Natural and engineered nucleic acids as tools to explore biology

RNA and DNA molecules can form complex, three-dimensional folded structures that have surprisingly sophisticated functions, including catalysing chemical reactions and controlling gene expression. Although natural nucleic acids make occasional use of these advanced functions, the true potential for sophisticated function by these biological polymers is far greater. An important challenge for biochemists is to take RNA and DNA beyond their proven use as polymers that form double-helical structures. Molecular engineers are beginning to harness the power of nucleic acids that form more complex three-dimensional structures, and apply them as tools for exploring biological systems and as therapeutics.     

Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I

In yeast, the cortical actin cytoskeleton seems to specify sites of growth of the cell surface. Because the actin-binding protein ABP1p is associated with the cortical cytoskeleton of Saccharomyces cerevisiae, it might be involved in the spatial organization of cell surface growth. ABP1p is localized to the cortical cytoskeleton and its overproduction causes assembly of the cortical actin cytoskeleton at inappropriate sites on the cell surface, resulting in delocalized surface growth. We have now cloned and sequenced the gene encoding ABP1p. ABP1p is a novel protein with a 50 amino-acid C-terminal domain that is very similar to the SH3 domain in the non-catalytic region of nonreceptor tyrosine kinases (including those encoded by the proto-oncogenes c-src and c-abl), in phospholipase C gamma and in alpha-spectrin. We also identified an SH3-related motif in the actin-binding tail domain of myosin-I. The identification of SH3 domains in a family of otherwise unrelated proteins that associate with the membrane cytoskeleton indicates that this domain might serve to bring together signal transduction proteins and their targets or regulators, or both, in the membrane cytoskeleton.     

Generation of cat retinal ganglion cells in relation to central pathways

The ganglion cells of the cat retina form classes distinguishable in terms of perikaryal size, dendritic morphology and functional properties. Further, the axons differ in their diameters, patterns of chiasmatic crossing and in their central connections. Here we define, by 3H-thymidine autoradiography, the order of production of cells of each class and relate the order of the 'birthdates' to the known axonal pathways. The ganglion cell classes are produced in broad waves, which overlap as cells are produced first for central then for peripheral retina. Medium-sized cells are produced before the largest cells, and small ganglion cells are produced throughout the period of cell generation. This sequence of cell production relates to the orderly arrangement of axons in the optic tract, and can also be related to the rules of chiasmatic crossing observed for each ganglion cell class.     

Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mps1 and Bub1/BubR1. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubR1 and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mps1. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.     

NK细胞免疫识别及其调节机制与免疫相关性疾病

自然杀伤受体(NKR)和Toll样受体(TLR)是天然免疫系统最重要的二群天然免疫识别受体家族,位于机体抵抗外来侵袭的第一道防线.二者各自具有独特的识别外来或内源性的危险信号、区分自我和非我的识别机制,是启动固有免疫和适应性免疫应答的关键链接分子.NK细胞是天然免疫系统的核心成员,具有早期识别和清除病毒感染和肿瘤细胞等功能,同时也是连接天然免疫和获得性免疫的桥梁.以NK细胞为载体将TLRs/NKRs连接起来,可以较好地反映机体内外环境变化或刺激时固有免疫对适应性免疫的调节作用,为有效控制感染、炎症、肿瘤及自身免疫性疾病提供崭新的治疗策略.     

出芽酵母蛋白激酶TOS3的过量表达及其在热胁迫应答中的作用

出芽酵母SNF1蛋白激酶参与葡萄糖阻遏和细胞胁迫应答.TOS3可通过磷酸化激活SNF1,参与SNF1调控的信号途径.本研究利用PCR方法扩增tos3基因的蛋白质编码序列,克隆到多拷贝表达载体pYES2/NTA上构建真核表达载体,转化酵母细胞并诱导TOS3过量表达.带HIS6标签的重组TOS3蛋白通过免疫印迹得以鉴定.进一步研究了过量表达TOS3对细胞热胁迫耐受性的影响,发现在热胁迫处理条件下,TOS3过量表达可恢复△snf1突变体细胞的生长缺陷,表明TOS3可能通过不依赖SNF1的其他信号途径参与细胞对热胁迫应答的调控.     

Alloantigen-specific suppressor t cells can also suppress the in vivo immune response to unrelated alloantigens

Delayed-type hypersensitivity (DTH) to both major histocompatibility complex (H-2) and non-H-2-coded antigens can be induced by subcutaneous immunization with allogeneic lymphoid cells in the mouse. While subcutaneous immunization with allogeneic cells preferentially induces DTH reactivity, intravenous immunization, especially with irradiated allogeneic cells, induces a state of suppression. Suppression is manifest both in direct host-versus-graft (HvG) assays and under graft-versus-host (GvH) conditions, where spleen cells of suppressed mice are used to reconstitute irradiated allogeneic hosts. The suppression is mediated by T cells. We have now studied the specificity of the suppressive effect by subcutaneous immunization of 'suppressed' mice with a combination of alloantigens comprising the antigen(s) used to induce the suppressor T cells as well as unrelated alloantigens. We report here that reaction against the third party alloantigens was effectively suppressed, provided these antigens were presented in combination with the antigen(s) that had induced the suppressor T cells. Both sets of alloantigens do not need to be physically associated.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.     

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.     

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.     

Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta

Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis and tumour development. Among the PKC isotypes, PKC-delta is unique in that its overexpression results in inhibition of cell growth. Here we show that mice that lack PKC-delta exhibit expansion of the B-lymphocyte population with the formation of numerous germinal centres in the absence of stimulation. The rate of proliferation in response to stimulation was greater for B cells from PKC-delta-deficient mice than for those from wild-type mice. Adoptive transfer experiments suggested that the hyperproliferation phenotype is B-cell autonomous. Production of interleukin-6 was markedly increased in B cells of PKC-delta-null mice as a result of an increase in the DNA-binding activity of NF-IL6. Furthermore, the PKC-delta-deficient mice contain circulating autoreactive antibodies and display immune-complex-type glomerulonephritis, as well as lymphocyte infiltration in many organs. These results suggest that PKC-delta has an indispensable function in negative regulation of B-cell proliferation, and is particularly important for the establishment of B-cell tolerance.