较强的细胞毒性能引起叶片气孔保卫细胞发生畸变,活性降低,进而导致幼苗气孔运动受阻,吸收CO2的能力下降,光合效率降低而影响幼苗生长发育.     

苜蓿皂苷对X射线照射小鼠造血系统的影响

为研究苜蓿皂苷(Alfalfa saponin)对X射线照射小鼠造血系统的影响,选取雄性SPF级昆明系小鼠90只,随机分为对照组和试验组,研究25、50和100mg/ml的苜蓿皂苷对X射线照射强度为4.0Gy小鼠造血系统的影响和不同剂量X射线对添加25mg/ml的小鼠造血系统的影响。结果表明:1)预防性给予25mg/mL苜蓿皂苷,可显著减轻4.0Gy的X射线对小鼠外周血白细胞(White blood cells,WBC)、红细胞(Red blood cells,RBC)和血小板(Platelets,PLT)数量的降低程度(P0.05),增加照射小鼠的骨髓有核细胞(Bone marrow nucleated cells,BMC)数(P0.05),显著降低微核率(P0.05)。2)预防性给予25mg/mL的苜蓿皂苷,相同照射剂量的试验组小鼠白细胞、红细胞、血小板和骨髓有核细胞数显著高于对照组(P0.05),微核率显著低于对照组(P0.05)。3)随照射强度增加,25mg/ml的苜蓿皂苷保护小鼠受辐射损伤的能力下降。综上,苜蓿皂苷可缓解X射线对小鼠造血系统和染色体的损伤,苜蓿皂苷浓度与射线强度存在量效关系,需要根据不同的照射强度添加不同浓度的苜蓿皂苷。     

拟南芥Rac/Rop GEF1基因功能研究（摘要）

[目的]对拟南芥Rac/RopGEF1（简称GEF1）基因在Rac/RopGTPse介导的生长素信号途径等方面所起的作用进行初步探索。[方法]利用实验室已构建的稳定表达的拟南芥GEF1基因启动子与GUS融合的转基因植株和GEF1基因过表达的转基因植株,通过GUS组织化学染色分析GEF1基因的表达模式,并对GEF1基因过表达植株的幼苗根系发育情况进行分析。[结果]GEF1基因主要在幼苗根的分生组织和维管组织细胞、侧根原基和根毛细胞表达;经生长素（NAA）诱导处理后,在上述细胞中的表达显著增强。过表达GEF1基因促进侧根的形成。[结论]GEF1基因的功能与根和根毛细胞的发育有关,GEF1基因可能参与对侧根发育的调控。     

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.