Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder,Paralichthys olivaceus

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon–intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two α-helices and eight β-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6 μg/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.     

Tributyltin-binding protein type 1 has a distinctive lipocalin-like structure and is involved in the excretion of tributyltin in Japanese flounder, Paralichthys olivaceus

Tributyltin-binding protein type 1 (TBT-bp1) is a newly discovered protein that binds with TBT in the blood of the Japanese flounder, Paralichthys olivaceus. We determined the genomic sequence of TBT-bp1 and found that this protein has a conserved exon–intron structure that is common to the lipocalin protein family. The secondary and tertiary structures of TBT-bp1, predicted from amino acid sequence, included at least two α-helices and eight β-sheets that are conserved in all lipocalins and form a barrel structure that may bind with ligands. Analysis of the gene structure, secondary structure, and tertiary structure demonstrated that TBT-bp1 could be classified as a lipocalin. A homology search revealed the presence of TBT-bp1-like proteins in eight species of teleost. When flounder were injected intraperitoneally with TBT-d27 at 11.6 μg/fish, TBT-d27 was detected in the blood and in the skin mucus. The concentration of TBT-d27 in mucus was approximately 1/100 of that in the serum. Western blotting analysis revealed that TBT-bp1 was present in the skin mucus. These results suggest that TBT-bp1 in Japanese flounder binds with TBT and is excreted from the body via the mucus.     

Lopap: A non-inflammatory and cytoprotective molecule in neutrophils and endothelial cells

Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L. obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte-endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and N^ω-nitro-l-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production.     

Reversibility of tributyltin-chloride-induced protein synthesis inhibition after ATP recovery in HEL-30 cells

The effect of tributyltin chloride (TBT) on ATP levels and protein synthesis was investigated in a murine epidermal cell line (HEL-30). Five minutes after exposure to 10(-5) M TBT, cellular levels of ATP decreased by 57% and protein synthesis was significantly inhibited. The effect of ATP was stable up to 2 h incubation, whereas inhibition of protein synthesis increased with time of treatment. Partial (1 h) or complete (2 h) recovery of the two cell functions was observed when dithiothreitol (DTT) was added to the medium, 5 min after the damage and in the absence of TBT. The sequence of the observed effects suggests that TBT inhibition of protein synthesis is due to impairment of ATP production.     

Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity

Non‐steroidal anti‐inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX‐inhibiting activity. To identify NSAIDs with COX‐independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre‐osteoblast cell line MC3T3‐E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX‐inhibiting activity. Moreover, 2,5‐dimethyl (DM)‐celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM‐celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)‐2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3‐E1 cells were not affected by celecoxib or DM‐celecoxib. Instead, celecoxib and DM‐celecoxib suppressed BMP‐2‐induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho‐dependent intracellular signalling, thereby modifying bone remodelling.     

Alteration of an essential NK cell signaling pathway by low doses of tributyltin in human natural killer cells

Tributyltin (TBT), a toxic and widespread environmental contaminant, has been shown to inhibit natural killer (NK) cell cytotoxic function significantly. Inhibition of NK cell cytotoxic function has the potential to increase viral infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells, an intracellular pathway is activated, which generally begins with activation of non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated protein kinase (MAPK)-mediated release of lytic granules toward the contacted target cell. In the current studies, we used a cytotoxicity assay to examine how low doses (200nM or lower) of TBT affect cytotoxic function. Additionally, we investigated how low doses of TBT modulate the signaling pathway that dictates lytic granule exocytosis. A 1h exposure to 200, 100, 50 and 25nM TBT significantly decreased cytotoxic function 6d later. We also saw significant activation of p38 and p44/42 by as low as 50nM TBT within ten minutes of exposure. The observed activation of MAPKs, p38 and p44/42, implicated their upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and MEK1/2, activation was seen within ten minutes. However, when the most upstream signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs) such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases (MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being inhibited by TBT. Taken together, these data suggest that TBT-induced activation of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6 and MEK1/2, respectively.     

Ligand recognition mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi functional protein acting as a PGD(2) synthesizing enzyme, a transporter or scavenger of various lipophilic ligands, and an amyloid β chaperon in the brain. L-PGDS is a member of the lipocalin superfamily and has the ability to bind various lipophilic molecules such as prostanoid, retinoid, bile pigment, and amyloid β peptide. However, the molecular mechanism for a wide variety of ligand binding has not been well understood. In this study, we determined by NMR the structure of recombinant mouse L-PGDS and L-PGDS/PGH(2) analog complex. L-PGDS has the typical lipocalin fold, consisting of an eight-stranded β-barrel and a long α-helix. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins and the cavity contained two pockets. Kinetic studies and molecular docking studies based on the result of NMR titration experiments provide the direct evidence for two binding sites for PGH(2) and retinoic acid in the large cavity of L-PGDS. Structural comparison of L-PGDS/U-46619 complex with apo-L-PGDS showed that the H2-helix, CD-loop, and EF-loop located at the upper end of the β-barrel change the conformation to cover the entry of the cavity upon U-46619 binding. These results indicated that the two binding sites in the large cavity and induced fit mechanism were responsible for the broad ligand specificity of L-PGDS.     

在Sf9昆虫细胞-杆状病毒系统中表达毒蕈碱型M_2及M_5受体重组突变体

目的为乙酰胆碱毒蕈碱(M)受体亚型特异性的变构调节剂及基因工程的研究提供实验平台。方法用PCR及搭桥PCR法对乙酰胆碱M2及M5受体作以下突变:①将N-糖基化位点Asp突变为Asn;②删除对蛋白酶敏感的M受体的第三个细胞内环;③在C端添加凝血酶识别位点(CMV)和6-His标记。将PCR扩增出重组嵌合蛋白基因亚克隆到杆状病毒转移载体,制备重组杆状病毒并感染昆虫细胞表达M2/M5受体蛋白。Western印迹及放射性配体受体结合实验验证受体的正确表达及功能。结果通过搭桥PCR,成功扩增出1018 bp的重组M2受体和1041 bp重组M5受体核酸序列;使用pUC/M13的扩增引物成功构建M2/M5重组转移载体。将重组载体质粒与线性化病毒DNA共转染昆虫细胞Sf9,制备重组杆状病毒并感染昆虫细胞,见细胞空泡样病变。Western印迹分析确定重组杆状病毒感染昆虫细胞M2/M5蛋白表达,放射性配体受体饱和实验结果表明,表达的重组受体蛋白与[3H]N-甲基-东莨菪碱具有特异性结合能力。结论 Sf9昆虫细胞能够表达M2及M5重组受体蛋白,M2及M5重组受体蛋白的病毒样颗粒可用于M受体的新药研究。     

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades.     

多巴胺D1受体在Sf9昆虫细胞中的表达及左旋氯代斯阔任对重组D1受体的激动作用

目的:在Sf9昆虫细胞中表达D_1受体,并研究左旋氯代斯阔任对重组D_1受体的激动作用。方法:构建含D_1受体cDNA的重组杆状病毒,以其感染Sf9昆虫细胞得到D_1受体表达。[~3H]SCH23390受体结合检测重组D_1受体的药理特性。[~3H]SCH23390受体结合和cAMP测定实验检测左旋氯代斯阔任对重组D_1受体的激动作用。结果:在Sf9昆虫细胞中成功表达D_1受体,[~3H]SCH23390与重组D_1受体最大结合量(B_(max))为(0.94±0.06)nmol/g蛋白,[~3H]SCH23390与重组D_1受体的结合解离常数(K_d)为(1.9±0.3)nmol/L,其药理特性与小牛纹状体脑匀浆所得结果一致。左旋氯代斯阔任对重组D_1受体有高亲和力,解离常数K_i为(6.3±1.4)nmol/L;并剂量依赖地引起胞内cAMP增加,EC_(50)为0.72μmol/L(95％可信限为0.67-0.77μmol/L),表现出D_1激动作用。结论:在杆状病毒/昆虫细胞Sf9中,成功建立了D_1受体异源表达系统。在细胞分子水平,直接证实了左旋氯代斯阔任对D_1受体的激动作用。     

Chronic effects of tributyltin on multiple biomarkers responses in juvenile common carp,Cyprinus carpio

In this study, the chronic toxic effects of tributyltin (TBT), an antifouling paints commonly present in surface and ground water, on morphological indices, reactive oxygen species (ROS) generation, and ATPase activity and heat shock protein (Hsp) 70 protein in tissues (liver, gill, and white muscle) of common carp were investigated. Fish were exposed at sublethal concentrations of TBT (75 ng/L, 0.75 μg/L, and 7.5 μg/L) for 60 days. When compared with the control, there was significant lower condition factor in fish exposed at the higher concentration of TBT. ROS levels in three tissues increased significantly at higher TBT concentrations (0.75 and 7.5 μg/L). The hepatic antioxidant enzymes (total antioxidative capacity and superoxide dismutase) activities were induced at higher concentrations (0.75 μg/L) of TBT. When compared with the hepatic antioxidant enzymes activities in fish exposed to 0.75 μg/L of TBT, there was a decreasing trend in those exposed to TBT with a concentration of 7.5 μg/L. However, all the antioxidant enzymes activities were significantly inhibited in gill and muscle of fish exposed to higher TBT concentrations (0.75 and 7.5 μg/L). Moreover, there was significant lower Na‐K‐ATPase in three tissues after long‐term exposure to higher concentration of TBT, but a significant higher Hsp70 protein levels was observed. In short, environmental concentrations of TBT could not induce obvious impacts on fish, but long‐term exposure to higher concentrations of TBT could affect seriously the health status of fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 937–944, 2016.     

Purification and functional reconstitution of the human P2Y12 receptor

The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y12-R. The G protein selectivity of the P2Y12-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. These results illustrate that the signaling properties of the P2Y12-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.     

Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

The primers were designed according to the gene sequence of lipocalin protein family, and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR. The random mutation lipocalin library was constructed using phagemid expression vector. Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells, and the lipocalin secondary library binding to mast cells was obtained. Then the lipocalin secondary library was enriched and screened with FcεRI-α receptor protein as target molecule, and specific binding phages were eluted. After three rounds of screening, eight recombinant phage clones were randomly selected from elution clones of the third round. ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-α receptor of mast cells. These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-α receptor, and also lay the foundation for the development of biological small molecule drugs to treat IgE associated allergic diseases.     

Pharmacological property of tributyltin in vivo and in vitro

Tributyltin (TBT), an assumed endocrine-disrupting chemical, is widely known to show harmful effects in invertebrates including the dioecious snails. As for mammals, there are several reports concerning TBT toxicology, but few indications about general pharmacology of TBT. In the present study, we comprehensively examined the pharmacological effects of TBT both in vivo and in vitro. TBT (0.3 or 1.0 mg/kg) attenuated the small intestinal propulsive activity measured by the charcoal method in vivo, and caused concentration-dependent relaxation of isolated guinea-pig ileum in vitro (1.0x10(-8)-3.0x10(-6) M). TBT induced concentration-dependent relaxation of guinea-pig trachea, which was not inhibited by pre-treatment with a beta-adrenoceptor antagonist. TBT caused a concentration-dependent contraction of rat aortae, and also evoked endothelium-dependent relaxation in the presence of an alpha-adrenoceptor antagonist. The relaxation was inhibited by a muscarinic receptor antagonist. TBT reduced the electrically evoked, sympathetic contractile responses of rat vas deferens, which were slightly prevented by an alpha(2)-adrenoceptor antagonist. These results suggest that TBT possesses diverse pharmacological properties in mammalian organs.     

Sensitization of vascular smooth muscle cell to TNF-alpha-mediated death in the presence of palmitate

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-alpha-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-alpha-mediated nuclear factor kappa B (NF-kappaB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-kappaB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-kappaB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-alpha-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.