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, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.     

结肠黑变病相关基因金属泛激蛋白-1cDNA和氨基酸序列分析

目的对结肠黑变病相关基因金属泛激蛋白-1(MPS-1)cDNA序列和氨基酸序列进行综合分析,为其结构和功能的研究奠定理论基础。方法从Pubmed数据库中获得目标cDNA序列,利用基因和蛋白质分析处理软件DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL等软件对该目标的基因序列和氨基酸序列进行综合分析。结果 MPS-1蛋白cDNA序列长为361 bp,编码84个氨基酸残基,基因序列比对显示该蛋白与核糖体蛋白S27(RPS27)mRNA的编码序列同源度最高,两者核苷酸的相似度为361/361(100%),氨基酸序列比对显示该蛋白与RPS27的氨基酸序列同源度最高,两者氨基酸的相似度为84/84(100%),目标序列中存在1段Ribosomal_S27e家族保守结构域,二级结构和三级结构预测显示目标蛋白中主要存在α-螺旋、β-折叠和无规卷曲的结构。结论 MPS-1蛋白与RPS27同源度高,与肿瘤的发生密切相关。     

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.     

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus)

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.     

Characteristics of bioconcentration and depuration of endocrine disruption chemicals in the flounder,Paralichthys olivaceus, under flow-through system

This study investigated the bioaccumulation effects of endocrine disruption chemicals (EDCs) such as Tributyltin (TBT), Nonylphenol (NP) and Bisphenol-A (BPA) on flounder,Paralichthys olivaceus. The exposure experiment with the flow through system was performed to examine the effects of bioconcentration for single or multi-chemicals. In the muscle of flounder exposed to TBT, the concentrations of TBT were significantly increased compared with the control tank after two months. It is markedly more accumulated in the liver of the flounder than in the muscle. TBT in muscle of the low level of EDCs were highly accumulated in single exposures of TBT compared to multi-chemical exposure with NP and/or BPA. The concentrations of TBT in muscle were increased in the multi-chemical exposure system. The concentration of TBT in the liver of flounder showed a slight decrease in the multi-chemical exposure system. The metabolites of TBT, DBT and MBT were also concentrated in the muscle and liver. NP and BPA in the muscle of flounder were accumulated with high concentration of EDCs. NP and BPA in muscle were significantly decreased compared with TBT after the depuration period.     

Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2 Different Regions in Cambodia

The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the skin contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish.     

Prediction of protein structure from amino acid sequence

Methods of predicting protein conformation from amino acid sequence are reviewed. Several widely used algorithms to predict local secondary structure are first discussed. Four general approaches to predict the tertiary structure are then described: (1) energy calculations from an open chain; (2) recognition of sequence homology with a known structure; (3) uses of a sequence template that dictates a particular fold; (4) docking alpha-helices and beta-strands. Throughout this review, the likely success of these methods is considered.     

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.     

Secondary structure of interleukin-2(Alal25) in unfolded state*

Secondary structure of interleukin-2(Alal25) in unfolded state was examined by circular dichroism (CD). Unfolding of tertiary structure of the protein, as determined by CD, was observed when the solvent pH was decreased below 3.0 or the disulfide bond was reduced. Consistent with the CD results, a stronger fluorescence enhancement of 1-anilinonaphthalene-8-sulfonic acid was observed on acidification or reduction of interleukin-2(Alal25) relative to that of the native protein, indicating a larger hydrophobic surface exposed to solvent. However, the secondary structure was fully retained in 5% acetic acid or aqueous HCl, pH 3.0. It seemed that α-helical content of the protein is even greater at pH 2.0. Reduced protein showed a far u.v. CD spectrum indistinguishable from the oxidized one at pH 4.0. These results suggest that the secondary structure of interleukin-2(Alal25) does not require tertiary structure.     

Regulation of Hsp27 and Hsp70 expression in human and mouse skin construct models by caveolae following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

Dermal exposure to the vesicant sulfur mustard causes marked inflammation and tissue damage. Basal keratinocytes appear to be a major target of sulfur mustard. In the present studies, mechanisms mediating skin toxicity were examined using a mouse skin construct model and a full-thickness human skin equivalent (EpiDerm-FT?). In both systems, administration of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide (CEES, 100-1000μM) at the air surface induced mRNA and protein expression of heat shock proteins 27 and 70 (Hsp27 and Hsp70). CEES treatment also resulted in increased expression of caveolin-1, the major structural component of caveolae. Immunohistochemistry revealed that Hsp27, Hsp70 and caveolin-1 were localized in basal and suprabasal layers of the epidermis. Caveolin-1 was also detected in fibroblasts in the dermal component of the full thickness human skin equivalent. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that Hsp27 and Hsp70 were localized in caveolae. Treatment of mouse keratinocytes with filipin III or methyl-β-cyclodextrin, which disrupt caveolar structure, markedly suppressed CEES-induced Hsp27 and Hsp70 mRNA and protein expression. CEES treatment is known to activate JNK and p38 MAP kinases; in mouse keratinocytes, inhibition of these enzymes suppressed CEES-induced expression of Hsp27 and Hsp70. These data suggest that MAP kinases regulate Hsp 27 and Hsp70; moreover, caveolae-mediated regulation of heat shock protein expression may be important in the pathophysiology of vesicant-induced skin toxicity.     

Association between a polymorphism in cysteinyl leukotriene receptor 2 on chromosome 13q14 and atopic asthma

OBJECTIVE: Cysteinyl leukotriene receptor 2 (CYSLTR2) is one of the receptors for the cysteinyl leukotrienes (CYSLTs), which cause bronchoconstrictions, vascular hyperpermeability and mucus hypersecretion in asthmatic patients. CYSLTR1 antagonists have been shown to be effective in the treatment of chronic asthma. CYSLTR2 is located approximately 300 kb from D13S153, which is reportedly linked to asthma in several populations. We characterized the genomic structure of humans CYSLTR2, determined the putative major promoter region and conducted association studies pertaining to polymorphisms in CYSLTR2 and asthma. METHODS AND RESULTS: We identified three novel exons in the 5' untranslated region of CYSLTR2 by rapid amplification of cDNA ends and identified eight novel polymorphisms in CYSLTR2 by direct sequencing. A transmission disequilibrium test with 137 Japanese asthmatic families revealed that the -1220A > C polymorphism is associated with the development of asthma (P = 0.0066). In addition, a polymorphism in the putative promoter region caused different promoter activities in vitro. CONCLUSION: Our results suggest that CYSLTR2 is one of the genes that contributes to susceptibility to asthma in the Japanese population.     

Lymnaea EGF and gigantoxin I, novel invertebrate members of the epidermal growth factor family

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.     

新肺癌相关基因hERGIC3的克隆与生物信息学分析

目的构建hERGIC3基因的原核表达载体,并对其进行生物信息学分析,以期全面地了解hERGIC3蛋白的生物学功能。方法采用实时半定量聚合酶链反应（RT-PCR）方法获取人类hERGIC3基因的开放阅读框（ORF）框DNA序列插入pGEM-T Easy载体;选择多个软件对hERGIC3蛋白进行生物信息学分析。结果酶切结果显示,插入序列为目标序列,成功克隆了hERGIC3基因;生物信息学分析结果显示：hERGIC3蛋白由383个氨基酸残基组成,相对分子质量为43.2×10^3,理论等电点为5.68,蛋白比较稳定;hERGIC3为跨膜蛋白,跨膜区为20～42和345～367,膜外区域为1～19和368～383,膜内区域为43～344;二级结构含有110个α螺旋、94条延伸链、184个随机卷曲、15个潜在的磷酸化位点,hERGIC3可能参与了氨基酸、辅酶的生物合成以及脂肪、能量代谢和蛋白质翻译、转运等功能;hERGIC3含有ERGIC_N和COPⅡcoated_ERV 2个蛋白保守结构域,hERGIC3可能与PPKCSH、ERGIC1、ERGIC2、COPA、PSMD11等蛋白有相互作用。结论成功克隆了hERGIC3基因;深入地分析了hERGIC3的结构与功能,为下一步研究hERGIC3基因在肺癌中的病理生理功能奠定了理论基础。     

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC₅₀ = 0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F₀, either in the form of free enzyme or of enzyme-substrate complex (Ki = K′i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg²⁺ cofactor, may bind strongly to F₁ subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation.     

Structural analysis on the abnormal elongated hemoglobin "hemoglobin Geneva"

Hemoglobin variants in which a frameshift results in chain elongation are not common. Hemoglobin Geneva (HbGeneva) is an unstable hemologin with abnormal elongation. This hemoglobinopathy is known for its high instability. Concerning the pathogenesis of HbGeneva, the data indicate a change in codon 114 from CTG (Leu) to -GG that results in a frame shift and the presumed synthesis of an abnormal beta-chain that is 156 residues long with a completely different C-terminal amino acid sequence. This abnormality causes a frame shift, which results in elongation of the beta-chain amino acids. A bioinformatic analysis was performed to study the secondary and tertiary structures of those abnormal amino acid sequences. A computer-based study for protein structure modeling was performed. According to this study, the secondary structure analysis of the Hb Geneva showed many defects in helix and strand of the Hb Geneva compared with normal beta-globin chains. On the basis of this information, the main alteration in the Hb Geneva might be due to these aberrations. With regard to the tertiary structure, the deterioration of folds, accompanied by the aberration in secondary structure of globin in Hb Geneva can be identified.     

Specialization of the sting venom and skin mucus of Cathorops spixii reveals functional diversification of the toxins

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.     

Alteration of monoamine concentrations in the brain of medaka, Oryzias latipes, exposed to tributyltin

We measured the concentrations of monoamines in the brain of Japanese medaka, Oryzias latipes, exposed to tributyltin (TBT). Fish were exposed to 0, 1, 5, 25, or 125 microg g(-1) of TBT via the diet for 21 days. After the administration period, six males and six females in each treatment group were dissected and their brains were collected. The following monoamines were analyzed: dopamine (DA), norepinephrine (NE), epinephrine (E), and 5-hydroxytryptamine (5-HT). The metabolites of DA, 3,4-dihydroxyphenylacetic acid and homovanilic acid, and the metabolite of 5-HT, 5-hydroxyindolacetic acid were also analyzed. The concentration of DA in the brain of male medaka and the concentrations of 5-HT and NE in the female brains were significantly decreased by exposure to 125 microg TBT g(-1). The concentrations of 5-HT and NE in males and of DA in females were slightly decreased by 125 micrg g(-1) of TBT, although the differences were not statistically significant. The present study demonstrates that TBT alters monoamine concentrations in the brain of medaka.     

Four Weeks' Inhalation Exposure of Long Evans Rats to 4‐tert‐Butyltoluene: Effect on Evoked Potentials,Behaviour, and Brain Neurochemistry

Long‐lasting central nervous system (CNS) neurotoxicity of 4‐tert‐butyltoluene (TBT) has been investigated using electrophysiology, behaviour, and neurochemistry in Long Evans rats exposed by inhalation to 0, 20, or 40 p.p.m. TBT 6 hr/day, 7 days/week for 4 weeks. Flash evoked potentials and somatosensory evoked potentials were not affected by TBT. In Auditory Brain Stem Response there was no shift in hearing threshold, but the amplitude of the first wave was increased in both exposed groups at high stimulus levels. Three to four months after the end of exposure, behavioural studies in Morris water maze and eight‐arm maze failed to demonstrate any TBT induced effects. Exposure was followed by a 5 months exposure‐free period prior to gross regional and subcellular (synaptosomal) neurochemical investigations of the brain. TBT reduced the NA concentration in whole brain minus cerebellum. Synaptosomal choline acetyltransferase activity increased and acetylcholinesterase activity was unchanged suggesting increased synaptosomal ability for acetylcholine synthesis. The relative and total yield of synaptosomal protein was reduced suggesting reduced density and total number of synapses in situ, respectively. We hypothesise that a reduced yield of synaptosomal protein reflects a more general effect of organic solvent exposure on the software of the brain. The synaptosomal concentration per mg synaptosomal protein and the total amount of 5‐hydroxytryptamine were not affected whereas the total amount of synaptosomal noradrenaline decreased. The concentration and the total amount of synaptosomal dopamine decreased. The noradrenergic and dopaminergic parts of CNS may be more vulnerable to TBT than the serotonergic, and these long‐lasting effects may cause or reflect TBT‐compromised CNS function.     

Prediction of primary structure deamidation rates of asparaginyl and glutaminyl peptides through steric and catalytic effects

Abstract: The primary sequence dependence of deamidation has been quantitatively explained on the basis of a simple steric and catalytic model. Application to the known deamidation rates of peptides produces a table of coefficients that permits calculation of the known deamidation rates and prediction of deamidation rates for peptide sequences that have not yet been measured. This work permits a better understanding of deamidation, provides a prediction procedure for protein engineering, and facilitates improved computation of peptide and protein primary, secondary, tertiary, and quaternary structure deamidation rates.