Regulation of human granulocyte function by products derived from murine tumors

Murine EL-4 thymoma was found to produce a factor that activated antibody-dependent cell-mediated cytotoxicity by human eosinophils and neutrophils. The eosinophil-activating factor had some properties similar to human eosinophil colony-stimulating factor in being heat stable, inactivated by 2-mercaptoethanol, and cochromatographing by gel filtration with a factor that promoted the growth of pure eosinophil colonies from human bone marrow. The neutrophil-activating factor from EL-4 was also heat stable but was not inactivated by 2-mercaptoethanol and was found in two peaks on chromatography, neither having human neutrophil-CSF activity but one cochromatographing with mouse granulocyte-macrophage CSF. EL-4 therefore secretes factors that activate two types of human granulocytes. Several other murine thymomas and macrophage cell lines produced a neutrophil-activating factor, the production of which could not be correlated with that of several other known lymphokines. Murine tumors produce factors that activate human granulocytes.     

Molecular regulation of phospholamban function and expression

Intracellular levels of cAMP regulated by the β-adrenergic actions of catecholamines play a key in the metabolic, electrical, and mechanical performance of the cardiac muscles. Among a number of biological actions of cAMP, the excitation–contraction coupling process in cardiac myocytes is markedly affected by cAMP through its stimulatory effect on cAMP-dependent protein kinase. Phospholamban, which is expressed in the sarcoplasmic reticulum of cardiac, slow-twitch skeletal, and smooth muscles, is one of the substrates for cAMP-dependent protein kinase. Phospholamban regulates the activity of Ca ATPase in the sarcoplasmic reticulum membranes in a manner dependent on the phosphorylation state of cAMP-dependent protein kinase, thereby changing the mechanical performance of the cardiac muscles. This Ca regulatory mechanism of phospholamban-Ca ATPase system is mediated by a direct protein–protein interaction between two proteins. This review focuses on recent advances in understanding the role of phospholamban molecule in the regulation of Ca transport by cardiac muscle sarcoplasmic reticulum.     

Dynamics of erythropoietin receptor expression on erythropoietin-responsive murine cell lines

We examined erythropoietin receptor expression in two murine cell lines, B6SUtA and DA-1, that respond to erythropoietin in different ways. While B6SUtA cells undergo erythroid differentiation with limited proliferation after addition of erythropoietin, DA-1 cells show only a proliferative response. Equilibrium binding experiments with 125I-erythropoietin revealed that both B6SUtA and DA-1 cells express a single class of erythropoietin receptors. In the absence of erythropoietin, B6SUtA cells exhibited 145 receptors per cell with a dissociation constant (kd) of 380 pmol/L. Six days after induction with erythropoietin, the B6SUtA cells displayed 310 receptors per cell without a change in binding affinity; exposure to erythropoietin also increased cellular hemoglobin content. DA-1 cells adapted to erythropoietin-dependent growth over a period of months and exhibited a progressive increase in erythropoietin receptor expression, from 85 per cell (kd = 540) to 550 per cell (kd = 530), although the cells remained uniformly benzidine-negative. We interpret the data with B6SUtA cells to indicate that early erythroid differentiation stages are attended by an increase in erythropoietin receptor display, coordinate with the initiation of expression of erythroid-specific genes. In contrast, the results with DA-1 cells are most compatible with clonal selection as the mechanism underlying enhanced receptor expression. Thus, display of the erythropoietin receptor is dynamic and can be modulated during the course of erythropoietin-induced differentiation.     

血管内皮生长因子在Ⅱ型胶原诱导的关节炎小鼠关节组织中的表达

目的 研究血管生成因子－血管内皮生长因子（VEGF）在Ⅱ型胶原诱导的关节炎（CIA）形成期的表达及功能。方法 于DBA／1J小鼠皮下注射Ⅱ型胶原制作关节炎模型并进行关节指数评价。用酶联免疫吸附法（ELISA）及免疫组织化学技术,检测关节组织内VEGF和vWF含量,以及通过RT－PCR,Southern blotting技术检测VEGF mRNA表达。结果 VEGF与vWF呈平行变化关系,均在关节炎发生后第4天达到最高,并与血管新生程度、关节炎严重程度呈正相关。VEGF mRNA在关节组织内表达形式为279、304bp。结论 VEGF在关节炎形成早期起着重要作用,影响着实验诱导关节炎血管新生及进展。     

Structure, expression, and regulation of the murine renin genes

It has long been known that the renin-angiotensin system plays an integral role in the regulation of blood pressure and electrolyte and fluid balance in mammals. The advent of molecular biologic techniques has afforded new insights into the genes regulating blood pressure. Laboratory mice and rats have been used as experimental models to examine the structural organization and expression of the renin gene. It is now well established that some mice, unlike rats and humans, contain a duplicated copy of the renin locus, which accounts for the high level of renin activity long known to be found in the submandibular gland of some mice. Indeed it is this fortuitous observation that facilitated the isolation of the first complementary DNA clones for renin and ultimately the many species-specific probes now available to analyze mammalian tissues for evidence of primary renin expression. The use of complementary DNAs as probes for primary renin expression helped confirm and further clarify earlier studies demonstrating the presence of renin activity in a number of extrarenal tissues. Although expression in some of these tissues is evolutionarily conserved, their significance has still been elusive. In this report we review the impact of molecular biology on our current understanding of renin gene structure and organization, tissue- and cell-specific expression and regulation, and the changes in renin expression throughout ontogeny. In addition, we describe how new developments in gene transfer technology have added important tools to our arsenal for examining renin gene regulation and how these technologies can be used to develop new tools for renin and hypertension research.     

氧化苦参碱对小鼠树突状细胞成熟和功能的影响

目的:研究氧化苦参碱(OXY)对小鼠树突状细胞(DC)成熟、表型及功能的影响。方法:流式细胞术检测DC表面分子CD40的表达;混合淋巴细胞反应(MLR)检测DC对T淋巴细胞的刺激能力;ELISA法检测MLR上清中细胞因子IFN-γ的分泌。结果:第0天OXY处理组较对照组DC表面分子CD40的表达明显升高(P<0.01),刺激T细胞能力增强,分泌细胞因子IFN-γ明显升高(P<0.05),对LPS诱导的DC成熟,与DC LPS组对照显著升高(P<0.05)。结果:OXY对DC的成熟和功能有一定的促进作用。     

Constitutive expression and function of cyclooxygenase-2 in murine gastric muscles

BACKGROUND & AIMS: Cyclooxygenase enzymes (COX) generate intermediates in the prostaglandin (PG) cascade. COX-1 is constitutively expressed in many cells, and COX-2 is typically thought to be an inducible isoform. METHODS: We evaluated constitutive expression and function of COX-2 in murine gastric muscles. RESULTS: Immunohistochemistry showed COX-2-like immunoreactivity (COX-2-LI) in myenteric neurons. Half the neurons with COX-2-LI expressed nitric oxide synthase (NOS). COX-2-LI was not observed in smooth muscle cells. Interstitial cells of Cajal within muscle layers (IC-IM) expressed COX-2-LI, suggesting a novel role for IC-IM. Molecular studies verified expression of COX-2 in gastric muscles. Quantitative polymerase chain reaction (PCR) showed equal expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased antral phasic contractions and potentiated responses to ACh. Indomethacin, but not GR253035X, increased contractions and potentiated responses in tissues of COX-2 knockout mice. Indomethacin and GR253035X reduced tone in the fundus. CONCLUSIONS: COX-2 is constitutively expressed by IC-IM and neurons in the stomach and at levels similar to COX-1. Prostanoids produced by COX-2 regulate mechanical activities of fundus and antral muscles.     

H-2-linked regulation of xenotropic murine leukemia virus expression.

A high proportion of lymphocytes from F/St mice produce infectious xenotropic murine leukemia virus (X-MuLV) and express high levels of cell surface antigens, termed XenCSA, related to the major glycoprotein of X-MuLV. In crosses of F/St with AKR, the high-virus phenotype of F/St was found to be recessive and was shown to be governed by a single locus, Cxv-1, less than 2 centimorgans from H-2K. The close association of Cxv-1 with the H-2 complex was confirmed by the observation that B10.F mice, congeneic for the H-2 region of F/St, expressed high levels of infectious X-MuLV and XenCSA, whereas C57BL/10 mice and other C57BL/10 H-2 congeneic strains did not. Studies of hybrid mice homozygous for Cxv-1s, but segregating for a chromosome 1 X-MuLV induction locus (V locus) of F/St, demonstrated that the high-virus phenotype of F/St was dependent on the interaction between Cxv-1 and the chromosome 1 V locus.     

MAdCAM-1 expression and regulation in murine colonic endothelial cells in vitro

BACKGROUND: Although the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is associated with the etiology of inflammatory bowel diseases, few studies have directly examined MAdCAM-1 using microvascular endothelium derived from the colon. This study measured the expression of MAdCAM-1 in a novel colon endothelial line MJC-1, as well as MAdCAM-1 regulation and function in vitro. METHODS: We cloned microvascular endothelial cells from primary colon cultures using ImmortoMice mice (whose cells express a temperature-sensitive SV40 large T antigen, H-2Kb-tsA58 mice). Expression of MAdCAM-1 after stimulation with cytokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, or interferon (IFN)-gamma] was determined by Western blotting. Signal paths regulating MAdCAM-1 expression were examined using pharmacological blockers before cytokines. We also examined lymphocyte adhesion using lymphocytes that constitutively express alpha4beta7 integrin. RESULTS: TNF-alpha induced MAdCAM-1 in a dose-dependent manner by 24 hours. MAdCAM-1 induction was protein kinase C, tyrosine kinase, p38 mitogen activated protein kinase, and nuclear-factor kappa-B/poly adenosine diphosphate ribose polymerase dependent. Lymphocyte adhesion was increased 2.6-fold after TNF-alpha stimulation and was inhibited by anti-MAdCAM-1 antibody before treatment (P < 0.05 control versus TNF-alpha). CONCLUSIONS: In vitro, MAdCAM-1 can be induced on colon endothelial cells by TNF-alpha stimulation and may represent a useful model to study microvascular injury in the large intestine.     

Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog

Enterotoxigenic Bacteroides fragilis is the most frequent disease-causing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of fragilysin-3 and found that its activity is repressed by a ～170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.     

CD1d expression on and regulation of murine hematopoietic stem and progenitor cells

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d(-/-) mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d(-/-), BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.     

Specific regulation of c-myc oncogene expression in a murine B-cell lymphoma.

The c-myc oncogene has been implicated in a wide spectrum of B-cell neoplasias. In normal cells, the level of expression of the c-myc gene correlates with growth status. In the present study, we examined the effect of receptor-mediated inhibition of growth on c-myc expression in a B-cell lymphoma. The murine lymphoma line WEHI 231 has been characterized as an early B cell; it bears surface-bound IgM and has unrearranged c-myc genes. Following treatment of a WEHI 231 culture with anti-mouse Ig antiserum, the cells undergo one round of division and further proliferation is inhibited. We observed that this treatment specifically affected cytoplasmic levels of c-myc mRNA. An initial early increase is followed by a precipitous drop such that by 4 hr (after exposure) the amount of c-myc mRNA is below control values by a factor of approximately equal to 10. The drop in c-myc precedes cessation of DNA synthesis. During the 2- to 4-hr period, c-myc mRNA had a maximal half-life of between 20 and 30 min. In contrast, even 24 hr after anti-Ig exposure, the amounts of most major mRNAs, including mu heavy chain and actin, were not significantly altered. These results indicate that expression of an unrearranged c-myc gene can be selectively responsive to receptor-mediated regulatory events.     

冠心病患者外周血单个核细胞源树突状细胞基因表达差异的研究

目的:体外培养冠心病患者外周血单个核细胞来源的树突状细胞(dendritic cells,DCs),并利用基因芯片技术对冠心病患者外周血培养的DC功能基因表达谱进行研究。方法:分别配对筛选急性冠状动脉综合征患者(ACS)及冠状动脉正常的胸痛综合征患者各5例,将人外周血分离的单个核细胞加入包含rhGM-CSF(20μg/L)和rhIL-4(20μg/L)的培养基中培养,使其分化为DCs。再用基因芯片技术检测DC特异性功能蛋白基因表达的变化。结果:ACS组功能蛋白表达基因中有5个基因呈明显上升,分别是G1P2、G1P3、IFIT4、IL-1β和MX1,这些都是高度相关的干扰素诱导蛋白基因。有17个基因呈明显下降表达,分别是ACPP、AIM2、ATM、CCR1、CCR5、CD1C、FCGR3A、IFI16、IL-16、IL-18、LY75、MAP4K3、TAP1、TAP2、TLR1、TNFRSF6和VCL等,分别属于抗原识别受体、细胞趋化因子受体、细胞因子和细胞内信号传导系统。结论:冠心病患者外周血单个核细胞来源的DC不但在形态和表型上存在明显差异,而且在功能蛋白表达方面也存在显著变化。     

Partial chemical characterization of Ia antigens derived from murine thymocytes.

Previous chemical studies attempting to demonstrate Ia antigens on mouse thymocytes have given contradictory results. We attempted to resolve the question of whether Ia antigens exist on thymocytes (defined as thymus cells that bear a T cell marker) by isolating strain C3H thymocytes free of other contaminating cells using the fluorescence-activated cell sorter, and then chemically testing the purified populations for Ia antigens. Immunoglobulin-negative thymus cells and thymus cells selected with a rabbit antiserum to mouse brain were the two populations of thymocytes labeled with [3H]leucine after sorting. Radiolabeled proteins were solubilized with the non-ionic detergent Nonidet P-40, reacted with anti-Ia antiserum, and analyzed by electrophoresis on discontinuous sodium dodecyl sulfate/polyacrylamide gels. Ia antigens were recovered from both cell populations. These antigens were synthesized by thymocytes and were found on molecules composed of two chains of molecular weight 33,000 and 25,000, respectively, similar to Ia antigens derived from spleen cells. Assuming that all thymocytes bear similar amounts of Ia antigens, we estimated that they have approximately 1/50 the amount of Ia antigens that spleen cells do.