表皮生长因子对正常细胞与转化细胞染色质蛋白激酶活性的影响

本文研究了正常C3H/10T1/2CI8细胞(一种源于小鼠胚胎的成纤维细胞,简称NC3H/10)与用~3H-TdR转化的C3H/10T1/2CI8细胞(简称TC3H/10)染色质蛋白激酶(CAPK)的作用物特异性,发现这两种细胞的CAPK均能以外源性酸性酪蛋白为作用物,而不以外源性碱性组蛋白为作用物,由此推测CAPK的天然作用物主要为非组蛋白。观察了表皮生长因子(EGF)对培养细胞的CAPK活性的影响,EGF能使NC_3H/10和TC3H/10的CAPK活性分别增加105.6％(P<0.01)和50.7％(P<0.01),后者增加的幅度仅为前者的1/2,说明转化细胞CAPK活性对EGF刺激的敏感性较正常细胞低。EGF和1-油酰-2-乙酰-消旋-甘油(OAG)对无细胞体系的CAPK活性无直接影响,提示EGF可能通过核内受体或OAG以外的其它第二信使来影响CAPK的活性。     

Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

五灵胶囊对LPS诱导鼠枯否细胞p38MAPK信号转导通路的影响

目的:探讨五灵胶囊对脂多糖(LPS)诱导的大鼠枯否细胞(Kupffer cells,KC)p38MAPK信号转导通路的影响。方法:分离纯化KCs,60ng/ml LPS刺激建立LPS的肝细胞损伤模型;40只SD大鼠药物处理后,分离制备含药血清。实验分为四组:空白血清组、LPS+空白血清组、含药血清Ⅰ组(10.0g/kg)+LPS、含药血清Ⅱ组(6.25g/kg)+LPS。KCs产生促炎因子(I125放免法测定TNF-α、IL-6、IL-8,比色法测定NO生成量),采用Western blot法检测ERK、p-ERK、p38、p-p38、TNF-α和STAT3的蛋白水平。结果:1、空白血清+LPS组,TNF-α、IL-6、IL-8和NO浓度明显高于空白血清组;2、同空白血清+LPS组比较,含药血清Ⅰ、Ⅱ组TNF-α、IL-6、IL-8和NO水平明显降低;3、与空白血清组比较,空白血清+LPS组能上调KCs对p-ERK、P38、p-P38、STAT3和TNF-α表达(p<0.01,p<0.05),对ERK表达无影响(p>0.05)4、同空白血清+LPS组比较,含药血清Ⅰ+LPS、含药血清Ⅱ+LPS组p-p38、S...     

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9 kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.     

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB

The effects of epigallocatechin-3-gallate (EGCG) on dendritic cells (DC) maturation were investigated. EGCG, in a dose-dependent manner, profoundly inhibited CD80, CD86, and MHC class I and II expression on bone marrow-derived murine myeloid DC. EGCG restored the decreased dextran-FITC uptake and inhibited enhanced IL-12 production by LPS-treated DC. EGCG-treated DC were poor stimulators of nai;ve allogeneic T-cell proliferation and reduced levels of IL-2 production in responding T cells. EGCG-pretreated DC inhibited LPS-induced MAPKs, such as ERK1/2, p38, JNK, and NF-kappaB p65 translocation. Therefore, the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation appeared to involve the inhibition of MAPK and NF-kappaB activation. These novel findings provide new insight into the immunopharmacological role of EGCG and suggest a novel approach to the manipulation of DC for therapeutic application of autoimmune and allergic diseases.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Okadaic acid,sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase Fa/GSK-3α in A431 cells

Exposure of A431 cells to a rapid and sudden increase from 37°C to 46°C for 30 min could induce an increase in protein level and cellular activity of protein (kinase Fa /GSK-3α) up to ∼200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37°C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37°C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46°C for another 30 min, the heat induction on kinase Fa /GSK-3α was found to be completed blocked. In sharp contrast, when cells were first treated with 1 μM TPA at 37°C for 24 h or with 5 μM sphingosine at 37°C for 30 min to down-regulate cellular PKC, the heat induction on kinase Fa /GSK-3α was found to be reversely promoted up to ∼ 250% of control level, demonstrating that kinase Fa /GSK-3α may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase Fa /GSK-3α in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase Fa /GSK-3α, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process. © 1996 Wiley-Liss, Inc.     

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase,phosphatidylinositol 3-kinase,Akt and mitogen-activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

Synthesis and anthelmintic activity of 7-substituted 3,4a-dimethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2-b]pyridine-6,8(7H)-diones

The racemic 7-substituted 3,4a-dimethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2-b]pyridine-6,8(7H)-diones represent novel tricyclic compounds with strong in vivo efficacy against the parasitic nematode Haemonchus contortus Rudolphi in sheep. Here we report on the synthesis of tricyclic endo-2,3-dihydro[3,2-b]pyridine-type cycloadducts and describe the separation of the racemic 3,4a-dimethyl-7-ethyl-4a,5a,8a,8b-tetrahydro-6H-pyrrolo[3',4':4,5]furo[3,2]pyridine-6,8(7H)-dione into the enantiomers by HPLC. The absolute configuration of the most anthelmintically active (4aS,5aS,8aS,8bR)-enantiomer was determined by single crystal X-ray analysis using its stable (4aS,5aS,8aS,8bR)-enantiomer-CuCl2 (2:1)-complex.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

Characterization of Protein Methyltransferases Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1 Reveals Extensive Post-Translational Modification

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.     

Stereoselective Synthesis of erythro-Serricornin, (4R, 6R, 7S)-, and (4S, 6R, 7S)-4,6-Dimethyl-7-hydroxynonan-3-one,Stereoisomers of the Sex Pheromone of Cigarette Beetle

A stereoselective synthesis of erythro-serricornin [(4RS,6R,7S)-4,6-dimethyl-7-hydroxynonan-3-one] was completed starting from l-(+)-tartaric acid. The relative configuration of C(6)-methyl and C(7)-hydroxyl groups in naturally occurring serricornin was threo.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.     

Synthesis and SAR of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one as NMDA/glycine site antagonists

A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [(3)H]5,7-dicholorokynurenic acid ([(3)H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C subunits. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC(50) value of 110 nM in [(3)H]DCKA binding and a K(b) of 11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse maximum electroshock-induced seizure test (ED(50)=2.3mg/kg, IP).