Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.     

A mutant protein kinase C that can transform fibroblasts

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.     

BABL/c突变卷毛小鼠免疫学特性指标的分析

目的 探讨BALB/c突变卷毛小鼠与BALB/c小鼠之间的免疫学指标是否存在差异。方法 选择6周龄的BALB/c突变卷毛小鼠和BALB/c小鼠各20只(雌雄各半),分别使用全自动血细胞分析仪检测6项血液免疫细胞指标;使用全自动生化仪检测4项抗体和补体指标;ELISA法检测血清细胞因子TNF-α、IL-2、IL-4水平;并对两组结果进行对比分析。结果 两组小鼠NE、NE#和MO的雌性及组间结果相比较具有显著性差异(P<0.05,P<0.01),两组小鼠LY的雌性结果相比较具有显著性差异(P<0.05);两组小鼠IgM的性别及组间结果相比具有显著性差异(P<0.05,P<0.01),两组小鼠的IgG和C3的雄性及组间结果相比较具有显著性差异(P<0.01),两组小鼠IL-4的雌性及组间结果相比较具有显著性差异(P<0.05)。结论 BALB/c突变卷毛小鼠和BALB/c小鼠的血液免疫学指标存在差异,提示突变基因可能对小鼠的免疫系统产生了一定的影响。     

Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.     

The class I major histocompatibility antigen gene activated in a line of SV40-transformed mouse cells is H-2Dd, not Qa/Tla

We have previously described several complementary DNA clones isolated because they correspond to messenger RNAs present at higher levels in the simian virus 40 (SV40)-transformed BALB/c 3T3 cell line SV3T3 Cl38 than in the normal, parental BALB/c 3T3 line. One of these clones, pAG64, hybridizes to RNAs which, while present in BALB/c 3T3 cells, are 10-20-fold more abundant in SV3T3 Cl38 and are found at high levels in a wide variety of transformed cell lines. Nucleotide sequence analysis showed that pAG64 encodes a class I antigen of the major histocompatibility complex. To ascertain the identity of pAG64, we compared its sequence with the available sequences of d haplotype class I antigen genes [K locus, L locus, D locus and the Qa gene defined by genomic clone 27.1] and found that it showed multiple clustered differences from each of these sequences. We therefore concluded that it was not derived from the H-2Kd, H-2Ld or H-2Dd genes and thus must correspond to one of the other class I antigen genes, namely those of the Qa/Tla complex, although it was clearly not the Qa gene defined by the genomic clone 27.1. We now report subsequent findings which indicate that pAG64 in fact corresponds to the H-2Dd gene and not to a Qa/Tla gene.     

PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.     

T cells with receptors for IgD

The role of IgD in the immune response has been elusive, although its predominance on the cell surface suggests a receptor function. We have shown previously that euthymic but not athymic BALB/c mice, injected with IgD before antigen, exhibit enhanced antibody responses which can be transferred by T cells. Isotype-specific T cells have been reported to have both upward and downward immunoregulatory effects. Here we demonstrate the existence of T cells with receptors for IgD, and show that exposure to IgD in vivo or in vitro significantly increases the number of T delta cells in the spleen and lymph nodes but not in the thymus. The kinetics of T delta-cell appearance in vivo parallels that of the immunoenhancing effect which occurs after injection of IgD. These T delta cells are of the Lyt 1+2- T-cell phenotype.     

De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii

Toxoplasma gondii is a ubiquitous protozoan parasite that is responsible for severe congenital birth defects and fatal toxoplasmic encephalitis in immunocompromized people. Fundamental aspects of obligate intracellular replication and pathogenesis are only now beginning to emerge for protozoan parasites. T. gondii has a fragmented pathway for salvaging pyrimidine nucleobases derived from the parasite or host cell, and this limited pyrimidine salvage capacity is funnelled exclusively through uracil phosphoribosyltransferase. Disrupting the function of this enzyme does not affect the growth of T. gondii tachyzoites, which suggests that the de novo pyrimidine biosynthesis pathway may be necessary for growth. We have examined the virulence of T. gondii mutants that lack carbamoyl phosphate synthetase II (uracil auxotrophs) to determine whether de novo pyrimidine biosynthesis is required in vivo. Here we show that T. gondii uracil auxotrophs are completely avirulent not only in immune-competent BALB/c mice but also in mice that lack interferon-gamma. A single injection of the uracil auxotroph into BALB/c mice induces long-term protective immunity to toxoplasmosis. Our findings indicate the significance of the de novo pyrimidine biosynthesis pathway for the virulence of parasitic protozoa, and suggest routes for developing vaccines and chemotherapy.     

Antisense oligodeoxynucleotides to GS protein alpha-subunit sequence accelerate differentiation of fibroblasts to adipocytes.

Fully-differentiated mouse 3T3-L1 fibroblasts accumulate large amounts of lipid at 7-10 days after induction by insulin or by dexamethasone and a methyl xanthine. G proteins mediate transmembrane signalling from a diverse group of cell-surface receptors to effector units that include phospholipase C, adenylyl cyclase and ion channels. They are also targets of regulation themselves. 3T3-L1 fibroblasts display marked changes in levels of G protein when induced to differentiate to adipocytes. Here we show that cholera toxin, which ADP-ribosylates and activates the G protein subunit Gs alpha, blocks the induction of differentiation, whereas increasing intracellular cyclic AMP directly with the dibutyryl analogue or indirectly with pertussis toxin or forskolin does not affect differentiation. Oligodeoxynucleotides antisense to the sequence encoding Gs alpha accelerate differentiation markedly. The time course of adipogenesis declined from 7-10 days in controls to roughly 3 days in cultures treated with antisense-Gs alpha oligodeoxynucleotides, whereas oligodeoxynucleotides, antisense to Gi alpha 1, Gi alpha 3, and sense and missense to Gs alpha, had no such effect. Antisense-Gs alpha alone induced differentiation by day 7, indicating that Gs alpha activity modulates differentiation in 3T3-L1 cells, acting in a new role which is independent of increased intracellular cAMP.     

芦荟的毒性研究

用体外细胞培养的方法分别研究了芦荟叶皮、芦荟凝胶和芦荟全叶的提取物对HepG2细胞和BALB/c3T3细胞的毒性作用,结果表明芦荟提取物对受试细胞没有产生毒性作用,由此可知芦荟产品是安全的.     

T cells can distinguish between allogeneic major histocompatibility complex products on different cell types

In the response of T cells to foreign antigens, the ligand for the T cell alpha/beta receptor is presented on a cell surface as a fragment of antigen complexed to one of the membrane molecules encoded in the major histocompatibility complex (MHC). The receptor apparently interacts via its variable elements (V beta, D beta, J beta, V alpha and J alpha) with residues within both the antigen and MHC portion of the ligand. The frequency of T cells responding to a conventional antigen plus self MHC is usually quite low, presumably reflecting the relative rarity of receptors with the particular combination of variable elements to match the antigen/MHC ligand. T cells also respond to allogeneic forms of MHC molecules in the absence of added antigen. In this case the frequency of responding T cells is very high. One hypothesis to explain this observation is that, in the absence of foreign antigen, MHC molecules are complexed to a large array of peptides derived from self-proteins. In this case the combination of the polymorphic MHC amino acid residues and many different self peptides presents so many possible ligands that the likelihood of recognition by a given T cell receptor is quite high. The recent crystallography experiments which revealed a dramatic binding cleft on the face of a human MHC molecule have given impetus to this view, but as yet there is no direct supporting evidence. We have recently described a close association between murine T cell receptors utilizing the V beta 17a element and reactivity to various allogeneic forms of the murine MHC molecule, I-E (ref. 8). In this paper, we show that this I-E ligand is detected on B cells, but not on I-E+ macrophages or fibroblasts expressing a transfected I-E gene. These results strongly suggest a B cell specific product combines with I-E to form the allogeneic ligand for V beta 17a+ receptors and thus support the concept of alloreactivity described above.     

The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.     

Regulations of thyroid hormone on cardiac protein kinase C signal pathway in vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin II (Ang II), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKC_α and PKC_ε expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKC_ε expression in cardiac myocytes. The AngII pretreatment led to an increase of PKC activity and PKC_ε expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKC_ε expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang II pretreatment, T3 could inhibit PKC activity and PKC_ε expression in cardiac myocytes. Foundation item: Supported by the Natural Science Foundation of Hubei Province (98091) Biography: WANG Bao-hua (1974-), female, Ph. D, research direction: cardiovascular pathophysiology.     

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules.

The alpha 1 and alpha 2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the alpha 3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CD8. CD8 is important in the selection of T cells as anti-CD8 antibody injected into perinatal mice interferes with this process. We previously used a hybrid class I molecule with the alpha 1/alpha 2 domains from Ld and the alpha 3 domain from Q7b and showed that this molecule binds an Ld-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-Ld cytotoxic T lymphocytes. In addition, positive selection of virus-specific Ld-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the alpha 1/alpha 2 domains of class I, the alpha 3 domain plays an important part in both positive and negative selection of antigen-specific cells.     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling.

The colony-stimulating factor-1 receptor (CSF-1R) mediates its pleiotropic effects through the coupling of its ligand-activated tyrosine kinase to multiple intracellular effector proteins, whose combined actions determine the magnitude and specificity of the biological response. The interaction of cytoplasmic signalling molecules with CSF-1R is mediated in part by sequence motifs flanking sites of receptor tyrosine phosphorylation. Mutation of an autophosphorylation site at tyrosine 809 in the cytoplasmic domain of human CSF-1R does not significantly reduce its ligand-stimulated tyrosine kinase activity, binding to phosphatidylinositol 3-kinase, or induction of the immediate early response genes, c-fos and junB (ref.2). Unlike cells bearing wild-type receptors, mouse NIH3T3 cells expressing mutant CSF-1R(Phe 809) were unable to grow in serum-free medium containing human recombinant CSF-1 and did not form colonies in semi-solid medium in its presence. CSF-1 induction of c-myc messenger RNA in these cells was impaired, but enforced expression of an exogenous c-myc gene restored their ability to proliferate in response to the growth factor. These studies demonstrate a receptor-mediated bifurcation of intracellular signal transduction pathways during the immediate early response and assign a central role for c-myc in CSF-1-induced mitogenesis.     

Suppression of c-ras transformation by GTPase-activating protein

The ras genes are required for normal cell growth and mediate transformation by oncogenes encoding protein tyrosine kinases. Normal ras can transform cells in vitro and in vivo, but mutationally activated ras does so much more efficiently, and highly transforming mutant versions of ras have been isolated from a variety of human and animal tumours. The ras genes encode membrane-associated, guanine nucleotide-binding proteins that are active when GTP is bound and inactive when GDP is bound. The slow intrinsic GTPase activity of normal mammalian Ras proteins can be greatly accelerated by the GTPase-activating protein (GAP), which is predominantly cytoplasmic. This activity of GAP, which can increase with cell density in contact-inhibited cells, suggests that it functions as a negative, upstream regulator of ras. Other studies, however, show that GAP interacts with a region of ras-encoded protein implicated in ras effector function, which raises the possibility that GAP might also be a downstream target of ras. Mutationally activated ras-encoded proteins also interact with GAP, although they are resistant to its catalytic activity. In an attempt to define the role of GAP in ras-mediated transformation, we examined the effects on transformation of normal or mutant ras when cells overexpress GAP. We found that GAP suppresses transformation of NIH 3T3 cells by normal Ha-ras (c-ras) but does not inhibit transformation by activated Ha-ras (v-ras). These results support the hypothesis that GAP functions as a negative regulator of normal ras and make it unlikely that GAP alone is the ras target.     

电化学法检测细胞中的胸腺嘧啶和胞嘧啶

用基于氧化石墨烯量子点/多壁碳纳米管/丝网印刷电极（GOQDs/MWCNTs/SPCE*）的电化学检测方法同时检测小鼠胚胎成纤维（BALB/c 3T3）细胞中的胸腺嘧啶和胞嘧啶， 并考察富集电位、 富集时间、 pH值对胸腺嘧啶和胞嘧啶标准品电化学行为的影响. 实验结果表明： 最佳检测条件为富集电位0， 富集时间150 s，  pH=7.4; 电极对胸腺嘧啶和胞嘧啶的最低检测限分别为0.69，0.95 μmol/L；  该方法可同时灵敏检测BALB/c 3T3细胞中的胸腺嘧啶和胞嘧啶.     

BALB／c突变无毛小鼠离体鼠皮透皮吸收的研究

本文用甲硝唑作为模型药物,Ａｚｏｎｅ为透皮吸收促进剂,对ＢＡＬＢ／ｃ突变无毛小鼠的离体皮肤的透皮吸收情况进行了初步研究,并与国内在透皮吸收研究常用的昆明小鼠的离体鼠皮进行了初步比较,为无毛小鼠在皮肤病学研究中的应用提供一些依据,实验结果表明,ＢＡＬＢ／ｃ突变无毛小鼠离体鼠对甲硝唑的透皮吸收明显低于昆明小鼠,而离散程度优于昆明小鼠。