Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

mmLDL通过p38 MAPK通路上调小鼠肠系膜动脉ET受体

目的:探讨弱氧化修饰低密度脂蛋白(mm LDL)是否通过激活p38 MAPK炎症通路上调小鼠肠系膜动脉内皮素(ET)A型(ETA)和B型(ETB)受体。方法:将昆明小鼠分为正常对照组(尾静脉注射生理盐水)、mm LDL组(尾静脉注射mm LDL)、LDL组(尾静脉注射LDL)、mm LDL+SB 203580组(尾静脉注射mm LDL及腹腔注射p38 MAPK抑制剂SB 203580)和mm LDL+DMSO组(尾静脉注射mm LDL及腹腔注射DMSO)。微血管张力描记仪记录ETB受体激动剂角蝰毒素6c和ET-1引起肠系膜动脉收缩的量效曲线;RT-q PCR检测ETB受体、ETA受体和白细胞介素(IL-6)的m RNA表达;ELISA检测血清IL-6的水平;Western blot检测ETB受体、ETA受体、IL-6、p38 MAPK、p-p38MAPK、NF-κB和p-NF-κB的蛋白水平。结果:mm LDL引起ETB受体和ETA受体介导的血管收缩反应显著增强(P<0.01),ETB受体、ETA受体和IL-6的m RNA和蛋白表达显著增加(P<0.01),p-p38 MAPK和p-NF-κB蛋白水平显著升高(P<0.01),血清中IL-6水平显著升高(P<0.01);腹腔注射SB 203580抑制了mm LDL的作用。mm LDL引起的IL-6血清浓度升高分别与ETB受体和ETA受体介导的最大收缩率呈正相关。结论:mm LDL通过激活p38 MAPK通路及下游NF-κB转录因子,提高炎症因子IL-6血清水平,增加小鼠肠系膜动脉IL-6、ETA受体和ETB受体表达,增强ETA受体和ETB受体介导的血管收缩功能。     

A chimeric T cell receptor with super-signaling properties

A key question yet to be resolved concerns the structure and function relationship of the TCR complex. How does antigen recognition by the TCR-alphabeta chains result in the activation of distinct signal transduction pathways by the CD3-gammadeltaepsilon/zeta complex? To investigate which part of the TCR-beta chain is involved in TCR signaling, we exchanged different domains of the constant regions of the TCR-beta chain with the corresponding TCR-gamma chain domains. We show here that hybridoma cells expressing a chimeric TCR-beta chain (betaIII) containing intracellular and transmembrane TCR-gamma amino acids, together with a wild-type TCR-alpha (alphawt) chain, were 10 times more sensitive to antigenic stimulation compared to cells expressing TCR-alphawt/betawt chains. This super-signaling phenotype of the betaIII chain was observed in two different TCRs. One specific for an alloantigen (I-A(bm12)) and one for an autoantigen (I-A(b)/MOG(35-55)). We found that this chimeric alphawt/betaIII TCR had normal association with CD3-gammadeltaepsilon and zeta chains. To investigate the effect of the chimeric betaIII chain in transgenic T cells, we made MOG(35-55)-specific TCR transgenic mice expressing either the alphawt/betawt or chimeric alphawt/betaIII TCR. Similar to what was observed in hybridoma cells, transgenic alphawt/betaIII T cells showed a super-signaling phenotype upon antigenic stimulation. Further studies may help us understand the effect of increased TCR signaling on autoimmunity and may lead to the identification of signaling molecules that can be targeted to stop the progression of autoimmune disorders such as multiple sclerosis.     

Avoidance conditioning in rats following changes in activity of the noradrenergic and cholinergic systems of the brain

Bilateral coagulation of the ventral noradrenergic pathways of the brain in male rats of the Wistar strain disrupted the conditioned passive avoidance response after unconditioned stimulation with a current of 0.75 mA. Injecting 10 mg/kg galantamine into these rats 20 min before training and increasing the unconditioned stimulus to 3 mA improved subsequent avoidance responses. Injecting galantamine under analogous conditions into normal rats impaired this reaction. Disruption of the conditioned avoidance response following the operation may be due to a change in the intracentral interrelation of the noradrenergic and cholinergic systems of the brain.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 68, No. 3, pp. 297–302, March, 1982.     

Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength

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紫草素通过PI3K/Akt通路抑制氧糖剥夺诱导的大鼠原代皮层神经元凋亡

目的:探讨紫草素对氧糖剥夺(OGD)损伤模型中大鼠原代皮层神经元的作用及机制。方法:用不同浓度(0. 02、0. 2、2和20μmol/L)紫草素对大鼠原代皮层神经元经进行预处理,再经OGD损伤处理,用乳酸脱氢酶(LDH)释放法和荧光素二乙酸酯/碘化丙啶(FDA/PI)双染法分别检测神经元活性和凋亡情况,选择最适紫草素浓度。然后,在加入紫草素之前提前加入LY294002(PI3K/Akt信号通路抑制剂,1μmol/L),用Wesern blot法检测神经元p-Akt(Ser473)水平变化,用LDH法和FDA/PI双染法检测神经元活性和凋亡率变化。结果:0. 2、2及20μmol/L的紫草素可显著提高神经元存活率(P 0. 05),同时还可使神经元内p-Akt(Ser473)水平显著升高(P 0. 05); LY294002可显著阻断紫草素对神经元p-Akt(Ser473)水平和凋亡率的影响(P 0. 05)。结论:紫草素可通过激活PI3K/Akt通路来减少OGD诱导的大鼠原代皮层神经元凋亡。     

GABAergic projections from the thalamic reticular nucleus to the anteroventral and anterodorsal thalamic nuclei of the rat

We have studied GABAergic projections from the thalamic reticular nucleus to the anterior thalamic nuclei of the rat by combining retrograde labelling with horseradish peroxidase and GABA-immunohistochentistry. Small iontophoretic injections of the tracer into subnuclei of the anterior thalamic nuclear complex resulted in retrograde labelling of cells in the rostrodorsal pole of the ipsilateral thalamic reticular nucleus. All of these cells were also GABA-positive. The projections were topographically organized. Neurons located in the most dorsal part of the rostral reticular nucleus projected to the dorsal half of both the posterior subdivision and the medial subdivision of the anteroventral thalamic nucleus, and to the rostral portion of the anterodorsal thalamic nucleus. Immediately ventral to this group of neurons, but still within the dorsal portion of the reticular nucleus, a second group of neurons, extending from the dorsolateral to the dorsomedial edge of the nucleus, projected to the ventral parts of the posterior and medial subdivisions of the anteroventral nucleus. Following injection of tracer into the dorsal part of the rostral anteroventral nucleus, retrograde labelled GABA-containing cell bodies were also found in the ipsilateral anterodorsal nucleus.     

脑缺血后TLR4作用与小胶质细胞的激活

脑缺血性损伤早期小胶质细胞即被激活。激活的小胶质细胞既有细胞毒性又有神经营养作用。小胶质细胞行使免疫功能的信号转导受体之一是TLR4（toll-like receptor 4）。TLR4在脑内主要表达在小胶质细胞，是一种模式识别受体（pattern recognition receptor，PRR）, 识别一些外源性和内源性的配体。最近的研究表明，TLR4信号通路在脑缺血再灌注损伤中起重要作用。TLR4通过激活小胶质细胞，大量表达炎症因子，加重脑缺血性损伤。     

Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness

The CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) α^δ+ and γ^λ+ cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR αβ-expressing IEL subsets such as CD4⁺8^? and CD4^?8α⁺β⁺ cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR αβ cells (including CD4^?8α⁺β^? and CD4⁺8αβ⁺β cells) as well as TcR γ^λ+ IEL fail to express CD28. Parallel experiments using aged BALB/c-nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28^? cells are considerably larger than CD28⁺ cells in the gut, although cycling cells were rare in both subsets. Finally CD28^? cells in the gut did not proliferate or produce IL-2 upon stimulation by anti-CD3 monoclonal antibodies (mAb) and phorbol 12-myristate 13-acetate, whereas CD28⁺ cells in the gut and lymph nodes responded to these stimuli. The response of the CD28⁺ cells was enhanced by anti-CD28 mAb. These results suggest that CD28^? IEL (CD4-8α⁺β^? cells, and some CD4⁺8α⁺β^?cells) may follow a different developmental pathway from that of CD28⁺ IEL in a thymus-independent environment, and that expression of CD28 correlates with responsiveness of the cells to triggering via the TcR-CD3 complex.