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Sample records for human cyp450 isoforms

  1. Effects of two novel sugar drug candidates on CYP450 isoforms in different sexed Chinese human liver microsome in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Jie; ZHANG Xin-hui; SU Jia-ru

    2008-01-01

    The sex-based differences between the effects of two novel sugar-based drug candidates, a sulfated polymannuroguluronate (SPMG-911) and an acidic oligosaccharide sugar chain compound (AOSC-971), on the enzymes CYP 1A2, CYP2E1 and CYP3A4 of Chinese human liver microsome were investigated. The results showed that neither SPMG-911 nor AOSC-971 have any effect on CYP3A4, AOSC-971 induced the CYP 2E1 in men but have no effect on CYP1A2, SPMG-911 inhibit the CYP1A2 also in men but have no effect on CYP2E1. The results are useful for their safety evaluation, as well as for the prediction of interdrug interactions associated with the two drugs.

  2. Docking studies of piperine - iron conjugate with human CYP450 3A4

    OpenAIRE

    2013-01-01

    Piperine, a major constituent of Piper nigrum (Black pepper), is one of the well known components in many Ayurvedic formulations. Piperine is most studied bioenhancer because it inhibits drug metabolizing enzymes in rodents and increases plasma concentrations of several drugs, including P-glycoprotein substrates. However, there areno evidences on piperine-iron conjugate to inhibit human CYP450 3A4. We therefore investigated the influence of piperine-Fe conjugate to study the metabolism of iro...

  3. Highly sensitive LC-MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human.

    Science.gov (United States)

    Grangeon, Alexia; Gravel, Sophie; Gaudette, Fleur; Turgeon, Jacques; Michaud, Veronique

    2017-01-01

    Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3μm PFP(2) 150×3mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small

  4. Cocktail 探针药物法评价辣椒素对大鼠CYP450亚型的影响%Effects of Capsaicin on Rat Cytochrome P450 Isoforms by Cocktail Probe Drug Method

    Institute of Scientific and Technical Information of China (English)

    舒舟; 翟学佳; 刘金梅; 吕永宁

    2014-01-01

    目的::采用cocktail 探针药物法研究辣椒素在体外对大鼠肝微粒体CYP 450四种亚型的影响。方法:分为试验组和对照组,试验组采用大鼠肝微粒体孵育体系、探针药物和系列浓度的辣椒素(对照组仅加入缓冲液)共同孵育20 min,反应终止后用HPLC方法检测代谢产物的生成量以代表酶的活性。采用Graphpad prism 5.0计算辣椒素对各亚型的IC50值。将辣椒素与大鼠肝微粒体分别预孵育0,5,10,15,20,30 min,计算不同预孵育时间下各亚型的相对活性百分比。结果:辣椒素对大鼠肝微粒体CYP1A2、CYP2C11、CYP2E1和CYP3A2的IC50值分别为36.21,17.19,51.64,18.86μmol·L-1。预孵育没有增强辣椒素对CYP450的抑制作用。结论:辣椒素在体外对大鼠肝微粒CYP1A2、CYP2C11、CYP2E1和CYP3A2具有抑制作用,且未呈现出预孵育时间依赖性。%Objective:To investigate the in vitro effect of capsaicin on four major rat cytochrome P450 ( CYP) enzymes using a cocktail probe drug method. Methods:The in vitro incubation was divided into capsaicin group and the control group. Rat liver micro-somes, probe drugs, capsaicin at various concentration ( buffer solution in the control group) and cofactors were cultured altogether for 20 min. After the culture, the concentration of metabolites was determined by HPLC to assess the activities of enzymes. IC50 value of capsaicin on every isoform was calculated using Graphpad prism 5. 0. Capsaicin and hepatic microsomess were pre-incubated respec-tively for 0, 5, 10, 15, 20 and 30 min, and then the relative activity of the four isoforms at different time was calculated. Results:The activity of the rat liver microsomes enzyme CYP450 isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2) was all inhibited by capsaicin in vitro with IC50 value of 36. 21, 17. 19, 51. 64 and 18. 86 μmol·L-1 , respectively. Pre-incubation could not increase cap-saicin inhibitory activity against the four CYP enzymes

  5. Cytochrome P450 (CYP450) Tests

    Science.gov (United States)

    Tests and Procedures Cytochrome P450 (CYP450) tests By Mayo Clinic Staff Your doctor may use cytochrome P450 (CYP450) tests to help determine how your ... find the right antidepressant. Genotyping tests, such as cytochrome P450 tests, may speed up the identification of ...

  6. 大鼠体外肝星状细胞活化过程中代谢活化相关 CYP450亚型的变化%MuItipIe xenobiotic metaboIism-associated cytochrome P450 isoforms might be invoIved in earIy activation of rat hepatic steIIate ceIIs in vitro

    Institute of Scientific and Technical Information of China (English)

    廖长秀; 吴勇; 平洁; 敖英; 印宪; 汪晖

    2014-01-01

    OBJECTIVE To investigate the role of several xenobiotic metabolism-associated CYP450 isoforms in the auto-activation of hepatic stellate cells(HSCs) invitro. METHODS HSCs were isolated from adult Wistar rats and cultured on plastic as an in vitroauto-activation model. Positive expression of α-smooth muscle actin(α-SMA)was used as an activated marker of HSCs and detected by immunocytochemical staining in HSCs cultured for 1,2,5 or 11 d. The expressions of CYP450 isoforms were analyzed by real-time RT-PCR in the HSCs. RESULTS The immunocytochemical stai-ning showed no α-SMA expression in HSCs cultured for 1 d,while its expression gradually increased during culture since day 2. ln terms of α-SMA expression,HSCs cultured for 1,2,5,and 11 d were classified as the quiescent,early,middle and later stages of activation,respectively. The RT-PCR results revealed that CYP1B1,CYP2B1/ 2,and CYP2E1 mRNA were expressed at high levels in the early stage of HSCs activation(at day 2),which were 2.1-,1.6-,and 23.9-fold those in the quiescent HSCs(day 1),respectively. Further study revealed that mRNA expressions of these up-regulated CYPs in the early stage of activation were diminished at the subsequent two stages. The levels of CYP1A1 and CYP1A2 mRNA decreased constantly throughtout the activation process. CONCLUSION Multiple xenobi-otic metabolism-associated CYP450 isoforms might be involved in the auto-activation of rat HSCs invitro.%目的:观察大鼠肝星状细胞(HSC)活化不同时期外源活化相关 CYP450亚型CYP1A1, CYP1B1,CYP1A2,CYP2B1/2和 CYP2E1 mRNA 表达水平的变化规律。方法分离大鼠 HSC,接种在无包被塑料培养瓶中作为 HSC 自氧化活化模型,免疫细胞化学法检测不同培养时间 HSC 活化标志物α-平滑肌肌动蛋白(α-SMA)表达。实时定量 RT-PCR 法测定 HSC 自氧化激活的不同时期多种外源物活化相关 CYP450亚型 mRNA 的表达变化。结果免疫细胞化学结果表明

  7. Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction

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    Pujan K. Desai

    2017-05-01

    Full Text Available There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450 induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2 indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

  8. In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity at CYP-450.

    Science.gov (United States)

    Labrie, Philippe; Maddaford, Shawn P; Lacroix, Jacques; Catalano, Concettina; Lee, David K H; Rakhit, Suman; Gaudreault, René C

    2006-12-01

    Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency of the P-glycoprotein (P-gp) transporter. The aromatic spacer group between nitrogen atoms (N1 and N2) in the known inhibitor XR9576 was replaced with a flexible alkyl chain of 2 to 6 carbon atoms in length. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline and their open-chain N-methylhomoveratrylamine counterparts were shown to be potent P-gp inhibitors. The maximal inhibition was obtained when using an ethyl or propyl spacer. Several compounds were more potent than verapamil and intrinsically less cytotoxic than XR9576. In addition, in vitro metabolism studies of 23a with a subset of human CYP-450 isoforms revealed that, unlike XR9576, 23a inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of anticancer drugs such as paclitaxel toward metabolism. In this context, 22a might be a suitable candidate for further drug development.

  9. Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

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    Limsrichamrern Somchai

    2011-09-01

    Full Text Available Abstract Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450 induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

  10. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    Science.gov (United States)

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  11. Cocktail探针药物法评价生、醋莪术对CYP450酶亚型的影响%Application of probe drugs for detecting influences of Rhizoma Curcumae and processed Rhizoma Curcumae on cytochrome P450 isoforms

    Institute of Scientific and Technical Information of China (English)

    王菊; 陆兔林; 毛春芹; 肖永庆; 顾娟娟

    2012-01-01

    Aim To research and compare the influences of Rhizoma Curcumae and processed Rhizoma Curcumae on the cytochrome P450 isoforms, and to explore the tropism processed of Rhizoma Curcumae to enhance or change the role. Methods Rats were randomly divided into three groups. The treatment groups were received Rhizoma Curcumae and processed Rhizoma Curcumae respectively. The other group were received saline as blank control group. Blood samples were collected aL different time points and the plasma concentration of cocktail probe drugs was determined by IIPLC. Results There was insignificant difference of the ophylline, dapsone, chlorzoxazone between the Rhizoma Curcumae group and the blank group. Compared with the blank group, the half-life time,Tmax and the area under curve increased significantly and the clearance lowered significantly of ophylline and chlorzoxazone in the processed Rhizoma Curcumae group; There was insignificant difference in the half-life time of dapsone , the area under curve lowered significantly and the clearance increased significantly in the processed Rhizoma Curcumae group. Conclusion Rhizoma Curcumae has no significant effect on the cytochrome P450 isoforms of CYP1A2, CYP3A4 and CYP2E1. But the processed Rhizoma Curcumae can in-hibit CYP1A2 and CYP2E1 and has an indue tive effect on the CYP3A4.%目的 用Cocktail探针药物法,研究生、醋莪术对大鼠CYP1A2、CYP3A4和CYP2E1活性的影响并进行比较,探讨其通过炮制增强或改变作用的趋向性.方法 将SD大鼠随机分组,生、醋莪术组分别给予生、醋莪术提取液(9 g·kg-1),以生理盐水为空白对照,在一定时间点采集血样,用HPLC检测探针药物在大鼠体内的代谢情况,以评价生、醋莪术对CYP450酶的影响.结果 生莪术的茶碱、氨苯酚和氯唑沙宗的代谢情况与空白组相比差异无显著性;但醋莪术与空白组相比,茶碱和氯唑沙宗的T12、Tmax 和AUC明显增加,CL/F明显降低;氨苯酚的T12 变

  12. Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays.

    Science.gov (United States)

    Nirogi, Ramakrishna; Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Srikakolapu, Surya Rao; Kalaikadhiban, Ilayaraja; Boggavarapu, Rajesh Kumar; Ponnamaneni, Ranjith Kumar; Ajjala, Devender Reddy; Bhyrapuneni, Gopinadh

    2015-02-01

    1. Chemical inhibition is the widely used method in reaction phenotyping assays for estimation of specific enzyme contribution to a given metabolic pathway. The results from phenotyping assays depend on the selectivity of chemical inhibitor and the concentration of inhibitor used in the incubation. 2. The higher protein concentrations used in the in vitro phenotyping assays will impact the inhibitory potency of chemical inhibitors. The objective of the study is to evaluate comprehensively the selectivity of chemical inhibitors and to guide in selecting appropriate concentration of the chemical inhibitors to be used in the phenotyping assays based on unbound fractions. 3. Selectivity of chemical inhibitors against nine major CYP450 isoforms was determined in liver microsomes using standard probe substrates. The unbound fractions of the selective inhibitors were determined in human liver microsomes using high-throughput equilibrium dialysis. Combining unbound inhibitor concentrations that are required to inhibit the CYP450 activities by 90% and unbound fractions of the chemical inhibitors in liver microsomes appropriate total concentrations of the inhibitors to be used in the phenotyping assays were reported. 4. The findings suggest that non-specific binding of the chemical inhibitors need to be taken into account while selecting concentrations for phenotyping assays.

  13. Inhibitory and inductive effects of Polygonum capitatum water extract on CYP450%头花蓼水提取物对CYP450酶诱导和抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    陆苑; 潘洁; 谢玉敏; 郑林; 黄勇; 王永林; 李勇军

    2015-01-01

    Aim To evaluate the inhibitive and induc-tive effects of Polygonum capitatum water extract on main cytochrome P450 isoforms in human and liver mi-crosomes of mouse in vitro for predicting the herb-drug interactions in clinical application. Methods The in vitro inhibitory effect was evaluated by incubating Po-lygonum capitatum water extract with the probe sub-strates of main phase I metabolic enzymes in human liver microsomes, including CYP1A2, CYP2E1, CYP2C9,CYP2C19 and CYP3A4. Mice were adminis-tered with Polygonum capitatum water extract at dosage of 0 . 58 g · kg-1 and 1 . 16 g · kg-1 by gastric lavage for successive 7 days and 14 days, then the cocktail-LC-MS/MS method was applied to assess the inductive effect of main CYP450 isoforms in mouse liver micro-somes. Results The IC50 values of Polygonum capita-tum water extract on main CYP450 isoforms in human liver microsomes were from 849 . 6 mg · L-1 to 2 287 mg·L-1 . Compared with the blank control group, the activites of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 7 d group were about 49 . 9 % and 21. 1 % higher ( P <0. 01, P < 0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 0. 58 g·kg-1 7 d group were 27. 6 % and 15. 5 % higher ( P <0. 01 , P <0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 14 d group were 67. 5 % and 32. 1 %higher (P<0. 01) respectively, while the activities of CYP1 A2 , CYP2 E1 and CYP2 C19 were not increased significantly in Polygonum capitatum treatment group. Conclusions Polygonum capitatum water extract do not show the inhibitory effect on main CYP450 in hu-man liver microsomes. There is induction on CYP2C9 and CYP3 A4 in mouse liver microsomes by Polygonum capitatum water extract.%目的:考察头花蓼水提取物对人肝微粒体CYP4505种亚型酶的体外抑制作用和对小鼠的体内诱导作用,从而预测发生药物相互作用的可能性,为临床合理用药提供科学依据。方法以探针底物代谢物生成法,考察头花

  14. Targeting CYP450 modulation to decrease the risk of induced cataract in the experimental model

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    Patel D

    2010-01-01

    Full Text Available Background: Diabetes is one of the major causes of cataract. Some drugs prescribed for the treatment of diabetes are the modulators of CYP450, which may alter the risk of cataract. Objective: To study the effect of CYP450 modulation in galactosemic cataract. Materials and Methods: Male Sprague-Dawley suckling rats were allotted to four groups (n = 6, as follows: Group 1: Normal control, Group 2: Galactose control, Group 3: CYP450 inhibitor pretreated and Group 4: CYP450 inducer pretreated. Cataract was induced in animals of all groups except group 1 by feeding them galactose (50%, 21 days after parturition. From the eighteenth day of life, CYP450 inhibitor (nifedipine; 8.1 mg/kg and CYP450 inducer (pioglitazone; 3.8 mg/kg were given orally to groups 3 and 4, respectively. The maturation pattern of the cataract was observed by an operating microscope, every third day. Biochemical changes in the lenses of all groups, for example, CYP450 activity expressed as ΅M NADPH oxidized / unit time, alterations in the levels of total proteins, soluble proteins, and reduced glutathione (GSH following the induction of cataract, were estimated. Results: The microscopic examination of the lenses indicated that CYP450 inhibitor pre-treatment delayed (fourteenth day the occurrence of cataract, while CYP450 inducer pretreatment demonstrated an early (ninth day cataract as compared to galactose control rats (twelfth day. A significant decrease and increase in CYP450 activity was observed with the CYP450 inhibitor and inducer pre-treatment, respectively. There was no alteration in the GSH level, but a significant increase in total and soluble protein was found in groups 3 and 4 as compared to group 2. Conclusion: CYP450 may have a role in the initiation of cataract without any effect on the maturation pattern, as revealed by the delayed occurrence of cataract with the CYP450 inhibitor and an early onset of cataract with the CYP450 inducer.

  15. In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2).

    Science.gov (United States)

    Labrie, Philippe; Maddaford, Shawn P; Lacroix, Jacques; Catalano, Concettina; Lee, David K H; Rakhit, Suman; Gaudreault, René C

    2007-06-01

    Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.

  16. Antidepressant-induced akathisia-related homicides associated with diminishing mutations in metabolizing genes of the CYP450 family

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    Lucire Y

    2011-08-01

    Full Text Available Yolande Lucire, Christopher CrottyEdgecliff Centre, Edgecliff, NSW, AustraliaPurpose: To examine the relation between variant alleles in 3 CYP450 genes (CYP2D6, CYP2C9 and CYP2C19, interacting drugs and akathisia in subjects referred to a forensic psychiatry practice in Sydney, Australia.Patients and methods: This paper concerns 10/129 subjects who had been referred to the first author’s practice for expert opinion or treatment. More than 120 subjects were diagnosed with akathisia/serotonin toxicity after taking psychiatric medication that had been prescribed for psychosocial distress. They were tested for variant alleles in CYP450 genes, which play a major role in Phase I metabolism of all antidepressant and many other medications. Eight had committed homicide and many more became extremely violent while on antidepressants. Ten representative case histories involving serious violence are presented in detail.Results: Variant CYP450 allele frequencies were higher in akathisia subjects compared with random primary care patients tested at the same facility. Ten subjects described in detail had variant alleles for one or more of their tested CYP450 genes. All but two were also on interacting drugs, herbals or illicit substances, impairing metabolism further. All those described were able to stop taking antidepressants and return to their previously normal personalities.Conclusion: The personal, medical, and legal problems arising from overuse of antidepressant medications and resulting toxicity raise the question: how can such toxicity events be understood and prevented? The authors suggest that the key lies in understanding the interplay between the subject’s CYP450 genotype, substrate drugs and doses, co-prescribed inhibitors and inducers and the age of the subject. The results presented here concerning a sample of persons given antidepressants for psychosocial distress demonstrate the extent to which the psychopharmacology industry has

  17. Effect of water-miscible organic solvents on CYP450-mediated metoprolol and imipramine metabolism in rat liver microsomes

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    T S Shah

    2015-01-01

    Full Text Available The catalytic activity of cytochrome P450 enzymes is known to be affected by presence of organic solvents in in vitro assays. However, these effects tend to be variable and depend on the substrate and CYP450 isoform in question. In the present study, we have investigated effect of ten water miscible organic solvents (methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, dimethylsulphoxide, dimethylformamide, dioxane and PEG400 on water soluble substrates of CYP450, metoprolol and imipramine, at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. Organic solvents studied had a concentration dependent inhibitory effect on the metoprolol and imipramine metabolism activity. Metoprolol metabolism was found to be more susceptible to the organic solvents, almost all the ten solvents had more or less inhibitory effect compared to imipramine metabolism. Except acetone, PEG400 and dimethylsulphoxide, all solvents had ~50% inhibition of total metoprolol metabolism activity, while in case of imipramine metabolism activity, only n-propanol, isopropanol and PEG400 had ~50% inhibition at 1% v/v. Interestingly, methanol, dimethylsulphoxide and acetonitrile had negligible effect on the imipramine metabolism (less than 10% inhibition at 1% v/v while, total metoprolol metabolism activity was substantially inhibited by these solvents (MeOH 52%, DMSO 29% and ACN 47% at 1% v/v. In both cases, dioxane was found to be the most inhibitory solvent (~90% inhibition at 1% v/v.

  18. Effect of Water-miscible Organic Solvents on CYP450-mediated Metoprolol and Imipramine Metabolism in Rat Liver Microsomes.

    Science.gov (United States)

    Shah, T S; Kamble, S H; Patil, Pranali G; Iyer, K R

    2015-01-01

    The catalytic activity of cytochrome P450 enzymes is known to be affected by presence of organic solvents in in vitro assays. However, these effects tend to be variable and depend on the substrate and CYP450 isoform in question. In the present study, we have investigated effect of ten water miscible organic solvents (methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, dimethylsulphoxide, dimethylformamide, dioxane and PEG400) on water soluble substrates of CYP450, metoprolol and imipramine, at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. Organic solvents studied had a concentration dependent inhibitory effect on the metoprolol and imipramine metabolism activity. Metoprolol metabolism was found to be more susceptible to the organic solvents, almost all the ten solvents had more or less inhibitory effect compared to imipramine metabolism. Except acetone, PEG400 and dimethylsulphoxide, all solvents had ~50% inhibition of total metoprolol metabolism activity, while in case of imipramine metabolism activity, only n-propanol, isopropanol and PEG400 had ~50% inhibition at 1% v/v. Interestingly, methanol, dimethylsulphoxide and acetonitrile had negligible effect on the imipramine metabolism (less than 10% inhibition at 1% v/v) while, total metoprolol metabolism activity was substantially inhibited by these solvents (MeOH 52%, DMSO 29% and ACN 47% at 1% v/v). In both cases, dioxane was found to be the most inhibitory solvent (~90% inhibition at 1% v/v).

  19. Cloning and expression of SgCYP450-4 from Siraitia grosvenorii

    Directory of Open Access Journals (Sweden)

    Dongping Tu

    2016-10-01

    Full Text Available CYP450 plays an essential role in the development and growth of the fruits of Siraitia grosvenorii. However, little is known about the SgCYP450-4 gene in S. grosvenorii. Here, based on transcriptome data, a full-length cDNA sequence of SgCYP450-4 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR and rapid-amplification of cDNA ends (RACE strategies. SgCYP450-4 is 1677 bp in length (GenBank accession No. AEM42985.1 and contains a complete open reading frame (ORF of 1422 bp. The deduced protein was composed of 473 amino acids, the molecular weight is 54.01 kDa, the theoretical isoelectric point (PI is 8.8, and the protein was predicted to possess cytochrome P450 domains. SgCYP450-4 gene was highly expressed in root, diploid fruit and fruit treated with hormone and pollination. At 10 days after treatment with pollination and hormones, the expression of SgCYP450-4 had the highest level and then decreased over time, which was consistent with the development of fruits of S. Grosvenorii. Hormonal treatment could significantly induce the expression of SgCYP450-4. These results provide a reference for regulation of fruit development and the use of parthenocarpy to generate seedless fruit, and provide a scientific basis for the production of growth regulator application agents.

  20. Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins.

    Science.gov (United States)

    Thomson, Scott; Edin, Matthew L; Lih, Fred B; Davies, Michael; Yaqoob, Muhammad M; Hammock, Bruce D; Gilroy, Derek; Zeldin, Darryl C; Bishop-Bailey, David

    2015-08-01

    Vascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors.

  1. Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Scott [Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU (United Kingdom); Edin, Matthew L.; Lih, Fred B. [Division of Intramural Research, NIEHS/NIH, Research Triangle Park, NC 27709 (United States); Davies, Michael [Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU (United Kingdom); Yaqoob, Muhammad M. [Barts and the London, Queen Mary University, Charterhouse Square, London EC1M 6BQ (United Kingdom); Hammock, Bruce D. [Department of Entomology and Comprehensive Cancer Center, University of California, Davies, CA 95616-8584 (United States); Gilroy, Derek [University College London, University Street, London (United Kingdom); Zeldin, Darryl C. [Division of Intramural Research, NIEHS/NIH, Research Triangle Park, NC 27709 (United States); Bishop-Bailey, David, E-mail: dbishopbailey@rvc.ac.uk [Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU (United Kingdom)

    2015-08-07

    Vascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors. - Highlights: • We examined oxylipin production in different

  2. 酒精性股骨头缺血性坏死与CYP450关系研究进展%Research progress in the relationship between alcohol-induced avascular necrosis of the femoral head and CYP450

    Institute of Scientific and Technical Information of China (English)

    李盛华; 孙凤歧; 周明旺; 郭铁峰; 穆欢喜

    2013-01-01

    Osteoarthrosis of the femoral head (ONFH),which is caused by long term over-dose alcohol drinking and happens mostly in young adults at the age of 20-50 years old,is a disease that severely threatens human health.Along with the changes of people's lifestyle,clinical cases of alcohol-induced ANFH (AIONFH) increase gradually.The pathogenesis of AIANFH remains unclear.Some scholars consider that this disease is related to some genes,but scholars from different countries have diverse reports and different opinions.This paper reviews the relationship between AIANFH and CYP450 from the national and international studies.%目的 长期过量饮酒引起的股骨头缺血性坏死(osteoarthrosis of femoral head,ONFH)是一种严重威胁人类健康的疾病,多发于20 ~ 50岁的青壮年.随着人们生活方式的改变,酒精性股骨头坏死的临床病例日渐增多,酒精性股骨头坏死(alcohol-induced osteoarthrosis of femoral head,AIONFH)的发病机制尚不完全清楚,很多学者认为主要与一些基因有关,各国学者报道情况不一,各持己见;本文就国内外酒精性股骨头坏死与CYP450的关系作一综述.

  3. CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Xiao-ping ZHAO; Jiao ZHONG; Xiao-quan LIU; Guang-ji WANG

    2007-01-01

    Aim: To study the metabolism of vinflunine and the effects of selective cyto-chrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. Methods: Individual selective CYP450 inhibitors were used to inves-tigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M1 and M2 in human liver microsomes.Results: Vinflunine was rapidly metabolized to 2 metabolites: M1 and M2 in human liver microsomes. M1 and M2 were tentatively presumed to be the N-oxide metabo-lite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M1, and competitively inhibited the formation of M2, while α-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M1 and M2. Conclusion: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.

  4. CYP450s analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development.

    Science.gov (United States)

    Ventura, Tomer; Bose, Utpal; Fitzgibbon, Quinn P; Smith, Gregory G; Shaw, P Nicholas; Cummins, Scott F; Elizur, Abigail

    2017-07-01

    Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  5. Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure.

    Science.gov (United States)

    Burkina, V; Zamaratskaia, G; Randak, T; Li, Z H; Fedorova, G; Pickova, J; Zlabek, V

    2012-05-01

    Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 μg l(-1)) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 羟基喜树碱的代谢与 CYP450的相关性研究%Correlation between Hydroxycamptothecin Metabolism and CYP450 Activity

    Institute of Scientific and Technical Information of China (English)

    胡巍; 胡文晋; 方芸

    2014-01-01

    Objective: To investigate the effect of hydroxycamptothecin (HCPT) on CYP450 in rat liver microsomes in vitro, providing reference for clinical rational use of drug combination therapy. Methods:Probe drugs of testosterone, tartrate, phenacetin, diclofenac sodium, acetaminophen, and omeprazole as well as HCPT were added to incubation systems of rat liver microsomes in vitro, the concentrations of the probe drugs in blank or with HCPT were determined by HPLC after incubation and the differences of metabolic rates were compared to evaluate the effects of HCPT on the activities of the six CYP450 subtypes. Results: L-HCPT at 50~200 μmol·L-1 inhibited the metabolism of testosterone and acetaminophen and had no effect on the metabolism of metoprolol tartrate, phenacetin, diclofenac sodium and omeprazole. Conclusion: We concluded that HCPT is affected by CYP3A4 and CYP2E1 in the metabolic process and competitively inhibited the metabolism of testosterone and acetaminophen in liver microsomes.%目的:研究羟基喜树碱(HCPT)在体外小鼠肝微粒体中对 CYP450活性的影响,为临床合理联合用药提供参考。方法:在小鼠体外肝微粒体中分别加入六种亚型酶的探针药物对乙酰氨基酚、非那西丁、双氯芬酸钠、睾酮、酒石酸美托洛尔、奥美拉唑和羟基喜树碱溶液,在优化的孵育体系下温孵。用 HPLC 法测定各空白对照组和羟基喜树碱溶液中各探针药物代谢产物的浓度并比较代谢率的差异,以评价羟基喜树碱对各亚型酶活性的影响。结果:在代谢反应过程中,在50~200μmol·L-1内,羟基喜树碱的浓度与孵育体系中 CYP3A4的特异性底物睾酮、CYP2E1的特异性底物对乙酰氨基酚的剩余药量呈正比例关系,且具显著性差异(P<0.05),与其他四种探针药物的剩余药量无明显的浓度依存关系(P>0.05)。结论:HCPT 在肝微粒体的代谢反应过程中受代谢酶 CYP3A4和 CYP2E1的

  7. [Compatibility of Banxia Houpo decoction on hepatic CYP450 and renal organicion transporters in mice].

    Science.gov (United States)

    Wang, Fumeng; Lu, Yan; Kong, Lingdong

    2011-01-01

    By analyzing the related indicators [hepatic CYP450 subtype and renal organic anion and cation transporters (OATs and OCTs)], the present study investigated the effects of formula Banxia Houpo decoction principal drug pinellia, assistant drug magnolia, their compatibility and the principle of the whole decoction on the metabolism ability in the liver and the transport change in the kidney of mice. Biochemical and molecular (RT-PCR and western blotting) results indicated that pinellia increased activity and expression of hepatic Cyp2e1 and Cyp3a11 in mice, respectively. Pinellia and magnolia increased expression of renal OAT1, OAT3, OCT1 and OCT2 in mice, respectively. The compatibility of pinellia and magnolia, as well as Banxia Houpo decoction synergistically restrained the activated effect of pinellia on hepatic Cyp2e1, therefore avoiding liver peroxidation and reducing toxicity potential. The compatibility of this drug pair and Banxia Houpo decoction not only reduced activity and expression of hepatic Cyp3a11 to control drug metabolism speed, but also balanced the expression of renal OAT1/3 and OCT1/2 to enhance drug efficacy. The effect of compatibility of Banxia Houpo decoction was better than that of pinellia and magnolia pair, and the normal dosage was better than the high dosage. The present study proved the advantage of the compatibility of pinellia combined with magnolia and the principle of Banxia Houpo decoction, which related to hepatic CYP450 and renal organic ion transporters, and guided the clinical use of Banxia Houpo decoction to exert its toxicity reduction and efficacy enhancement.

  8. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  9. Impact of cardiovascular drug Quick-Acting Heart Reliever and Tongxinluo capsules on CYP450s in rats after successive administration%心血管药物速效救心丸和通心络胶囊对大鼠细胞色素P450酶的影响

    Institute of Scientific and Technical Information of China (English)

    卜明华; 郑咏秋; 张颖; 李利群; 刘建勋; 刘东春

    2011-01-01

    AIM: To investigate the influence of Quick-Acting Heart Reliever and Tongxinluo capsules on drug metablic enzyme cytochrome P450(CYP450) system in rats. METHODS: Rat liver and intestine microsome were prepared after a ten-day continuous administration of QuickActing Heart Reliever and Tongxinluo capsules.The content of total CYP450s in rat liver microsome was measured by UV-visible absorption spectrometry. Meanwhile, the protein expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A in the liver microsome and CYP3A in the small intestine microsome was detected by western blotting assay. RESULTS: No significant difference of total CYP450 content was observed between each treatment group and control group(P>0.05). Western blot analysis showed that Quick-Acting Heart Reliever induced the CYP1A2 in liver microsome, but inhibited the liver CYP2C11 and CYP3A in intestine microsome; Tongxinluo capsules induced the expression of CYP1A2 and CYP2E1 in liver microsome. CONCLUSION: Quick-Acting Heart Reliever and Tongxinluo capsules exhibit induction/inhibition on the expression of CYP450 isoforms. The results suggest that the related possible side effects of drug-drug interaction could not be neglected in the period of clinical therapy.%目的:观察速效救心丸和通心络胶囊对大鼠药物代谢酶细胞色素P450(CYP450)的影响.方法:给予大鼠试验药物后制备肝及小肠微粒体,CO还原差示光谱法测定肝微粒体CYP450含量,蛋白免疫印迹法检测肝微粒体中CYP1A2、CYP2C11、CYP2E1、CYP3A和肠微粒体中CYP3A蛋白的表达量.结果:速效救心丸组和通心络胶囊组与空白对照组比较,大鼠肝脏CYP450含量无显著变化(P>0.05);但是West-ern blotting实验对肝脏和肠道中多种CYP450酶表达量测定的结果显示,速效救心丸可诱导肝脏CYP1A2表达,但抑制肝脏CYP2C11和肠道CYP3A表达(P<0.05);通心络胶囊可诱导大鼠肝脏CYP1A2、CYP2E1蛋白表达(P<0.05).结论:速效救心丸和通心络胶囊对大鼠肝脏CYP

  10. Drugs impact on CYP-450 enzyme family: A pharmacogenetical study of response variation

    Directory of Open Access Journals (Sweden)

    Kalra Kapil

    2009-01-01

    Full Text Available Pharmacogenetics is the study of genetic basis in the individual response to drugs. A thorough knowledge of this will lead to a future where tailor-made drugs, suiting an individual, can be used. Scandinavian countries have been known for wide usage of pharmacogenetics and the most widely used application is for genotyping CYP2D6 in treating psychiatric illness. The CYP-450 enzyme, a super family of microsomal drug-metabolizing enzymes, is the most important of enzymes that catalyzes phase-I drug metabolism reaction. CYP2D6 is a member of this family and it has been most intensively studied and the best example of pharmacogenetics variation in drug metabolism. Neuro-transmitter and drug acting CNS viz. codeine, dextromethorphan, metoprolol and tryptyline etc. are well metabolized by this enzyme. Thus, CYP2D6 is one of the most important and responsible enzymes which regulates bioavailability and metabolism of drug. Presently 75 alleles of CYP2D6 have been described which are responsible for variance of metabolism and toxicity of drugs. Thus, by determining variance of CYP2D6 using molecular approaches viz., PCR, real-time PCR, DNA micro-array and molecular docking can determine the adverse effects, drug toxicity, bioavailability and therapeutic potential of new drug.

  11. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  12. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  13. Chronic unpredicted mild stress-induced depression alter saxagliptin pharmacokinetics and CYP450 activity in GK rats

    Directory of Open Access Journals (Sweden)

    Zhengchao Xia

    2016-01-01

    Full Text Available Background. This study was to explore the pharmacokinetics of saxagliptin (Sax in Goto–Kakizaki (GK rats complicated with depression induced by chronic unpredicted mild stress (CUMS. The comorbidity of diabetic patients with depression is becoming more and more epidemic. Whether depression mental disorder alters the pharmacokinetics of hypoglycemic drugs in diabetes patients is not clear.Methods. Five-week-old male GK rats were kept in the cage for 7 weeks in a specific pathogen free (SPF-grade lab until the emergence of diabetes and were then divided into two groups: control group and depression model group. Rats in the CUMS-induced depression group were exposed to a series of stressors for 8 weeks. Plasma serotonin and dopamine levels and behavior of open-field test were used to confirm the establishment of the depression model. All rats were given 0.5 mg/kg Sax orally after 8 weeks and blood samples were collected at different time points. The Sax concentration was assayed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS. The CYP450 activity of the liver microsomes was determined by using cocktails of probe drugs in which the activities of CYP enzymes were assessed through the determination of the production of the probe drugs.Results. Statistically significant differences in Sax pharmacokinetics were observed for area under curve, clearance, peak concentration, peak time and mean residence time between the depression rats and the control rats, while no statistical differences were observed for half-time and distribution volume by HPLC-MS/MS analysis. The CYP450 activity had different changes in the depression group.Conclusions. These results indicated that CUMS-induced depression alters the drug metabolic process of Sax and CYP450 activity of the liver microsomal enzymes in GK rats.

  14. Antidepressant-induced akathisia-related homicides associated with diminishing mutations in metabolizing genes of the CYP450 family

    OpenAIRE

    Lucire Y; Crotty C

    2011-01-01

    Yolande Lucire, Christopher CrottyEdgecliff Centre, Edgecliff, NSW, AustraliaPurpose: To examine the relation between variant alleles in 3 CYP450 genes (CYP2D6, CYP2C9 and CYP2C19), interacting drugs and akathisia in subjects referred to a forensic psychiatry practice in Sydney, Australia.Patients and methods: This paper concerns 10/129 subjects who had been referred to the first author’s practice for expert opinion or treatment. More than 120 subjects were diagnosed with akathisia/...

  15. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

    Directory of Open Access Journals (Sweden)

    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  16. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross...

  17. Simultaneous determination of four probe drugs of CYP450 in rat plasma by RP-HPLC%RP-HPLC法同时测定大鼠血浆中4种CYP450探针药物的浓度

    Institute of Scientific and Technical Information of China (English)

    张香凝; 李想; 刘高峰

    2012-01-01

    Objective: To establish a method for simultaneously determining the concentrations of four probe drugs of CYP450 including caffeine,tolbutamide,metoprolol and dapsone in rat plasma. Methods:The four drugs were extracted with one solvent system and simultaneously determined by RP - HPLC on a Diamonsil C18 column (4. 6 mm×200 mm,5 μm) with the mobile phase consisting of methanol - water - glacial acetic acid -triethylamine(40:60:0.4:0.5) at the flow rate of 0 -16 min 0.5 mL · min-1,after 16 min 1.4 mL · min-1.The detection wavelength for caffeine,tolbutamide, metoprolol and dapsone were at 271 ran,229 nm,225 nm and 295 ran respectively,and the column temperature was 35 ℃. Results: The four drugs and the internal standard(phenacetin) were separated well under the above RP - HPLC conditions. There were good linearity of caffeine,tolbutamide,metoprolol ,and dapsone in the ranges of 0.10 -100.0 μg·mL-1 (r=0.9956),0.25 -25.0 μg · mL-1(r =0.9997) ,0.050-20.0 μg · mL-1(r =0.9995),0.050 -50.0 μg · mL-1 (r=0.9997). The recoveries were 71.43% - 87.17% , 86. 22% - 97. 80% ,77. 29% -91.26% ,67.46% -98.47%,respectively. Conclusion: The method is reliable,accurate,sensitive,and suitable for determining plasma concentrations of the four probe drugs (caffeine, tolbutamide, metoprolol and dapsone) in rats in a single run, which could be employed in evaluating the effects of drugs on CYP450 subtype enzymes activities and in relatived studies.%目的:建立大鼠血浆中4种细胞色素P450亚型酶(CYP1A2、CYP2C9、CYP2D6和CYP3A4)的探针药物咖啡因、甲苯磺丁脲、美托洛尔和氨苯砜同时测定的方法,为相关研究提供方法学参考.方法:用一种提取溶剂系统对4种药物同时提取,并用反相高效液相色谱法(RP-HPLC)同时测定,选用Diamonsil C18色谱柱(4.6 mm× 200 mm,5μm);流动相为甲醇-水-冰乙酸-三乙胺(40∶60∶0.4∶0.5);检测波长:咖啡因271 nm,甲苯磺丁脲229 nm,美托洛尔225 nm,氨苯砜295

  18. 麻醉药物代谢酶CYP450酶系基因组学研究进展%Cytochrome P450 pharmacogenomics of anesthetics

    Institute of Scientific and Technical Information of China (English)

    汪国香; 方向明; 郑汉光

    2005-01-01

    遗传多态性是造成个体对药物反应差异的重要因素之一,其中CYP450酶系的基因多态性可能导致种族间及个体间对同一底物代谢能力的不同.现就镇静安定药、吸入麻醉药、局麻药、镇痛药的药物效应与CYP450基因多态性的相关性进行综述,为临床个体化给药,实现个体优化治疗提供新思路.

  19. [Screen potential CYP450 2E1 inhibitors from Chinese herbal medicine based on support vector regression and molecular docking method].

    Science.gov (United States)

    Chen, Xi; Lu, Fang; Jiang, Lu-di; Cai, Yi-Lian; Li, Gong-Yu; Zhang, Yan-Ling

    2016-07-01

    Inhibition of cytochrome P450 (CYP450) enzymes is the most common reasons for drug interactions, so the study on early prediction of CYPs inhibitors can help to decrease the incidence of adverse reactions caused by drug interactions.CYP450 2E1(CYP2E1), as a key role in drug metabolism process, has broad spectrum of drug metabolism substrate. In this study, 32 CYP2E1 inhibitors were collected for the construction of support vector regression (SVR) model. The test set data were used to verify CYP2E1 quantitative models and obtain the optimal prediction model of CYP2E1 inhibitor. Meanwhile, one molecular docking program, CDOCKER, was utilized to analyze the interaction pattern between positive compounds and active pocket to establish the optimal screening model of CYP2E1 inhibitors.SVR model and molecular docking prediction model were combined to screen traditional Chinese medicine database (TCMD), which could improve the calculation efficiency and prediction accuracy. 6 376 traditional Chinese medicine (TCM) compounds predicted by SVR model were obtained, and in further verification by using molecular docking model, 247 TCM compounds with potential inhibitory activities against CYP2E1 were finally retained. Some of them have been verified by experiments. The results demonstrated that this study could provide guidance for the virtual screening of CYP450 inhibitors and the prediction of CYPs-mediated DDIs, and also provide references for clinical rational drug use. Copyright© by the Chinese Pharmaceutical Association.

  20. Characterization of the Human LPIN1-encoded Phosphatidate Phosphatase Isoforms*

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M.

    2010-01-01

    The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210–9218). In this work, we characterized human lipin 1 α, β, and γ isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the α, β, and γ isoforms were dependent on Mg2+ or Mn2+ ions at pH 7.5 at 37 °C. The activities were inhibited by concentrations of Mg2+ and Mn2+ above their optimums and by Ca2+, Zn2+, N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 °C. The α, β, and γ activities followed saturation kinetics with respect to the molar concentration of PA (Km values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number ∼2) kinetics with respect to the surface concentration of PA (Km values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (kcat) for the α, β, and γ isoforms were 68.8 ± 3.5, 42.8 ± 2.5, and 5.7 ± 0.2 s−1, respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity. PMID:20231281

  1. Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay%CYP450酶与中药代谢相互作用及酶活性测定的研究进展

    Institute of Scientific and Technical Information of China (English)

    陆兔林; 苏联麟; 季德; 顾薇; 毛春芹

    2015-01-01

    药物对于人体属于外源性化合物,进入人体后将被许多酶所代谢.CYP450酶作为主要的代谢酶,且是重要的药物Ⅰ相代谢酶,人体约75%的药物代谢是通过CYP450酶进行,而且CYP450酶是引起中药-中药(药对)、中药-西药等联合用药时药物相互作用产生的关键因素.为明确代谢酶与中药代谢的相互作用关系,普遍以酶活性作为评价指标,即以CYP450酶活性的增强和降低来推断中药有效成分及提取物等对酶的诱导或抑制作用.目前,代谢酶活性测定的常用方法是Cocktail探针药物法,其中探针底物的选择是关键.这对研究药物在机体内的吸收、分布、代谢及排泄(ADME)过程有着重大意义.该文就中药、中药有效成分、中药提取物及Cocktail探针底物等与CYP450酶的相互作用进行了综述,以期对将来的相关研究有借鉴和指导意义.

  2. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  3. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  4. Cloning and characterization of the neural isoforms of human dystonin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, A.; Dalpe, G.; Mathieu, M.; Kothary, R. [Univ. of Montreal, Quebec (Canada)

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.

  5. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Directory of Open Access Journals (Sweden)

    Marina Ilicic

    2017-01-01

    Full Text Available Background. Regulation of myometrial progesterone receptor (PR expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture.

  6. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    Science.gov (United States)

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

  7. CYP450氧化还原酶的药物基因组学研究进展%Advances in pharmacogenomics of cytochrome P450 oxidoreductase

    Institute of Scientific and Technical Information of China (English)

    胡蕾; 高利臣; 卓伟; 周宏灏; 范岚

    2012-01-01

    The redox reaction of the cytochrome P450 enzymes ( CYP) are important physiological and biochemical reactions in the human body, involved in the metabolism of endogenous and exogenous compounds and steroids synthesis. POR (cytochrome P450 oxidoreductase) is the only electron donor for all the hepatic microsomal CYP enzymes. Not only acts as an electron donor involved in drug metabolism mediated by CYP enzymes, POR also directly induces the transformation and metabolism of some anti-tumor precursors. Therefore POR plays an important role in drug metabolism. The gene encoding human POR is highly polymorphic, which is of great clinical significance by having an significant effect on the metabolism and even curative effects of clinically used drugs. Studies on the pharmacogenomics of POR in recent years are summarized as follows.%细胞色素P450氧化酶(cytochrome P450enzymes,CYP)的氧化还原反应是人体内重要的生理生化反应,参与许多内、外源化合物的代谢和激素类化合物的合成.CYP450氧化还原酶(cytochrome P450 oxidoreductase,POR)是所有肝微粒体内CYP酶的唯一电子供体.POR不仅可作为电子供体参与由CYP介导的药物代谢,而且可通过1-电子还原反应直接介导一些抗肿瘤前体药物的代谢和转化.可见,POR在药物代谢过程中发挥着极其重要的作用.众多研究证实,编码人POR的基因具有遗传多态性,对临床药物代谢乃至疗效有着显著影响,具有重要的临床意义.下面对近年来POR的药物基因组学最新研究进展作一综述.

  8. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  9. Isoenzimas do CYP450 e biotransformação de drogas Biotransformation of drugs

    Directory of Open Access Journals (Sweden)

    Flávia Daniela Pussi

    2000-05-01

    Full Text Available A administração de uma droga em seres humanos, muitas vezes, gera uma resposta farmacológica inesperada, que pode ser determinada pela concentração plasmática anormal da mesma, podendo produzir, como conseqüência, vários efeitos indesejáveis. A avaliação deste fato revela que tais efeitos podem estar diretamente relacionados ao processo de biotransformação. A biotransformação de drogas envolve reações bioquímicas específicas, em que cada etapa conta com a participação de seqüências enzimáticas altamente ordenadas. A produção dessas enzimas, por sua vez, é determinada geneticamente através de processos de tradução e transcrição. Uma anormalidade nos genes codificadores da produção enzimática responsável pela biotransformação de drogas pode gerar alterações na resposta terapêutica. Além disso, inúmeras substâncias podem interferir no processo de biotransformação, através da inibição ou da indução de enzimas responsáveis pelo mesmo. O propósito deste trabalho é ressaltar a biotransformação como uma das principais etapas farmacocinéticas responsáveis pela atividade terapêutica das drogas e analisar os fatores genéticos que possam interferir neste processoDrug administration to human beings often generates an unexpected pharmacological response that may be determined by its abnormal plasmatic concentration and may consequently produce several undesirable effects. The analysis of this fact reveals that these effects are directly connected with the biotransformation process. Biotransformation of drugs generally involves specific biochemical reactions in each of whose stages highly ordered enzymatic sequences participate. The production of these enzymes in its turn is genetically determined through translation and transcription processes. An abnormality in the codifying genes of the enzyme production responsible for the biotransformation of drugs may generate alterations in the therapeutical

  10. Expression of aquaporin isoforms during human and mouse tooth development.

    Science.gov (United States)

    Felszeghy, S; Módis, L; Németh, P; Nagy, G; Zelles, T; Agre, P; Laurikkala, J; Fejerskov, O; Thesleff, I; Nielsen, S

    2004-04-01

    Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

  11. CYP450酶翻译后修饰对其活性影响的研究进展%Effects of posttranslational modification on the activity of cytochrome P450: current progress

    Institute of Scientific and Technical Information of China (English)

    黎玉华; 毕惠嫦; 黄民

    2011-01-01

    药物代谢酶,特别是CYP450酶的活性调节一直是药物代谢的研究热点,大多数现有的研究都集中在转录和翻译水平上探讨化合物对其基因表达和蛋白水平的影响.但是随着研究的深入,发现在翻译后水平的修饰作用也可能对代谢酶的活性有重要影响.近年来,翻译后修饰作用对酶活性的影响越来越受到重视,并发现了一些对代谢酶活性有重要影响的修饰方式,如磷酸化、硝基化、泛素化等.本文对近年来翻译后修饰对CYP450酶活性影响的研究进行综述.%Regulation of the activity of CYP450 has always been research focus of drug metabolism. The effect of compounds on the mRNA and protein expression level of CYP450 is the main purpose of most of the existing reports. In recent years, the protein modification in the posttranslation level has been found to participate in maintaining the proper function of CYP450, thus effect of posttranslational modification on the enzyme activity has been paid more and more attention. Postkanslational modifications including phosphorylation nitration and ubiquitination have been described to regulate the activity of CYP450. In this paper, recent developments in the effects of posttranslational modifications on the activity of CYP450 have been reviewed.

  12. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  13. Does Pharmacogenetic Testing for CYP450 2D6 and 2C19 among Patients with Diagnoses within the Schizophrenic Spectrum Reduce Treatment Costs?

    DEFF Research Database (Denmark)

    Herbild, Louise; Andersen, Stig Ejdrup; Werge, Thomas;

    2013-01-01

    The effect of pharmacogenetic testing for CYP450 2D6 and 2C19 on treatment costs have not yet been documented. This study used Danish patient registers to calculate health care costs of treating patients with diagnoses within the schizophrenic spectrum for one year with or without pharmacogenetic...... testing for polymorphisms in the genes for the CYP2D6 and CYP2C19 enzymes. In a randomized, controlled trial, stratified with respect to metabolizer genotype, 104 patients were assigned to treatment based on pharmacogenetic testing and 103 patients to treatment as usual. Random exclusion of extensive...

  14. The landscape of isoform switches in human cancers

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

    2017-01-01

    developed methods for identification and visualization of isoform switches with predicted functional consequences. Using these methods, we characterized isoform switching in RNA-seq data from >5,500 cancer patients covering 12 solid cancer types. Isoform switches with potential functional consequences were...... highly predictive of patient survival independent of cancer types. Our data constitute an important resource for cancer researchers, available through interactive web tools. Moreover, our methods, available as an R package, enable systematic analysis of isoform switches from other RNA-seq datasets...

  15. Insight into the binding interactions of CYP450 aromatase inhibitors with their target enzyme: a combined molecular docking and molecular dynamics study.

    Science.gov (United States)

    Galeazzi, Roberta; Massaccesi, Luca

    2012-03-01

    CYP450 aromatase catalyzes the terminal and rate-determining step in estrogen synthesis, the aromatization of androgens, and its inhibition is an efficient approach to treating estrogen-dependent breast cancer. Insight into the molecular basis of the interaction at the catalytic site between CYP450 aromatase inhibitors and the enzyme itself is required in order to design new and more active compounds. Hence, a combined molecular docking-molecular dynamics study was carried out to obtain the structure of the lowest energy association complexes of aromatase with some third-generation aromatase inhibitors (AIs) and with other novel synthesized letrozole-derived compounds which showed high in vitro activity. The results obtained clearly demonstrate the role of the pharmacophore groups present in the azaheterocyclic inhibitors (NSAIs)-namely the triazolic ring and highly functionalized aromatic moieties carrying H-bond donor or acceptor groups. In particular, it was pointed out that all of them can contribute to inhibition activity by interacting with residues of the catalytic cleft, but the amino acids involved are different for each compound, even if they belong to the same class. Furthermore, the azaheterocyclic group strongly coordinates with the Fe(II) of heme cysteinate in the most active NSAI complexes, while it prefers to adopt another orientation in less active ones.

  16. Inhibitory effect of salvianolate on human cytochrome P450 3A4 in vitro involving a noncompetitive manner.

    Science.gov (United States)

    Qin, Chong-Zhen; Ren, Xian; Zhou, Hong-Hao; Mao, Xiao-Yuan; Liu, Zhao-Qian

    2015-01-01

    Salvianolic acid B (Sal B), which is purified from Danshen, is a popular herb extract. Sal B has anti-oxidative, anti-inflammatory, anti-hypoxic, anti-arteriosclerotic and anti-apoptotic properties. This substance can also ameliorate brain injury or neurodegenerative diseases. The listed drug Salvianolate, which contains a substantial amount of Sal B, has been used for the treatment of coronary heart disease. Our present work aimed to evaluate the inhibitory effect of salvianolate on seven cytochrome P450 isoforms (CYP450), namely, CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, in human liver microsomes (HLMs) and recombinant enzymes through high-performance liquid chromatography (HPLC) assay. Salvianolate have a potent inhibitory effect on CYP3A4 activity with IC50 values of 1.438 (HLMs) and 3.582 (recombinant cDNA-expressed CYP3A4) mg/L, respectively. Salvianolate strongly dose, but not time-dependently decreased CYP3A4 activity in HLMs. The typical Lineweaver-Burk plots showed that Salvianolate inhibited CYP3A4 activity noncompetitively, with a Ki value of 2.27 mg/L in HLMs. Other CYP450 isoforms are not markedly affected by Salvianolate. These findings indicate that salvianolate may be involved in potential drug interactions when co-administrated with CYP3A4 substrates.

  17. p53 Family: Role of Protein Isoforms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  18. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

  19. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  20. Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer

    Science.gov (United States)

    2009-02-01

    TITLE: Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer PRINCIPAL INVESTIGATOR: Jianghua Wang, M.D...6 JAN 2009 / / /4. TITLE AND SUBTITLE Biological Effects of TMPRSS2/ERG Fusion Isoforms in Human Prostate Cancer 5a. CONTRACT NUMBER W81XWH...quantitative RT-PCR arrays we have identified candidate mediators of these phenotypic effects . We propose to extend these studies to primary prostate epithelial

  1. Detection of VEGF-A(xxx)b isoforms in human tissues.

    Science.gov (United States)

    Bates, David O; Mavrou, Athina; Qiu, Yan; Carter, James G; Hamdollah-Zadeh, Maryam; Barratt, Shaney; Gammons, Melissa V; Millar, Ann B; Salmon, Andrew H J; Oltean, Sebastian; Harper, Steven J

    2013-01-01

    Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

  2. 西尼地平在人肝微粒体内代谢及代谢抑制%Metabolism and metabolic inhibition of cilnidipine in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    柳晓泉; 赵阳; 李丹; 钱之玉; 王广基

    2003-01-01

    目的:在体外研究西尼地平在人肝微粒体内的代谢及选择性细胞色素P-450(CYP450)酶抑制剂对其代谢的影响.方法:在体外用人肝微粒体研究西尼地平的代谢,并用CYP450酶的选择性抑制剂探讨其对西尼地平代谢的影响及人肝微粒体中参与西尼地平二氢吡啶环脱氢代谢的CYP450酶.结果:西尼地平在人肝微粒体内被迅速代谢为三个代谢物,分别是西尼地平二氢吡啶环脱氢代谢物M1,二氢吡啶环侧链脱甲基代谢物M2,二氢吡啶环脱氢及其侧链脱甲基代谢物M3.酮康唑竞争性地抑制西尼地平二氢吡啶环的脱氢代谢,同时降低西尼地平的代谢速率.而其它抑制剂,奎尼丁,α-Naphthoflavone,diethyldithiocarbamate,sulfaphenazole和tra-nylcypromine对西尼地平二氢吡啶环的脱氢代谢没有明显的影响.结论:西尼地平在人肝微粒体内被迅速代谢,其二氢吡啶环的脱氢代谢是其代谢的关键性的步骤.CYP3A作为主要的CYP酶参与了西尼地平二氢吡啶环的脱氢代谢,CYP3A的抑制剂可能会与西尼地平发生代谢相互作用.%AIM: To study the metabolism of cilnidipine and the effects of selective cytochrome P-450 (CYP450) inhibitors onthe metabolism of cilnidipine in human liver microsomes in vitro. METHODS: Human liver microsomes were usedto perform metabolism studies. Various selective CYP450 inhibitors were used to investigate their effects on themetabolism of cilnidipine and the principal CYP450 isoform involved in dehydrogenation of dihydropyridine ring ofcilnidipine in human liver microsomes. RESULTS: Cilnidipine was rapidly metabolized to three metabolites. Theyare dehydrogenated metabolite of dihydropyridine ring of cilnidipine (M1), demethylation metabolite of lateral chainof dihydropyridine ring of cilnidipine (M2), and the dehydrogenation and demethylation metabolite of cilnidipine(M3). Ketoconazole (Ket) competitively inhibited the dehydrogenation of dihydropyridine ring

  3. Androgen receptor isoforms in human prostatic cancer tissue and LNCaP cell line

    Institute of Scientific and Technical Information of China (English)

    Shu-Jie XIA; Xiao-Da TANG; Qing-Zheng MA

    2001-01-01

    Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three types of AR isoforms were detected with pI values at 6.5,6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3H-DHT) to these three isoforms was inhibited by the addition ofl00-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isofonns is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.

  4. Enhanced expression of two discrete isoforms of matrix metalloproteinase-2 in experimental and human diabetic nephropathy

    Science.gov (United States)

    Bae, Sun Sik; Lee, Min Young; Rhee, Harin; Kim, Il Young; Seong, Eun Young; Lee, Dong Won; Lee, Soo Bong; Kwak, Ihm Soo; Lovett, David H.

    2017-01-01

    Background We recently reported on the enhanced expression of two isoforms of matrix metalloproteinase-2 (MMP-2) in human renal transplantation delayed graft function. These consist of the conventional secreted, full length MMP-2 isoform (FL-MMP-2) and a novel intracellular N-Terminal Truncated isoform (NTT-MMP-2) generated by oxidative stress-mediated activation of an alternate promoter in the MMP-2 first intron. Here we evaluated the effect of hyperglycemia and diabetes mellitus on the in vitro and in vivo expression of the two MMP-2 isoforms. Methods We quantified the abundance of the FL-MMP-2 and NTT-MMP-2 transcripts by qPCR in HK2 cells cultured in high glucose or 4-hydroxy-2-hexenal (HHE) and tested the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). The streptozotocin (STZ) murine model of Type I diabetes mellitus and renal biopsies of human diabetic nephropathy were used in this study. Results Both isoforms of MMP-2 in HK2 cells were upregulated by culture in high glucose or with HHE. PDTC treatment did not suppress high glucose-mediated FL-MMP-2 expression but potently inhibited NTT-MMP-2 expression. With STZ-treated mice, renal cortical expression of both isoforms was increased (FL-MMP-2, 1.8-fold; NTT-MMP-2, greater than 7-fold). Isoform-specific immunohistochemical staining revealed low, but detectable levels of the FL-MMP-2 isoform in controls, while NTT-MMP-2 was not detected. While there was a modest increase in tubular epithelial cell staining for FL-MMP-2 in STZ-treated mice, NTT-MMP-2 was intensely expressed in a basolateral pattern. FL-MMP-2 and NTT-MMP-2 isoform expression as quantified by qPCR were both significantly elevated in renal biopsies of human diabetic nephropathy (12-fold and 3-fold, respectively). Conclusions The expression of both isoforms of MMP-2 was enhanced in an experimental model of diabetic nephropathy and in human diabetic nephropathy. Selective MMP-2 isoform inhibition could offer a novel approach for

  5. 人肝微粒体中尼莫地平代谢的酶动力学及其抑制%Enzyme kinetics and inhibition of nimodipine metabolism in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    柳晓泉; 任亚丽; 钱之玉; 王广基

    2000-01-01

    AIM:To study the enzyme kinetics of nimodipine (NDP) metabolism and the effects of selective cytochrome P-450 (CYP-450) inhibitors on the metabolism of NDP in human liver microsomes in vitro. METHODS: Microsomes from six individual human liver specimens were used to perform enzyme kinetic studies and the kinetic parameters were estimated by Eadie-Hofstee equation. Various selective CYP-450 inhibitors were used to investigate their effects on the metabolism of NDP and the principal CYP-450 isoform involved in dehydrogenation of dihydropyridine ring of NDP in human liver microsomes. RESULTS: There was an important intersubject variability in NDP metabolism in human liver microsomes. For NDP dehydrogeuase activity, the Km value was (36 + 11 ) μmol and the Vm value was ( 17 + 7) μmol · g- 1· min- 1. The dehydrogenation of dihydropyridine ring of NDP was competitively inhibited by ketoconazole (Ket) and troleandomycin (TAO), and the Ki values for Ket and TAO were 0.59 and 122.2 μmol, respectively.Phenacetin ( Pnt ), quinidine ( Qui ), diethyldithiocarbamate (DDC), sulfaphenazole (Sul), and tranylcypromine (Tta) had a little or no inhibitory effects on the dehydrogenation of NDP. CONCLUSION: The intersubject variability of NDP pharmacokinetics was attributed to the metabolic polymorphism of NDP in liver.Cytochrome P-4503A (CYP3A) is involved in the dehydrogenation of dihydropyridine ring of NDP.%目的:在体外研究人肝微粒体中尼莫地平代谢的酶动力学及选择性的细胞色素P-450(CYP450)酶抑制剂对尼莫地平代谢的影响.方法:采用人肝微粒体研究尼莫地平脱氢酶的代谢动力学,运用Eadie-Hof-stee方程估算其动力学参数.在体外运用CYP450酶的选择性抑制剂探讨其对尼莫地平代谢的影响及人肝微粒体中参与尼莫地平二氢吡啶环脱氢代谢的CYP450酶.结果:尼莫地平在人肝微粒体中的代谢存在较大的个体差异,尼莫地平脱氢酶的Km为(36±11)

  6. Two novel human NUMB isoforms provide a potential link between development and cancer.

    Science.gov (United States)

    Karaczyn, Aldona; Bani-Yaghoub, Mahmud; Tremblay, Roger; Kubu, Chris; Cowling, Rebecca; Adams, Tamara L; Prudovsky, Igor; Spicer, Douglas; Friesel, Robert; Vary, Calvin; Verdi, Joseph M

    2010-12-01

    We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  7. Two novel human NUMB isoforms provide a potential link between development and cancer

    Directory of Open Access Journals (Sweden)

    Prudovsky Igor

    2010-12-01

    Full Text Available Abstract We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif GTPase activating protein 1. Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5 and filopodia (NUMB6 in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.

  8. Differential Regulation of Human Thymosin Beta 15 Isoforms by Transforming Growth Factor Beta 1

    Science.gov (United States)

    Banyard, Jacqueline; Barrows, Courtney; Zetter, Bruce R.

    2009-01-01

    We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080 and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322 and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility. PMID:19296525

  9. 半夏泻心汤及不同配伍组对大鼠肝脏CYP450酶活性的影响%In vitro effect of Banxiaxiexin Tang and different conbinations on hepatic CYP450 in rats

    Institute of Scientific and Technical Information of China (English)

    蔡巧玲; 崔波; 王莹; 安叡; 王新宏; 马越鸣

    2012-01-01

    目的:研究半夏泻心汤及不同配伍组对大鼠肝微粒体CYP450亚型酶的影响,从肝脏代谢角度评价半夏泻心汤组方的合理性.方法:将大鼠随机分为全方组、辛开组、苦降组、甘补组及空白对照组,分别灌胃水煎液,运用肝微粒体体外温孵法,对探针底物进行孵育,并结合超高效液相检测方法,测定各探针底物代谢产物的含量,计算代谢速率,以反映各给药组的肝微粒体CYP2C6,CYP2E1,CYP3A1/2亚型酶活性.结果:与空白组相比,全方组,苦降组对各亚型酶均起抑制作用(P<0.01);辛开组对CYP2C6有抑制作用(P<0.01),而对CYP2E1和CYP3A1/2无显著抑制作用;甘补组对CYP2C6和CYP2E1亚型起抑制作用(P<0.01),而对CYP3A1/2无抑制作用.结论:全方对大鼠肝微粒体CYP450酶起抑制作用,比较各配伍组间差异,苦降组抑制作用较其他配伍组显著.%To study the effect of Banxiaxiexin Tang and different conbinations on CYP450 in rat liver microsomes, from the point of liver metabolism, evaluate significance of Banxiaxiexin Tang compatibility. The rats were randomly divided into five groups: Banxiaxiexin Tang group, pungent-swelling group, bitter-descending group, sweet-invigorating group and control group, which were all given decoction by gavage. Using liver microsomes in vitro incubation method, probe substrate were incubated and their metabolites was detected by ultra-high performance liquid chromatography, then was calculated metabolic rate to reflect the drug-treated liver microsomes CYP2C6, CYP2E1 , CYP3A1/2 activity. The results showed that comparing with the control group, both Banxiaxiexin Tang group and bitter-descending group showed inhibition on all enzyme subtype (P <0. 01) , pungent-swelling group showed significant inhibition on CYP2C6, but no inhibition on CYP2E1 and CYP3A1/2, sweet-invigorating group showed inhibition on CYP2C6 and CYP2E1, but no inhibition on CYP3A1/2. Compared the inhibition on CYP

  10. 参麦注射液和注射用血塞通对大鼠肝脏及肠道药物代谢酶CYP450的影响%Impact of Shenmai injection and Xuesaitong injection on CYP450s in rat liver and intestine after successive administration

    Institute of Scientific and Technical Information of China (English)

    卜明华; 郑咏秋; 张颖; 李利群; 刘建勋; 刘东春

    2012-01-01

    Objective To investigate the influence of Shenmai injection (SM) and Xuesaitong injection (XST) on drag metablic enzyme of cyto-chrome P450(CYP450) system in rat. Methods Microsome of rat liver and intestine was prepared after intravenous injection of SM and XST for 10 days. The content of total CYP450s in rat liver microsome was measured by UV - visible absorption spectrometry. Meanwhile, the protein expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A in the liver micro-some and CYP3A in the small intestine microsome were detected by western blotting analysis. Results Although no significant difference of total P450 content was observed between each treatment group and control group (P > 0.05), but obvious difference was exhibited in western bloting analysis on CYPs. In addition to both SM and XST induced the CYP2E1 in liver microsome significantly, SM inhibited the CYP3A in intestine microsome while XST induced the expression of CYP1A2, CYP2C11 and CYP3A in liver microsome significantly. Conclusion Since the expression of CYPs subtypes in liver and intestine was significantly changed after successive administration, the possible side effect of drug - drug interaction could not be neglected in the period of clinical therapy.%目的 考察参麦注射液和注射用血塞通对大鼠药物代谢酶细胞色素P450(CYP450)的影响.方法 连续给予大鼠试验药物后,制备肝及小肠微粒体;用CO还原差示光谱法,测定肝微粒体CYP450含量;用蛋白免疫印迹法,检测肝微粒体中CYP1A2、CYP2C11、CYP2E1、CYP3A和肠微粒体中CYP3A蛋白的表达量.结果 参麦注射液和注射用血塞通组与空白对照组大鼠的肝脏总P450含量无显著性差异(P>0.05).但参麦注射液可显著诱导肝脏CYP2E1表达,但抑制肠微粒体中CYP3A表达;肝CYP1A2、CYP2C11水平均有所上升.血塞通可显著诱导大鼠肝脏CYP1A2、CYP2E1、CYP2C11、CYP3A蛋白表达,肠CYP3A的表达水平显示出下降趋势.结论 参麦注

  11. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Directory of Open Access Journals (Sweden)

    Niels Jacob Aachmann-Andersen

    Full Text Available The membrane-assisted isoform immunoassay (MAIIA quantitates erythropoietin (EPO isoforms as percentages of migrated isoforms (PMI. We evaluated the effect of recombinant human EPO (rhEPO on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13; high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13; or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3 % (mean (SD. High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2% (p<0.00001 and 45.2 (7.3% (p<0.00001. Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8% (p<0.00001 and 46.1 (10.4% (p<0.00001. In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4% (p=0.029; low-dose Epoetin beta: 73.1 (17.8% (p=0.039. In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  12. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution.

    Science.gov (United States)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p=0.029); low-dose Epoetin beta: 73.1 (17.8)% (p=0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal.

  13. A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

    Directory of Open Access Journals (Sweden)

    Evangelos D Karousis

    Full Text Available Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02 which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

  14. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

    OpenAIRE

    Erickson, J.D.; Schafer, M K; Bonner, T I; Eiden, L. E.; Weihe, E.

    1996-01-01

    A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpr...

  15. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    Science.gov (United States)

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  16. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Directory of Open Access Journals (Sweden)

    Dipak K. Dube

    2016-01-01

    Full Text Available In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4 each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

  17. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Science.gov (United States)

    Abbott, Lynn; Alshiekh-Nasany, Ruham; Mitschow, Charles

    2016-01-01

    In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart. PMID:27703814

  18. alpha isoforms of soluble and membrane-linked folate-binding protein in human blood

    DEFF Research Database (Denmark)

    Hoier-Madsen, M.; Holm, J.; Hansen, S.I.

    2008-01-01

    supported the hypothesis that serum FBP (29 kDa) mainly originates from neutrophils. The presence of FBP/FR alpha isoforms were established for the first time in human blood using antibodies specifically directed against human milk FBP alpha. The alpha isoforms identified on erythrocyte membranes...... a non-functional FR beta on the surface, and, in addition, nanomolar concentrations of a secretory functional FBP (29 kDa) can be present in the secondary granules. A statistically significant correlation between the concentrations of functional FBP, probably a gamma isoform, in granulocytes and serum......, and in granulocytes and serum, only constituted an almost undetectable fraction of the functional FBP The FBP alpha in neutrophil granulocytes was identified as a cytoplasmic component by indirect immunofluorescence. Gel filtration of serum revealed a peak of FBP alpha (>120 kDa), which could represent receptor...

  19. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  20. Does pharmacogenetic testing for CYP450 2D6 and 2C19 among patients with diagnoses within the schizophrenic spectrum reduce treatment costs?

    Science.gov (United States)

    Herbild, Louise; Andersen, Stig E; Werge, Thomas; Rasmussen, Henrik B; Jürgens, Gesche

    2013-10-01

    The effect of pharmacogenetic testing for CYP450 2D6 and 2C19 on treatment costs have not yet been documented. This study used Danish patient registers to calculate healthcare costs of treating patients with diagnoses within the schizophrenic spectrum for 1 year with or without pharmacogenetic testing for polymorphisms in the genes for the CYP2D6 and CYP2C19 enzymes. In a randomized, controlled trial, stratified with respect to metabolizer genotype, 104 patients were assigned to treatment based on pharmacogenetic testing and 103 patients to treatment as usual. Random exclusion of extensive and intermediate metabolizers was used to increase the frequency of extreme metabolizers (poor metabolizers and ultrarapid metabolizers for CYP2D6) to 20% in both groups. Cost differences were analysed at several levels including (i) overall healthcare expenditure, (ii) psychiatric hospital cost (iii) nonpsychiatric hospital cost, (iv) primary care spending and (v) pharmaceuticals. Statistically significant differences in costs of psychiatric care dependent on metabolizer status were found between intervention groups. Pharmacogenetic testing significantly reduced costs among the extreme metabolizers (poor metabolizers and ultrarapid metabolizers) to 28%. Use of primary care services and pharmaceuticals was also affected by the intervention.This study confirms earlier findings that extreme metabolizers (poor and ultrarapid metabolizers) incur higher costs than similar patients with a normal metabolizer genotype. However, this study shows that these excess costs can be reduced by pharmacogenetic testing. Pharmacogenetic testing for CYP2D6 and CYP2C19 could thus be considered as a means of curtailing high psychiatric treatment costs among extreme metabolizers.

  1. Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors.

    Science.gov (United States)

    Valve, E; Martikainen, P; Seppänen, J; Oksjoki, S; Hinkka, S; Anttila, L; Grenman, S; Klemi, P; Härkönen, P

    2000-12-01

    FGF-8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF-8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF-8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF-8 is over-expressed in human breast and prostate cancers. Expression has also been found in RT-PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF-8 isoforms are expressed in ovarian cancer and whether FGF-8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT-PCR and immunohistochemistry. FGF-8 isoform b was expressed in all ovarian tumors and in all 7 ovarian-cancer cell lines studied. Isoform a was co-expressed in 9 malignant ovarian tumors. FGF-8 mRNA was not detected by RT-PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF-8 protein to cancer cells. In general, the increased intensity of FGF-8 staining was associated with loss of differentiation within the tumors (Bowker's test, p = 0.37). FGF-8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT-PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF-8 receptors expressed in normal ovaries and in ovarian tumors. FGF-8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF-8 have acquired the capacity of expressing FGF-8. This suggests that FGF-8 has an important role in ovarian tumorigenesis.

  2. A computational approach predicting CYP450 metabolism and estrogenic activity of an endocrine disrupting compound (PCB-30).

    Science.gov (United States)

    Harris, Jason B; Eldridge, Melanie L; Sayler, Gary; Menn, Fu-Min; Layton, Alice C; Baudry, Jerome

    2014-07-01

    Endocrine disrupting chemicals influence growth and development through interactions with the hormone system, often through binding to hormone receptors such as the estrogen receptor. Computational methods can predict endocrine disrupting chemical activity of unmodified compounds, but approaches predicting activity following metabolism are lacking. The present study uses a well-known environmental contaminant, PCB-30 (2,4,6-trichlorobiphenyl), as a prototype endocrine disrupting chemical and integrates predictive (computational) and experimental methods to determine its metabolic transformation by cytochrome P450 3A4 (CYP3A4) and cytochrome P450 2D6 (CYP2D6) into estrogenic byproducts. Computational predictions suggest that hydroxylation of PCB-30 occurs at the 3- or 4-phenol positions and leads to metabolites that bind more strongly than the parent molecule to the human estrogen receptor alpha (hER-α). Gas chromatography-mass spectrometry experiments confirmed that the primary metabolite for CYP3A4 and CYP2D6 is 4-hydroxy-PCB-30, and the secondary metabolite is 3-hydroxy-PCB-30. Cell-based bioassays (bioluminescent yeast expressing hER-α) confirmed that hydroxylated metabolites are more estrogenic than PCB-30. These experimental results support the applied model's ability to predict the metabolic and estrogenic fate of PCB-30, which could be used to identify other endocrine disrupting chemicals involved in similar pathways.

  3. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Science.gov (United States)

    Ihnatovych, Ivanna; Sielski, Neil L; Hofmann, Wilma A

    2014-01-01

    Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  4. CYP450-dependent biotransformation of the insecticide fipronil into fipronil sulfone can mediate fipronil-induced thyroid disruption in rats.

    Science.gov (United States)

    Roques, Béatrice B; Lacroix, Marlène Z; Puel, Sylvie; Gayrard, Véronique; Picard-Hagen, Nicole; Jouanin, Isabelle; Perdu, Elisabeth; Martin, Pascal G; Viguié, Catherine

    2012-05-01

    In rats, the widely used insecticide fipronil increases the clearance of thyroxine (T(4)). This effect is associated with a high plasma concentration of fipronil sulfone, the fipronil main metabolite in several species including rats and humans. In sheep, following fipronil treatment, fipronil sulfone plasma concentration and thyroid disruption are much lower than in rats. We postulated that fipronil biotransformation into fipronil sulfone by hepatic cytochromes P450 (CYP) could act as a potential thyroid disruptor. The aim of this study was to determine if fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and CYP induction in rats. Fipronil and fipronil sulfone treatments (3.4 μmol/kg/day per os, 14 days) increased total and free T(4) clearances to the same extent in THX + T(3), euthyroid-like rats. Both treatments induced a 2.5-fold increase in Ugt1a1 and Sult1b1 messenger RNA (mRNA) expressions and a twofold increase in UGT1A activity suggesting that T(4) elimination was mediated, at least in part, by hepatic uridine 5'-diphospho-glucuronosyltransferases (UGT) and/or sulfotransferases (SULT) induction. Both treatments induced a 10-fold increase in Cyp3a1 and Cyp2b2 mRNA expressions concomitant with a threefold increase in CYP3A immunoreactivity and a 1.7-fold increase in antipyrine clearance, a biomarker of CYP3A activity. All these results showed that fipronil sulfone treatment could reproduce the fipronil treatment effects on T(4) clearance and hepatic enzyme induction in rats. The potential of fipronil sulfone to act as a thyroid disruptor is all the more critical because it persists much longer in the organism than fipronil itself.

  5. Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450-independent mitochondrial toxicity of acetaminophen and amiodarone.

    Science.gov (United States)

    Prill, Sebastian; Bavli, Danny; Levy, Gahl; Ezra, Elishai; Schmälzlin, Elmar; Jaeger, Magnus S; Schwarz, Michael; Duschl, Claus; Cohen, Merav; Nahmias, Yaakov

    2016-05-01

    Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 μm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.

  6. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    Science.gov (United States)

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

    Directory of Open Access Journals (Sweden)

    Kristin R Wildsmith

    Full Text Available Apolipoprotein E (ApoE is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4 each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD. Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS, we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

  8. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    Science.gov (United States)

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. The human-specific invariant chain isoform Iip35 modulates Iip33 trafficking and function.

    Science.gov (United States)

    Sand, Kine Marita Knudsen; Landsverk, Ole J B; Berg-Larsen, Axel; Bakke, Oddmund; Gregers, Tone F

    2014-10-01

    The invariant chain (Ii) is a multifunctional protein, which has an essential role in the assembly and transport of major histocompatibility complex class II (MHC II) molecules. From a single gene, Ii is synthesized as four different isoforms: Iip33, Iip35, Iip41 and Iip43. Iip35 and Iip43 are specific to humans, and are formed due to an upstream alternative translation site, resulting in an N-terminal extension of 16 amino acids. This extension harbors a strong endoplasmic reticulum (ER) retention motif. Consequently, Iip35 or Iip43 expressed alone are retained in the ER, whereas Iip33 and Iip41 rapidly traffic to the endosomal pathway. Endogenously expressed, the four isoforms form mixed heterotrimers in the ER; however, mainly due to the absence of the Iip35/p43 isoforms in mice, little is known about how they influence general Ii function. In this study, we have co-expressed Iip33 and Iip35 in human cells with and without MHC II to gain a better understanding of how Iip35 isoform influences the cellular properties of Iip33. We find that Iip35 significantly affects the properties of Iip33. In the presence of Iip35, the transport of Iip33 out of the ER is delayed, its half-life is dramatically prolonged and its ability to induce enlarged endosomes and delayed endosomal maturation is abrogated.

  10. 对乙酰氨基酚合用琥乙红霉素对大鼠肝微粒体CYP450的影响%Effect of paracetamol combined with erythromycin ethylsuccinate on CYP450 in rat liver microsomes

    Institute of Scientific and Technical Information of China (English)

    欧扬; 姜佳希; 周文

    2009-01-01

    OBJECTIVE To study the effect of paracetamol with erythromycin ethylsuccinate on drug-metabolizing enzyme in rats liver, and evaluate their availability and safety for rational administration in clinic. METHODS Using CO reduction differ-ential spectrum to detect the content of cytochrome P450 and cytochrome b5. The activity of GST, ERD and ADM were meas-ured by UV. RESULTS Paracetamol co-administration with erythromycin ethylsuccinate could inhibit the activity of cyto-chrome P450 in rat liver microsomes. CONCLUSION The co-administration of paracetamol with erythromycin ethylsuccinate can influence the activity of drug-metabolizing enzymes, and change its metabalism characteristics resulting in drug metabalism interaction and poor efficacy.%目的:通过研究对乙酰氨基酚合用琥乙红霉素对大鼠肝药物代谢酶的影响,对二者合用后的有效性和安全性作出评价,为临床合理用药提供理论依据.方法:采用CA)还原差示光谱法,检测细胞色素P450(CYP450)及细胞色素b5(Cytb5)的含量;采用紫外-可见光分光光度法测定谷胱甘肽转移酶(GST)、红霉素N-脱甲基酶(ERD)和氨基比林N-脱甲基酶(ADM)的活性.结果:对乙酰氨基酚合用琥乙红霉素对大鼠肝微粒体CYP450的含量和活性有不同程度的影响.结论:对乙酰氨基酚合用琥乙红霉素对药物代谢酶有影响,可能使药物的体内代谢特征发生改变,产生药物代谢性相互作用,从而影响药物疗效,增加肝脏损伤.

  11. Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

    Science.gov (United States)

    U, M; Miyashita, T; Shikama, Y; Tadokoro, K; Yamada, M

    2001-11-30

    Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

  12. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...

  13. The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.

    Directory of Open Access Journals (Sweden)

    Maléne E Lindholm

    2016-09-01

    Full Text Available Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  14. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zorka Milićević

    2014-01-01

    Full Text Available In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa and Δ133p53β (35 kDa, known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.

  15. Pharmacokinetics of mangiferin and its metabolite-norathyriol, Part 2: Influence of UGT, CYP450, P-gp, and enterobacteria and the potential interaction in Rhizoma Anemarrhenae decoction with timosaponin B2 as the major contributor.

    Science.gov (United States)

    Tian, Xiaoting; Xu, Zhou; Li, Zhixiong; Ma, Yuanjie; Lian, Shan; Guo, Xiaozhen; Hu, Pei; Gao, Yu; Huang, Chenggang

    2016-09-10

    The poor bioavailability of mangiferin (MGF) is a major obstacle on its further development. Aimed to illustrate the underlying mechanism and improve its poor exposure, the compared PK profiles of MGF and norathyriol (NTR) after different MGF preparation were performed: pure MGF, the Rhizoma Anemarrhenae (Zhi-mu) decoction, MGF, and timosaponin B2 (TB-2) combination. Furthermore, the potential contributing factors, including uridine diphosphoglucuronosyltransferase (UGT), cytochrome P450 (CYP450), P-gp, and enterobacterial were investigated by comparing the PK profiles with and without the corresponding inhibitors or in different rat models. After taking MGF, CYP450 and UGT inhibition could decrease MGF and NTR exposure; P-gp inhibition slightly enhanced (48%) MGF exposure, whereas more apparent for the improved NTR exposure (302%); enterobacterial inhibition almost completely stopped the NTR production, but no such effect was observed for MGF. Compared with the limited improvement by the abovementioned inhibition, the MGF and NTR exposure could significantly increase by 11.5- and 5.9-fold in the Zhi-mu decoction compared with the MGF treatment, probably contributed to TB-2 as an absorption enhancer because the MGF and TB-2 combination produced a similar level of improvement on the PK paremeters of MGF and NTR to the herb treatment. Likewise, most of the effects by UGT, CYP450, P-gp, and enterobacteria followed a similar variation tendency between them. Therefore, the poor bioavailability of MGF possibly mainly attributed to its poor membrane permeability, but not transporters or metabolic enzymes, and the compatibility of MGF and TB-2 could probably expand the prospective application of MGF by improving its bioavailability. © 2016 BioFactors, 42(5):545-555, 2016.

  16. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    Science.gov (United States)

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 micro

  17. Multiple isoforms of the human pentraxin serum amyloid P component

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH;

    1995-01-01

    Human serum amyloid P component (SAP) isolated from 20 healthy individuals was analyzed by anion exchange chromatography and isoelectric focusing (IEF) in order to investigate the existence of multiple forms of SAP and interindividual structural differences. Anion exchange chromatography showed one...... major and several minor subpopulations of SAP. IEF of all SAP isolates showed a previously unreported degree of heterogeneity with six isoelectric forms (pKi range 5.5-6.1) and with minor interindividual differences in respect of isoelectric points. Total enzymatic deglycosylation of SAP reduced...

  18. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  19. 甘遂半夏汤加减甘遂及不同品种甘草对腹水模型大鼠肝脏CYP450mRNA的影响%Comparing the effect of Gansui Banxia decoction plus or reduce kansui and varieties of glycyrrhiza on the CYP450 mRNA of liver of ascites model rats

    Institute of Scientific and Technical Information of China (English)

    许皖; 柳海艳; 钟赣生; 于雪; 张建美; 郭岩松; 王思睿; 修琳琳

    2016-01-01

    目的:观察含不同品种甘草的甘遂半夏汤加减甘遂甘草反药组合对腹水模型大鼠肝脏CYP450 mRNA的影响。方法采用Walker-256细胞制造癌性腹水模型,将Wistar大鼠按体质量随机分为空白组、模型组、阳性对照组、全方炙甘草组、全方炙光果甘草组、全方炙胀果甘草组、去遂炙甘草组、去遂炙光果甘草组、去遂炙胀果甘草组、去炙甘草组、去炙甘草醋甘遂组,共计11组。空白组和模型组灌胃蒸馏水,其余各给药组灌胃相应药物。给药11天后,摘取肝脏,液氮保存,进行PCR实验。结果在CYP2E1 mRNA表达方面,全方炙甘草组、全方炙胀果甘草表达下调,且优于全方去掉一味或两味反药。三个甘草品种的甘遂半夏汤的CYP3A1 mRNA、CYP3A2 mRNA表达上调,且优于全方去掉一味或两味反药。结论(1)全方炙甘草组、全方炙胀果甘草组在CYP450表达方面表现出一定的药效作用,其中全方炙甘草组优于全方炙胀果甘草,且均优于全方去掉一味或两味反药组。(2)全方炙光果甘草组则未表现出一定的药效作用,但也未表现出毒性作用,弱于去掉一味或两味反药。%Objective To observe the effect of different varieties of glycyrrhiza and kansui incompatible herbs in Gansui Banxia decoction on liver CYP450 mRNA ascites model rat. Methods The Walker-256 cell was used to manufacture carcinomatous ascites model. The Wistar rats were randomly divided into blank group, model group, positive control group, complete formula with Glycyrrhiza uralensis Fisch. group, complete formula with Glycyrrhiza glabra L. group, complete formula with Glycyrrhiza inflata Bat. group, the Glycyrrhiza uralensis Fisch. Without kansui group , the Glycyrrhiza glabra L. without kansui group, the Glycyrrhiza inflata Bat. without kansui group, prescription without glycyrrhiza group, prescription without glycyrrhiza and kansui group, a total of 11

  20. Effects of the compatibility of Huangyaozi and Gancao on the CYP450 mRNA expression in rat liver%黄药子及配伍甘草对大鼠肝脏CYP450基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    华碧春; 黄智锋; 刘娇; 程心玲; 陈小峰; 王英豪; 卓实

    2014-01-01

    目的:考察甘草对黄药子引起的肝损伤的CYP450酶基因表达的影响.方法:采用逆转录聚合酶链反应(RT-PCR)技术检测大鼠肝脏CYP1A2、CYP2C 19的mRNA的表达.结果:在mRNA水平上,28天、35天黄药子组均显著诱导CYP1A2的基因表达,配伍甘草后表达降低,具有统计学意义.甘草组可微弱下调CYP2C 19的表达,但与配伍组相比无统计学意义.结论:甘草通过抑制CYP1A2的mRNA表达,对黄药子致肝损伤具有一定保护作用.

  1. Effects of halophenol LM49 on microsomal CYP450 in Rats%卤酚化合物LM49对大鼠肝微粒体细胞色素P450的影响

    Institute of Scientific and Technical Information of China (English)

    白玉; 张丽锋; 冯秀娥; 梁泰刚; 李青山

    2012-01-01

    目的 研究LM49对大鼠肝微粒体蛋白及细胞色素P450含量的影响.方法 将大鼠分成空白对照组、溶剂对照组、阳性对照组(苯巴比妥组、地塞米松组、β-萘黄酮组)、LM49低、中、高剂量组.给药后采用超速离心法制备大鼠肝微粒体;BCA法测定大鼠肝微粒体蛋白浓度;Omura and Sato法测定大鼠肝微粒体细胞色素P450的含量.结果 给予不同剂量的LM49后,大鼠肝微粒体的蛋白及细胞色素P450含量均明显降低.低、中、高剂量组与对照组比较差异有统计学意义(P<0.05).结论 LM49对大鼠肝微粒体细胞色素P450具有一定的抑制作用,可能引起肝药酶对某些药物代谢的改变.%Objective To determine the effects of LM49 on microsomal protein and CYP450 in rats. Methods Rats were assigned into blank control group, solvent control group, positive control groups (phenobarbital, dexametha-sone and (3-nephthoflavone) and LM49-treated (low, medium and high dosage) groups, respectively. Hepatic micro-somes were prepared using ultracentrifuge after administration of interventions. The concentration of microsomal proteins was determined using BAG approach, and the content of microsomal CYP450 was assayed via Omura and Sato method. Results Marked reduction in the concentration of microsomal proteins and CYP450 was associated with administration of LM49 with various (low, medium and high) dosages as compared with control group (all P<0.05). Conclusion LM49 inhibits hepatic microsomal CYP450 and may result in changes of metabolism of certain medicines induced by liver drug enzymes in rats.

  2. Secreted Isoform of Human Lynx1 (SLURP-2)

    DEFF Research Database (Denmark)

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we......-evoked currents at concentrations via...... interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs....

  3. Human septin isoforms and the GDP-GTP cycle.

    Science.gov (United States)

    Zent, Eldar; Wittinghofer, Alfred

    2014-02-01

    Septins form oligomeric complexes consisting of septins from different subgroups, which form filaments that are involved in a number of biological processes. They are GTP-binding proteins that contain all the necessary elements to perform the general GDP-to-GTP conformational switch. It is however unclear whether or not such a switch is important for the dynamics of septin filaments. Here we investigate the complex GTPase reaction of members of each of the four human septin groups, which is dominated by the stability of dimer formation via the nucleotide binding or so-called G-interface. The results also show that the actual hydrolysis reaction is very similar for three septin groups in the monomeric state while the Sept6 has no GTPase activity. Sept7, the only member of the Sept7 subgroup, forms a very tight G-interface dimer in the GDP-bound state. Here we show that the stability of the interface is dramatically decreased by exchanging GDP with a nucleoside triphosphate, which is believed to influence filament formation and dynamics via Sept7.

  4. Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.

    Science.gov (United States)

    Fukuda, Mitsunori; Ogata, Yukie; Saegusa, Chika; Kanno, Eiko; Mikoshiba, Katsuhiko

    2002-07-01

    Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

  5. Multiple, but Concerted Cellular Activities of the Human Protein Hap46/BAG-1M and Isoforms

    Directory of Open Access Journals (Sweden)

    Ulrich Gehring

    2009-03-01

    Full Text Available The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types.

  6. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    National Research Council Canada - National Science Library

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    .... There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms...

  7. Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Soraya Scuderi

    2014-01-01

    Full Text Available Parkinson protein 2, E3 ubiquitin protein ligase (PARK2 gene mutations are the most frequent causes of autosomal recessive early onset Parkinson’s disease and juvenile Parkinson disease. Parkin deficiency has also been linked to other human pathologies, for example, sporadic Parkinson disease, Alzheimer disease, autism, and cancer. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification. To date several PARK2 splice variants have been identified; however, the expression and distribution of parkin isoforms have not been deeply investigated yet. Here, the currently known PARK2 gene transcripts and relative predicted encoded proteins in human, rat, and mouse are reviewed. By analyzing the literature, we highlight the existing data showing the presence of multiple parkin isoforms in the brain. Their expression emerges from conflicting results regarding the electrophoretic mobility of the protein, but it is also assumed from discrepant observations on the cellular and tissue distribution of parkin. Although the characterization of each predicted isoforms is complex, since they often diverge only for few amino acids, analysis of their expression patterns in the brain might account for the different pathogenetic effects linked to PARK2 gene mutations.

  8. Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2*

    Science.gov (United States)

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. PMID:20139082

  9. Temporal changes of sleep disturbances and their associations with CYP450 2B6 polymorphism and plasma drug level in HIV patients on efavirenz

    Directory of Open Access Journals (Sweden)

    K To

    2012-11-01

    Full Text Available Neurological and sleep disturbances were commonly reported among HIV patients on efavirenz (EFV, the pharmacokinetic pattern varies with different CYP450 2B6 G516T genotypes. This prospective study aims to detect temporal changes and differences in the profile of these adverse reactions and their relation to plasma EFV level and host genotypes. HIV patients of Chinese ethnicity on an EFV-containing HAART regimen were recruited from a specialist clinic. Blood for CYP2B6 G516T genotypes was taken. A questionnaire assessing adverse neurological problems and sleep disturbance was administered, alongside testing for plasma EFV levels at baseline, 4 weeks, 8 months and 1 year intervals after treatment. Analysis was performed using χ2 and t-test. A total of 64 patients (31 GG, 27 GT, 6 TT genotypes, 59 male, and 5 female, mean age of 41±9.9 were recruited. At 4 weeks after EFV, 49 (76% gave a history of any one of the neurological side effects: dizziness, headache and drunk feeling. Sleep disturbances were common: bizarre dream (45%, nightmares (35%, waking at night (73%, poor sleep quality (31%, nocturia (84% and difficulty in falling asleep (67%. The mean plasma EFV level of GG genotype was 2.8 µg/ml and 3 µg/ml, GT genotype was 3.8 µg/ml and 3.9 µg/ml, at 4 weeks and 1 year respectively. The mean plasma EFV level of TT genotype was 11.9 µg/ml and 9.7 µg/ml at 4 weeks and 1 year respectively. There was no significant variation of drug level within each genotype over time (p>0.08, while EFV level of TT was significantly higher at all time points (p<0.01. Overall, nightmares and difficulty to fall asleep were significantly related to the plasma EFV level (p=0.021 and 0.017 respectively. However, the sleep quality, nocturnal awakening, nocturia or requirement of sleeping pills was not significantly associated with EFV level (p=0.28, 0.06, 0.1 and 0.5 respectively. When the side effects were separately evaluated according to time points, they all

  10. Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

    Directory of Open Access Journals (Sweden)

    Katherine J D A Excoffon

    Full Text Available Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7 and CAR(Ex8. We found low-level expression of the CAR(Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7, CAR(Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7, CAR(Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8 providing a potential mechanism for the apical localization of CAR(Ex8 in airway epithelial. In summary, apical localization of CAR(Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.

  11. Isoform-specific targeting and interaction domains in human nicotinamide mononucleotide adenylyltransferases.

    Science.gov (United States)

    Lau, Corinna; Dölle, Christian; Gossmann, Toni I; Agledal, Line; Niere, Marc; Ziegler, Mathias

    2010-06-11

    Several important signaling pathways require NAD as substrate, thereby leading to significant consumption of the molecule. Because NAD is also an essential redox carrier, its continuous resynthesis is vital. In higher eukaryotes, maintenance of compartmentalized NAD pools is critical, but so far rather little is known about the regulation and subcellular distribution of NAD biosynthetic enzymes. The key step in NAD biosynthesis is the formation of the dinucleotide by nicotinamide/nicotinic acid mononucleotide adenylyltransferases (NMNATs). The three human isoforms were localized to the nucleus, the Golgi complex, and mitochondria. Here, we show that their genes contain unique exons that encode isoform-specific domains to mediate subcellular targeting and post-translational modifications. These domains are dispensable for catalytic activity, consistent with their absence from NMNATs of lower organisms. We further demonstrate that the Golgi-associated NMNAT is palmitoylated at two adjacent cysteine residues of its isoform-specific domain and thereby anchored at the cytoplasmic surface, a potential mechanism to regulate the cytosolic NAD pool. Insertion of unique domains thus provides a yet unrecognized enzyme targeting mode, which has also been adapted to modulate subcellular NAD supply.

  12. PPARG in Human Adipogenesis: Differential Contribution of Canonical Transcripts and Dominant Negative Isoforms

    Directory of Open Access Journals (Sweden)

    M. Aprile

    2014-01-01

    Full Text Available The nuclear receptor PPARγ is a key regulator of adipogenesis, and alterations of its function are associated with different pathological processes related to metabolic syndrome. We recently identified two PPARG transcripts encoding dominant negative PPARγ isoforms. The existence of different PPARG variants suggests that alternative splicing is crucial to modulate PPARγ function, underlying some underestimated aspects of its regulation. Here we investigate PPARG expression in different tissues and cells affected in metabolic syndrome and, in particular, during adipocyte differentiation of human mesenchymal stem cells. We defined the transcript-specific expression pattern of PPARG variants encoding both canonical and dominant negative isoforms and identified a novel PPARG transcript, γ1ORF4. Our analysis indicated that, during adipogenesis, the transcription of alternative PPARG variants is regulated in a time-specific manner through differential usage of distinct promoters. In addition, our analysis describes—for the first time—the differential contribution of three ORF4 variants to this process, suggesting a still unexplored role for these dominant negative isoforms during adipogenesis. Therefore, our results highlight crucial aspects of PPARG regulation, suggesting the need of further investigation to rule out the differential impact of all PPARG transcripts in both physiologic and pathologic conditions, such as metabolism-related disorders.

  13. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

    Science.gov (United States)

    Shafqat, Naeem; Turnbull, Andrew; Zschocke, Johannes; Oppermann, Udo; Yue, Wyatt W

    2010-05-14

    3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.

  14. Functional role of human NK cell receptor 2B4 (CD244) isoforms.

    Science.gov (United States)

    Mathew, Stephen O; Rao, Krithi K; Kim, Jong R; Bambard, Nowland D; Mathew, Porunelloor A

    2009-06-01

    2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

  15. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  16. Compatibility of Banxia Houpo decoction on hepatic CYP450 and renal organicion transporters in mice%半夏厚朴汤及君臣配伍对小鼠肝脏CYP450和肾脏离子转运子的影响

    Institute of Scientific and Technical Information of China (English)

    王抚梦; 卢燕; 孔令东

    2011-01-01

    By analyzing the related indicators [hepatic CYP450 subtype and renal organic anion and cation transporters ( OATs and OCTs)], the present study investigated the effects of formula Banxia Houpo decoction principal drug pinellia, assistant drug magnolia, their compatibility and the principle of the whole decoction on the metabolism ability in the liver and the transport change in the kidney of mice.Biochemical and molecular ( RT-PCR and western blotting) results indicated that pinellia increased activity and expression of hepatic Cyp2e1 and Cyp3a11 in mice, respectively.Pinellia and magnolia increased expression of renal OAT1, OAT3,OCT1 and OCT2 in mice, respectively.The compatibility of pinellia and magnolia, as well as Banxia Houpo decoction synergistically restrained the activated effect of pinellia on hepatic Cyp2e1, therefore avoiding liver peroxidation and reducing toxicity potential.The compatibility of this drug pair and Banxia Houpo decoction not only reduced activity and expression of hepatic Cyp3a11 to control drug metabolism speed, but also balanced the expression of renal OAT1/3 and OCT1/2 to enhance drug efficacy.The effect of compatibility of Banxia Houpo decoction was better than that of pinellia and magnolia pair, and the normal dosage was better than the high dosage.The present study proved the advantage of the compatibility of pinellia combined with magnolia and the principle of Banxia Houpo decoction, which related to hepatic CYP450 and renal organic ion transporters, and guided the clinical use of Banxia Houpo decoction to exert its toxicity reduction and efficacy enhancement.%目的:研究半夏厚朴汤中君药半夏、臣药厚朴、君臣药对以及伞方对小鼠体内肝脏微粒体CYP450亚型和肾脏离子转运子的影响,以期从肝脏代谢和肾脏排泄能力的角度,评价半夏厚朴汤的配伍意义及合理性.方法:将小鼠随机分为半夏组、厚朴组、半夏厚朴配伍药对组、复方的原方剂量组和高

  17. The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

    Directory of Open Access Journals (Sweden)

    Junting Hu

    2014-12-01

    Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

  18. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  19. Isoform switch of pyruvate kinase M1 indeed occurs but not to pyruvate kinase M2 in human tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Cheng Zhan

    Full Text Available Muscle type of pyruvate kinase (PKM is one of the key mediators of the Warburg effect and tumor metabolism. Due to alternative splicing, there are at least 12 known isoforms of the PKM gene, of which PKM1 and PKM2 are two major isoforms with only a 23 amino acid sequenced difference but quite different characteristics and functions. It was previously thought the isoform switch from PKM1 to PKM2 resulted in high PKM2 expression in tumors, providing a great advantage to tumor cells. However, this traditional view was challenged by two recent studies; one study claimed that this isoform switch does not occur during the Warburg effect; the other study asserted that the isoform switch is tissue-specific. Here, we re-analyzed the RNA sequencing data of 25 types of human tumors from The Cancer Genome Atlas Data Portal, and confirmed that PKM2 was the major isoform in the tumors and was highly elevated in addition to the entire PKM gene. We further demonstrated that the expression level of PKM1 significantly declined even though there was substantially increased expression of the entire PKM gene. The proportion of PKM1 in total transcript variants also significantly declined in tumors but the proportion of PKM2 did not change accordingly. Therefore, we conclude that the isoform switch of PKM1 does indeed occur, but it switches to other isoforms rather than PKM2. Considering the change in the expression levels of PKM1, PKM2 and the entire PKM gene, we propose that the upregulation of PKM2 is primarily due to elevated transcriptional levels of the entire PKM gene, instead of the isoform switch.

  20. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  1. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  2. Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability*

    Science.gov (United States)

    Wegmann, Susanne; Jung, Yu Jin; Chinnathambi, Subashchandrabose; Mandelkow, Eva-Maria; Mandelkow, Eckhard; Muller, Daniel J.

    2010-01-01

    Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility. PMID:20566652

  3. Human Tau isoforms assemble into ribbon-like fibrils that display polymorphic structure and stability.

    Science.gov (United States)

    Wegmann, Susanne; Jung, Yu Jin; Chinnathambi, Subashchandrabose; Mandelkow, Eva-Maria; Mandelkow, Eckhard; Muller, Daniel J

    2010-08-27

    Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the "thin twisted" and "thin smooth" fibrils resemble flat ribbons (cross-section approximately 10 x 15 nm) with diverse twist periodicities. "Thick fibrils" show periodicities of approximately 65-70 nm and thicknesses of approximately 9-18 nm such as routinely reported for "paired helical filaments" but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of approximately 17-19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures ( approximately 2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the "fuzzy coat" of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.

  4. Design and synthesis of benzodiazepine analogs as isoform-selective human lysine deacetylase inhibitors.

    Science.gov (United States)

    Reddy, D Rajasekhar; Ballante, Flavio; Zhou, Nancy J; Marshall, Garland R

    2017-02-15

    A comprehensive investigation was performed to identify new benzodiazepine (BZD) derivatives as potent and selective human lysine deacetylase inhibitors (hKDACis). A total of 108 BZD compounds were designed, synthesized and from that 104 compounds were biologically evaluated against human lysine deacetylases (hKDACs) 1, 3 and 8 (class I) and 6 (class IIb). The most active compounds showed mid-nanomolar potencies against hKDACs 1, 3 and 6 and micromolar activity against hKDAC8, while a promising compound (6q) showed selectivity towards hKDAC3 among the different enzyme isoforms. An hKDAC6 homology model, refined by molecular dynamics simulation was generated, and molecular docking studies performed to rationalize the dominant ligand-residue interactions as well as to define structure-activity-relationships. Experimental results confirmed the usefulness of the benzodiazepine moiety as capping group when pursuing hKDAC isoform-selectivity inhibition, suggesting its continued use when designing new hKDACis.

  5. Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos.

    Science.gov (United States)

    Joo, Hyun; Choi, Kyoungju; Rose, Randy L; Hodgson, Ernest

    2007-01-01

    Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.

  6. Contribution of NADH increases to ethanol’s inhibition of retinol oxidation by human ADH isoforms

    Science.gov (United States)

    Chase, Jennifer R.; Poolman, Mark G.; Fell, David A.

    2010-01-01

    Background A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free NADH in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. Method To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of four reactions: (1) ADH oxidation of ethanol and NAD+ (2) ADH oxidation of retinol and NAD+ (3) oxidation of ethanol by a generalized Ethanoloxidase that uses NAD+ (4) NADHoxidase which carries out NADH turnover. Results Using the metabolic modeling package SCRUMPY, we have shown that the ethanol-induced increase in NADH contributes from 0–90% of the inhibition by ethanol, depending on [ethanol] and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanoloxidase and the NADHoxidase contribute as well. Discussion Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases. PMID:19183134

  7. CYP450氧化还原酶的遗传多态对药物代谢的影响%Genetic polymorphisms of cytochrome P450 oxidoreductase and its effect on drug metabolism

    Institute of Scientific and Technical Information of China (English)

    范岚; 彭向东; 郭志华; 周宏灏

    2009-01-01

    CYP450氧化还原酶(cytochrome P450 oxidoreductase, POR)是所有肝微粒体的细胞色素P450氧化酶(cytochrome P450 monooxygenases, CYP)的唯一电子供体,其中一些CYP是I相药物代谢酶,负责临床上超过80%药物的氧化代谢.另外,POR直接介导了一些抗肿瘤前体药物的代谢.因此,POR的遗传多态引起其活性的改变,对临床药物代谢具有非常重要的临床意义.该文总结了近年来POR的遗传多态影响药物代谢的最新研究进展.

  8. Myosin heavy chain isoform expression in human extraocular muscles: longitudinal variation and patterns of expression in global and orbital layers.

    Science.gov (United States)

    Park, Kyung-Ah; Lim, Jeonghee; Sohn, Seongsoo; Oh, Sei Yeul

    2012-05-01

    We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs. Copyright © 2012 Wiley Periodicals, Inc.

  9. Interaction of meropenem with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and molecular docking study.

    Science.gov (United States)

    Rehman, Md Tabish; Ahmed, Sarfraz; Khan, Asad U

    2016-09-01

    Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both 'N' and 'B' isoforms of HSA (ΔG < 0 and binding constant ~10(4) M(-1)). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with 'N' and 'B' isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow's site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in Km and an increase in kcat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.

  10. V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

    Science.gov (United States)

    Miquel-Serra, Laia; Serra, Montserrat; Hernández, Daniel; Domenzain, Clelia; Docampo, María José; Rabanal, Rosa M; de Torres, Inés; Wight, Thomas N; Fabra, Angels; Bassols, Anna

    2006-09-01

    Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.

  11. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    Science.gov (United States)

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions.

  12. [Isolation of isoforms of apolipoprotein CIII from human serum by chromatofocusing].

    Science.gov (United States)

    Fang, D; Gong, R; O, K

    1999-03-01

    This study aimed to isolate isoforms of apolipoprotein (apo) C III from human serum. 24-hour fasting serum from normal and hyperlipidemic subjects was pooled and subjected to ultracentrifugation at plasma density for 20 hours. Very low density lipoprotein (VLDL) was collected at density of d < 1.006 g/ml, and it was delipidated by ethanol and ether. The delipidated apo-VLDL was dissolved in a solution containing 7.2 mol/L urea and 20 mmol/L dithiothreitol. The insoluble apo B was removed by centrifugation. The soluble apo-VLDL was applied to PBE94 column, and eluted with elution buffer containing polybuffer 74 and 8 mol/L urea (1:8, pH4.0). After pooled, the eluted peaks of apolipoproteins were applied to column chromatography of hydroxylapatite to remove the polybuffer. The purified isoforms of apoC III and the purified apo C I, C II and E, were characterized by isoelectrofocusing and west blot. The results showed that the purified apoC III1, C III2, and C II were pure.

  13. Temporal, Diagnostic, and Tissue-Specific Regulation of NRG3 Isoform Expression in Human Brain Development and Affective Disorders.

    Science.gov (United States)

    Paterson, Clare; Wang, Yanhong; Hyde, Thomas M; Weinberger, Daniel R; Kleinman, Joel E; Law, Amanda J

    2017-03-01

    Genes implicated in schizophrenia are enriched in networks differentially regulated during human CNS development. Neuregulin 3 (NRG3), a brain-enriched neurotrophin, undergoes alternative splicing and is implicated in several neurological disorders with developmental origins. Isoform-specific increases in NRG3 are observed in schizophrenia and associated with rs10748842, a NRG3 risk polymorphism, suggesting NRG3 transcriptional dysregulation as a molecular mechanism of risk. The authors quantitatively mapped the temporal trajectories of NRG3 isoforms (classes I-IV) in the neocortex throughout the human lifespan, examined whether tissue-specific regulation of NRG3 occurs in humans, and determined if abnormalities in NRG3 transcriptomics occur in mood disorders and are genetically determined. NRG3 isoform classes I-IV were quantified using quantitative real-time polymerase chain reaction in human postmortem dorsolateral prefrontal cortex from 286 nonpsychiatric control individuals, from gestational week 14 to 85 years old, and individuals diagnosed with either bipolar disorder (N=34) or major depressive disorder (N=69). Tissue-specific mapping was investigated in several human tissues. rs10748842 was genotyped in individuals with mood disorders, and association with NRG3 isoform expression examined. NRG3 classes displayed individually specific expression trajectories across human neocortical development and aging; classes I, II, and IV were significantly associated with developmental stage. NRG3 class I was increased in bipolar and major depressive disorder, consistent with observations in schizophrenia. NRG3 class II was increased in bipolar disorder, and class III was increased in major depression. The rs10748842 risk genotype predicted elevated class II and III expression, consistent with previous reports in the brain, with tissue-specific analyses suggesting that classes II and III are brain-specific isoforms of NRG3. Mapping the temporal expression of genes

  14. In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4

    Directory of Open Access Journals (Sweden)

    Qiao-Li Lv

    2015-12-01

    Full Text Available Glycyrrhetinic acid (GA has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450 cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

  15. Differential ligand binding affinities of human estrogen receptor-α isoforms.

    Directory of Open Access Journals (Sweden)

    Amanda H Y Lin

    Full Text Available Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66 and the truncated isoforms, estrogen receptor-α46 (ER46 and estrogen receptor-α36 (ER36. However, the binding affinities of the membrane estrogen receptors (mERs remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

  16. Functional refolding and characterization of two Tom40 isoforms from human mitochondria.

    Science.gov (United States)

    Mager, Frauke; Gessmann, Dennis; Nussberger, Stephan; Zeth, Kornelius

    2011-07-01

    Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

  17. Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

    Science.gov (United States)

    Vemu, Annapurna; Atherton, Joseph; Spector, Jeffrey O; Szyk, Agnieszka; Moores, Carolyn A; Roll-Mecak, Antonina

    2016-06-17

    Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

  18. Isoforms of Hsp70-binding human LDL in adult Schistosoma mansoni worms.

    Science.gov (United States)

    Pereira, Adriana S A; Cavalcanti, Marília G S; Zingali, Russolina B; Lima-Filho, José L; Chaves, Maria E C

    2015-03-01

    Schistosoma mansoni is one of the most common parasites infecting humans. They are well adapted to the host, and this parasite's longevity is a consequence of effective escape from the host immune system. In the blood circulation, lipoproteins not only help to conceal the worm from attack by host antibodies but also act as a source of lipids for S. mansoni. Previous SEM studies showed that the low-density lipoprotein (LDL) particles present on the surface of adult S. mansoni worms decreased in size when the incubation time increased. In this study, immunocytochemical and proteomic analyses were used to locate and identify S. mansoni binding proteins to human plasma LDL. Ultrathin sections of adult worms were cut transversely from the anterior, medial and posterior regions of the parasite. Immunocytochemical experiments revealed particles of gold in the tegument, muscle region and spine in male worms and around vitelline cells in females. Immunoblotting and 2D-electrophoresis using incubations with human serum, anti-LDL antibodies and anti-chicken IgG peroxidase conjugate were performed to identify LDL-binding proteins in S. mansoni. Analysis of the binding proteins using LC-MS identified two isoforms of the Hsp70 chaperone in S. mansoni. Hsp70 is involved in the interaction with apoB in the cytoplasm and its transport to the endoplasmic reticulum. However, further studies are needed to clarify the functional role of Hsp70 in S. mansoni, mainly related to the interaction with human LDL.

  19. Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

    DEFF Research Database (Denmark)

    Redlich, Gorden; Zanger, Ulrich M; Riedmaier, Stephan;

    2008-01-01

    In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based...

  20. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  1. 题目:CYP450代谢酶和一些抗氧化信号参与调控了绿原酸抑制对乙酰氨基酚的肝毒性%Chlorogenic acid prevents acetaminophen-induced liver injury:the involvement of CYP450 metabolic enzymes and some antioxidant signals

    Institute of Scientific and Technical Information of China (English)

    Chun PANG; Yu-chen SHENG; Ping JIANG; Hai WEI; Li-li JI

    2015-01-01

    目的:本研究旨在观察绿原酸对乙酰氨基酚诱导肝损伤的解毒作用及其机理。创新点:发现CYP450代谢酶和一些重要的抗氧化信号分子(如Prx家族蛋白等)参与调控了绿原酸抑制对乙酰氨基酚的肝毒性。方法:检测小鼠血清转氨酶含量,检测体外 CYP2E1、CYP3A4和CYP1A2酶活性,检测肝组织中丙二醛(MDA)、谷胱甘肽(GSH)和活性氧(ROS)含量,用实时聚合酶链反应(real-time PCR)检测肝组织中Prx1-6、Ephx2、Polr2k、Fmo5、Nrf2等的mRNA表达情况。结论:绿原酸可以明显抑制对乙酰氨基酚造成的急性肝损伤。给药组小鼠血清中的转氨酶与模型组相比均有显著下降,绿原酸在体外可以微弱抑制CYP2E1和CYP1A2代谢酶的活性,通过MDA、GSH和 ROS分析发现绿原酸可以抑制对乙酰氨基酚造成的氧化应激损伤。通过real-time PCR分析发现对乙酰氨基酚降低了抗氧化酶 Prx家族、Ephx2、Polr2k和 Nrf2的基因表达,而绿原酸可以逆转对乙酰氨基酚降低的这些基因的表达。%Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cel viability induced by AP in L-02 celsin vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST)in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin

  2. Differential subcellular localizations of two human Sgo1 isoforms: implications in regulation of sister chromatid cohesion and microtubule dynamics.

    Science.gov (United States)

    Wang, Xiaoxing; Yang, Yali; Dai, Wei

    2006-03-01

    Sgo1 is an evolutionarily conserved protein that functions as a protector of centromeric cohesin during mitosis. Recent studies show that Sgo1 is kinetochorelocalized and required for accurate segregation of mitotic chromosomes because depletion of Sgo1 in mammalian cells results in precocious initiation of anaphase and mis-segregation of chromosomes. Through analysis of GFP fusion proteins, we observe that two major isoforms of human Sgo1 exhibit entirely different subcellular localization patterns. The short isoform of Sgo1 (sSgo1) that lacks exon 6 does not localize to kinetochores during any stages of the cell cycle. Instead, it is enriched at mitotic spindles. On the other hand, the longer isoform of Sgo1 primarily localizes to kinetochores during G(2) phase and mitotic prophase, metaphase, and anaphase. During late mitosis, Sgo1 does not appear to be associated with kinetochores. Intriguingly, the longer isoform of Sgo1 forms discrete foci during S phase, some of which are apparently in the nucleoli. However, a majority of these foci colocalize with CREST, a kinetochore antigen, indicating that Sgo1 is loaded onto kinetochores during or immediately after DNA replication. Together, our studies suggest that different isoforms of Sgo1 may play distinct roles during the cell cycle and that Sgo1 may have an interphase function as well.

  3. Significance of redox-active cysteines in human FAD synthase isoform 2.

    Science.gov (United States)

    Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

    2014-12-01

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

  4. Statistical evaluation of the isoform patterns of N-acetyl-beta-hexosaminidase from human renal cancer tissue separated by isoelectrofocusing.

    Science.gov (United States)

    Borzym-Kluczyk, Malgorzata; Radziejewska, Iwona; Olszewska, Ewa; Szajda, Sławomir; Knaś, Małgorzata; Zwierz, Krzysztof

    2007-03-01

    Isoenzymes of HEX from human renal carcinoma and neighbouring macroscopically normal renal tissue can show different patterns on isoelectrofocusing gels. The aim of our work was to elaborate the method for statistical evaluation of differences. Isoenzymes of N-acetyl-beta-hexosaminidase were separated from renal (control and cancerous) tissues of 15 patients. Isoenzymes were electrofocused in Multiphor II, with ampholine pH 3.5-9.0 (2%) and then evaluated densitometrically and analysed statistically. A similar pattern in activity of isoforms of isoenzymes A and B in normal and cancerous renal tissue was observed. The proposed method of statistical evaluation of differences in isoforms of N-acetyl-beta-glucosaminidase can also be adapted to estimate the isoforms of other enzymes in different tissues.

  5. Discoidin domain receptor 1: isoform expression and potential functions in cirrhotic human liver.

    Science.gov (United States)

    Song, Sunmi; Shackel, Nicholas A; Wang, Xin M; Ajami, Katerina; McCaughan, Geoffrey W; Gorrell, Mark D

    2011-03-01

    Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

  6. Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

    The differential availability of thyroxine (T/sub 4/) and 3,5,3'-triiodothyronine (T/sub 3/) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of (/sup 125/I)T/sub 4/ and (/sup 125/I)T/sub 3/ to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, (/sup 125/I)T/sub 4/ was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. (/sup 125/I)T/sub 3/ was localized to only two isoforms of TBG, TBG-III, and -IV, with pI's that were identical with those for (/sup 125/I)T/sub 4/. In normal female serum, (/sup 125/I)T/sub 4/ was localized to the same four isoforms of TBG as those of normal male serum, while (/sup 125/I)T/sub 3/ was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, (/sup 125/I)T/sub 4/ was localized to five isoforms, whereas (/sup 125/I)T/sub 3/ was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T/sub 3/. The K/sub i/ values of T/sub 3/ binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and the chromatofocusing (pH range of 3.5-5.0). T/sub 4/ preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T/sub 4/ and T/sub 3/. In conclusion, this study shows that T/sub 4/ and T/sub 3/ do not bind to a single competitive binding site on TBG. Instead, T/sub 4/ is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T/sub 3/ for these proteins.

  7. Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

    Science.gov (United States)

    Rodriguez, R J; Miranda, C L

    2000-09-01

    N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (DAK to N-hydroxy-DAK (15.9%) and metabolite 1 (6.6%). Last, DAK did not appear to be a substrate for human or rabbit FMO5. Heat inactivation of cDNA-expressed FMOs abolished DAK metabolite formation. These results suggest that DAK is a substrate for human and rabbit FMO1 and FMO3, rabbit FMO2, but not human or rabbit FMO5.

  8. Human invariant chain isoform p35 restores thymic selection and antigen presentation in CD74-deficient mice.

    Science.gov (United States)

    Genève, Laetitia; Chemali, Magali; Desjardins, Michel; Labrecque, Nathalie; Thibodeau, Jacques

    2012-10-01

    The invariant chain (Ii) has pleiotropic functions and is a key factor in antigen presentation. Ii associates with major histocompatibility complex class II molecules in the endoplasmic reticulum (ER) and targets the complex in the endocytic pathway to allow antigenic peptide loading. The human Iip35 isoform includes a cytoplasmic extension containing a di-arginine motif causing ER retention. This minor isoform does not exist in mice and its function in humans has not been thoroughly investigated. We have recently generated transgenic mice expressing Iip35 and these were crossed with Ii-deficient mice to generate animals (Tgp35/mIiKO) expressing exclusively the human isoform. In these mice, we show that Iip35 is expressed in antigen presenting cells and is inducible by interferon gamma (IFN-γ). Despite the low constitutive expression of the protein and some minor differences in the Vβ repertoire of Tgp35/mIiKO mice, Iip35 restored thymic selection of CD4(+) T cells and of invariant natural killer T cells. In vitro functional assays using purified primary macrophages treated with IFN-γ showed that Iip35 allows presentation of an Ii-dependent ovalbumin T-cell epitope. Altogether, our results suggest that Iip35 is functional and does not require co-expression of other isoforms for antigen presentation.

  9. [Isoforms of the human histamine H3 receptor: Generation, expression in the central nervous system and functional implications].

    Science.gov (United States)

    García-Gálvez, Ana Maricela; Arias-Montaño, José Antonio

    2016-01-01

    Histamine plays a significant role as a neuromodulator in the human central nervous system. Histamine-releasing neurons are exclusively located in the tuberomammillary nucleus of the hypothalamus, project to all major areas of the brain, and participate in functions such as the regulation of sleep/wakefulness, locomotor activity, feeding and drinking, analgesia, learning, and memory. The functional effects of histamine are exerted through the activation of four G protein-coupled receptors (H1, H2, H3 and H4), and in the central nervous system the first three receptors are widely expressed. The H3 receptor (H3R) is found exclusively in neuronal cells, where it functions as auto- and hetero-receptor. One remarkable characteristic of the H3R is the existence of isoforms, generated by alternative splicing of the messenger RNA. For the human H3R, 20 isoforms have been reported; although a significant number lack those regions required for agonist binding or receptor signaling, at least five isoforms appear functional upon heterologous expression. In this work we review the evidence for the generation of human H3R isoforms, their expression, and the available information regarding the functionality of such receptors.

  10. Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase.

    Science.gov (United States)

    Laurent, Laetitia; Deroy, Kathy; St-Pierre, Joey; Côté, Francine; Sanderson, J Thomas; Vaillancourt, Cathy

    2017-09-01

    The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Co-expression of SERCA isoforms, phospholamban and sarcolipin in human skeletal muscle fibers.

    Directory of Open Access Journals (Sweden)

    Val A Fajardo

    Full Text Available Sarcolipin (SLN and phospholamban (PLN inhibit the activity of sarco(endoplasmic reticulum Ca(2+-ATPases (SERCAs by reducing their apparent affinity for Ca(2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg to vastus lateralis homogenates increased the apparent Ca(2+ affinity of SERCA (K Ca, pCa units (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02 and maximal SERCA activity (μmol/g protein/min (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11 demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.

  12. Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To study the gene expression of metallothionein 1 (MT-1) isoforms in human peripheral blood lymphocytes (HPBLs). Methods The expression of mRNA representing the seven active MT-1 genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-1X, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT-1H, 1F, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference (P<0.05). in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, 1F, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1G, MT-1H, MT-1F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

  13. Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

    Science.gov (United States)

    Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

    2017-01-01

    Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features.

  14. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane, tumor-associated isoforms IX and XII with boronic acids.

    Science.gov (United States)

    Winum, Jean-Yves; Innocenti, Alessio; Scozzafava, Andrea; Montero, Jean-Louis; Supuran, Claudiu T

    2009-05-15

    A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K(I) of 3.7-4.5 microM, whereas the remaining derivatives showed inhibition constants in the range of 6.0-1560 microM for hCA I and of 6.0-1050 microM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K(I)s of 7.6-12.3 microM) whereas the arylalkenyl and aryl-alkyl derivatives generally showed weaker inhibitory properties (K(I)s of 34-531 microM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)(2) moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.

  15. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    Directory of Open Access Journals (Sweden)

    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  16. Charged residues distribution modulates selectivity of the open state of human isoforms of the voltage dependent anion-selective channel.

    Science.gov (United States)

    Amodeo, Giuseppe Federico; Scorciapino, Mariano Andrea; Messina, Angela; De Pinto, Vito; Ceccarelli, Matteo

    2014-01-01

    Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

  17. Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology.

    Science.gov (United States)

    Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A

    2008-06-01

    The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.

  18. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

    Science.gov (United States)

    Farr, Christine J; Antoniou-Kourounioti, Melissa; Mimmack, Michael L; Volkov, Arsen; Porter, Andrew C G

    2014-04-01

    As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

  19. Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

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    Robert A Eagle

    Full Text Available BACKGROUND: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. METHODOLOGY/PRINCIPAL FINDINGS: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have

  20. 冬虫夏草及人工虫草丝菌提取物对四氯化碳致急性化学性肝损伤小鼠的保护作用%Protective effects of cordyceps sinensis and mycelium of cultured sinensis on acute liver injury and expression of CYP450 induced by Carbon Tetrachloride

    Institute of Scientific and Technical Information of China (English)

    杨夕霞; 陈小红; 陈方军; 胡蓉; 曹庄; 陈晓宇

    2010-01-01

    目的 研究冬虫夏草及人工虫草丝菌对四氯化碳(CCl_4)致急性化学性肝损伤小鼠细胞色素P450(CYP450)活性的影响.探讨其对肝损伤的保护作用机制.方法 昆明种雄性小鼠CCl_4(0.1%,20 ml/kg)一次性腹腔注射建立急性化学性肝损伤模型,冬虫夏草及人工虫草丝菌(100 mg/ml)每6 h灌胃一次,共3次,随后在3个不同时间段(6 h、12 h、24 h)处死小鼠,检测血清ALT、GSH及肝微粒体CYP450含量,Westernbolt检测CYP450蛋白表达.结果 冬虫夏草及人工虫革丝菌在各个不同时间段明显降低ALT水平,以24 h降低更为明显,且GSH、CYP450活性提高.结论 冬虫夏草及人工虫草丝菌对化学性肝损伤治疗作用,可能与其提高CYP450等肝药酶活性,增加其抗氧化作用有关.%Objective To investigate the effect of Cordyceps Sinensis and Mycelium of Cultured Sinensis on acute injury in this test and expression of CYP450. Methods Mice received a single dose of 0. 1% CCl_4 i. p, after CCl_4 intoxication,mice received Cordyceps Sincnsis and Mycelium of Cultured Sinensis (100mg/ml) 3 times per six hour,then the mice sacrificed at 6h,12h,24h,the levels of aminotransferase (ALT) and glutathione (GSH) in ser-um,CYP45 content in hepatic microsomal activity in fiver homogenate were measured. Results Mice given Cordyceps Sinensis and Mycelium of Cultured Sinensis supplement had less elevation of serum ALT concentration, Cordyceps Sinensis and Mycelium of Cultured Sinensis administration restored the reduction of hepatic microsomal P450 protein content as well as inducing an increase in hepatic GSH concentration. Additionally, Cordyceps Sinensis and Mycelium of Cultured Sincnsis treatment inhibited the elevation of hepatic CYP450 protein after CCl_4 intoxication. Condusion The protect effect of Cordyceps Sinensis and Mycelium of Cultured Sinensis may be due to its abilitity to increase CYP450 content which decreased in CCl_4-induced liver in combination with increasing

  1. Effects of Wansheng Huafeng Dan on mRNA expression of hepatic cytochrome P450 in rats%万胜化风丹对大鼠肝脏 CYP450酶系亚型基因的影响

    Institute of Scientific and Technical Information of China (English)

    王丽娜; 刘富鑫; 吴芹; 石京山; 刘杰

    2015-01-01

    Objective:To investigate the effects of Wansheng Huafeng Dan (WSHFD)and HgCl2 on the expression of cytochrome P450 mRNA in SD rat liver.Method:A total of 25 SD male rats were randomly divided into 5 groups including the control group (distilled water),WSHFD0 group (1.0 g/Kg),WSHFD2 group (1.0 g/Kg),WSHFD3 group (1.0 g/Kg)and HgCl2 group (10 mg/Kg).Rats were orally adminis-tered with drugs for consecutive 7 days.The expression of CYP450 mRNA was detected by real time quantita-tive RT -PCR.Results:Compared with the control group,there were no obvious changes of the Cyp1a1, Cyp2e1,Cyp3a1,and Cyp4a10 expression in WSHFD0,WSHFD2 and WSHFD3 groups,but significantly re-duced in the HgCl2 group (P <0.05).The expression of Cyp3a2 and Cyp7a1 was decreased in WSHFD0, WSHFD2 and WSHFD3 groups (P <0.05),but it was unaltered in HgCl2 group.Conclusion: (1 )The effects of WSHFD on Cyp1a1,Cyp2e1,Cyp3a1,Cyp3a2,Cyp4a10 and Cyp7a1 mRNA expression were dif-ferent from the HgCl2 group;(2)The effects of WSHFD0,WSHFD2 and WSHFD3 on Cyp1a1,Cyp2e1,Cyp3a1,Cyp3a2,Cyp4a10 and Cyp7a1 mRNA expression were not obvious;(3 )WSHFD obviously in-creased the expression of Cyp3a2 mRNA,suggesting it could have potential influence on the drug metabolism;(4)WSHFD obviously increased the expression of Cyp7a1 mRNA,which suggests it may affect the metabo-lism of bile acid.%目的:研究万胜化风丹(WSHFD)和氯化汞对大鼠肝脏组织细胞色素 P450酶系亚型基因的影响。方法:雄性 SD 大鼠25只,随机分为5组,每组5只,分别为空白组,WSHFD0组(不含朱砂、雄黄的万胜化风丹配方,剂量1.0 g/kg),WSHFD2组(减量2/3朱砂雄黄的万胜化风丹现代配方,剂量1.0 g/kg),WSHFD3组(万胜化风丹古方组,剂量1.0 g/kg),氯化汞组(剂量10 mg/kg)。连续灌胃给药7 d 后麻醉,取大鼠肝脏组织提取 RNA,采用 real time RT -PCR 检测 CYP450酶系亚型基因的表达。结

  2. Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

    OpenAIRE

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Michael H Gelb; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h...

  3. High molecular weight FGF2 isoforms demonstrate canonical receptor-mediated activity and support human embryonic stem cell self-renewal

    Directory of Open Access Journals (Sweden)

    Denis Kole

    2017-05-01

    Full Text Available Basic fibroblast growth factor (FGF2 is a highly pleiotropic member of a large family of growth factors with a broad range of activities, including mitogenesis and angiogenesis (Ornitz et al., 1996; Zhang et al., 2006, and it is known to be essential for maintenance of balance between survival, proliferation, and self-renewal in human pluripotent stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011. A single FGF2 transcript can be translated into five FGF2 protein isoforms, an 18 kDa low molecular weight (LMW isoform and four larger high molecular weight (HMW isoforms (Arese et al., 1999; Arnaud et al., 1999. As they are not generally secreted, high molecular weight (HMW FGF2 isoforms have predominantly been investigated intracellularly; only a very limited number of studies have investigated their activity as extracellular factors. Here we report over-expression, isolation, and biological activity of all recombinant human FGF2 isoforms. We show that HMW FGF2 isoforms can support self-renewal of human embryonic stem cells (hESCs in vitro. Exogenous supplementation with HMW FGF2 isoforms also activates the canonical FGFR/MAPK pathway and induces mitogenic activity in a manner similar to that of the 18 kDa FGF2 isoform. Though all HMW isoforms, when supplemented exogenously, are able to recapitulate LMW FGF2 activity to some degree, it appears that certain isoforms tend to do so more poorly, demonstrating a lesser functional response by several measures. A better understanding of isoform-specific FGF2 effects will lead to a better understanding of developmental and pathological FGF2 signaling.

  4. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

    Directory of Open Access Journals (Sweden)

    Nathalie Esber

    Full Text Available The progesterone receptor (PR with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4 and ulipristal acetate (UPA, a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.

  5. Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain.

    Directory of Open Access Journals (Sweden)

    Phillip B Jones

    Full Text Available We applied a novel application of FLIM-FRET to in situ measurement and quantification of protein interactions to explore isoform specific differences in Aβ-ApoE interaction and ApoE tertiary conformation in senile plaques in human Alzheimer brain. ApoE3 interacts more closely with Aβ than ApoE4, but a greater proportion of Aβ molecules within plaques are decorated with ApoE4 than ApoE3, lending strong support to the hypothesis that isoform specific differences in ApoE are linked with Aβ deposition. We found an increased number of ApoE N-terminal fragments in ApoE4 plaques, consistent with the observation that ApoE4 is more easily cleaved than ApoE3. In addition, we measured a small but significant isoform specific difference in ApoE domain interaction. Based on our in situ data, supported by traditional biochemical data, we propose a pathway by which isoform specific conformational differences increase the level of cleavage at the hinge region of ApoE4, leading to a loss of ApoE function to mediate clearance of Aβ and thereby increase the risk of AD for carriers of the APOEε4 allele.

  6. Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein.

    Science.gov (United States)

    Ramos, Adrián M; Duschak, Vilma G; Gerez de Burgos, Nelia M; Barboza, Mariana; Remedi, María S; Vides, Miguel A; Chiabrando, Gustavo A

    2002-02-01

    Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.

  7. Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation

    Directory of Open Access Journals (Sweden)

    Hartmann-Fatu Cristina

    2006-03-01

    Full Text Available Abstract Background The peptidyl prolyl cis/trans isomerase (PPIase Parvulin (Par14/PIN4 is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro. Results In this study we confirm by RT-PCR the existence of a longer Parvulin isoform expressed in all tissues examined so far. This isoform contains a 5' extension including a 75 bp extended open reading frame with two coupled SNPs leading to amino acid substitutions Q16R and R18S. About 1% of all Parvulin mRNAs include the novel extension as quantified by real-time PCR. The human Parvulin promoter is TATA-less and situated in a CpG island typical for house keeping genes. Thus, different Parvulin mRNAs seem to arise by alternative transcription initiation. N-terminally extended Parvulin is protected from rapid proteinaseK degradation. In HeLa and HepG2 cell lysates two protein species of about 17 and 28 KDa are detected by an antibody against an epitope within the N-terminal extension. These two bands are also recognized by an antibody towards the PPIase domain of Parvulin. The longer Parvulin protein is encoded by the human genome but absent from rodent, bovine and non-mammalian genomes. Conclusion Due to its molecular weight of 16.6 KDa we denote the novel Parvulin isoform as Par17 following the E. coli Par10 and human Par14 nomenclature. The N-terminal elongation of Par17-QR and Par17-RS suggests these isoforms to perform divergent functions within the eukaryotic cell than the well characterized Par14.

  8. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    Science.gov (United States)

    Girolamo, Francesco; Dallatomasina, Alice; Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  9. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    Directory of Open Access Journals (Sweden)

    Francesco Girolamo

    Full Text Available NG2/CSPG4 is a complex surface-associated proteoglycan (PG recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS

  10. Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity

    Energy Technology Data Exchange (ETDEWEB)

    Lepont, Pierig; Stickney, John T.; Foster, Lauren A.; Meng, Jin-Jun; Hennigan, Robert F. [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Ip, Wallace [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States)], E-mail: wallace.ip@uc.edu

    2008-01-01

    Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2{sup -/-} mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

  11. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  12. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  13. Differential ligand binding affinities of human estrogen receptor-α isoforms

    OpenAIRE

    Amanda H.Y. Lin; Li, Rachel W. S.; Ho, Eva Y. W.; George P H Leung; Susan W S Leung; Paul M Vanhoutte; Man, Ricky Y K

    2013-01-01

    Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cel...

  14. Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation*

    Science.gov (United States)

    Su, Shifeng; Blackwelder, Amanda J.; Grossman, Gail; Minges, John T.; Yuan, Lingwen; Young, Steven L.; Wilson, Elizabeth M.

    2012-01-01

    Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH2-terminal 110LLXXVLXXLL119 motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium. PMID:22891251

  15. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

    NARCIS (Netherlands)

    Rennel, E.; Waine, E.; Guan, H.; Schuler, Y.; Leenders, W.P.J.; Woolard, J.; Sugiono, M.; Gillatt, D.; Kleinerman, E.; Bates, D.; Harper, S.

    2008-01-01

    Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the

  16. CYP450-Dependent Biotransformation of the Insecticide Fipronil into Fipronil Sulfone Can Mediate Fipronil-Induced Thyroid Disruption in Rats (Full paper and erratum)

    OpenAIRE

    ROQUES, Beatrice; Lacroix, Marlène; Puel, Sylvie; Gayrard, Véronique; Picard-Hagen, Nicole; Jouanin, Isabelle; Perdu, Elisabeth; Martin, Pascal

    2012-01-01

    In rats, the widely used insecticide fipronil increases the clearance of thyroxine (T-4). This effect is associated with a high plasma concentration of fipronil sulfone, the fipronil main metabolite in several species including rats and humans. In sheep, following fipronil treatment, fipronil sulfone plasma concentration and thyroid disruption are much lower than in rats. We postulated that fipronil biotransformation into fipronil sulfone by hepatic cytochromes P450 (CYP) could act as a pote...

  17. Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

    Science.gov (United States)

    Wanga, Shaynah; Ceron, Carla S; Delgado, Cynthia; Joshi, Sunil K; Spaulding, Kimberly; Walker, Joy P; Song, Sangheon; Olson, Jean L; Lovett, David H

    2015-01-01

    Delayed graft function (DGF) is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2), as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2) that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC), in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are associated with

  18. Two Distinct Isoforms of Matrix Metalloproteinase-2 Are Associated with Human Delayed Kidney Graft Function.

    Directory of Open Access Journals (Sweden)

    Shaynah Wanga

    Full Text Available Delayed graft function (DGF is a frequent complication of renal transplantation, particularly in the setting of transplantation of kidneys derived from deceased donors and expanded-criteria donors. DGF results from tubular epithelial cell injury and has immediate and long term consequences. These include requirement for post-transplantation dialysis, increased incidence of acute rejection, and poorer long-term outcomes. DGF represents one of the clearest clinical examples of renal acute ischemia/reperfusion injury. Experimental studies have demonstrated that ischemia/reperfusion injury induces the synthesis of the full length secreted isoform of matrix metalloproteinase-2 (FL-MMP-2, as well as an intracellular N-terminal truncated MMP-2 isoform (NTT-MMP-2 that initiates an innate immune response. We hypothesized that the two MMP-2 isoforms mediate tubular epithelial cell injury in DGF. Archival renal biopsy sections from 10 protocol biopsy controls and 41 cases with a clinical diagnosis of DGF were analyzed for the extent of tubular injury, expression of the FL-MMP-2 and NTT-MMP-2 isoforms by immunohistochemistry (IHC, in situ hybridization, and qPCR to determine isoform abundance. Differences in transcript abundance were related to tubular injury score. Markers of MMP-2-mediated injury included TUNEL staining and assessment of peritubular capillary density. There was a clear relationship between tubular epithelial cell expression of both FL-MMP-2 and NTT-MMP-2 IHC with the extent of tubular injury. The MMP-2 isoforms were detected in the same tubular segments and were present at sites of tubular injury. qPCR demonstrated highly significant increases in both the FL-MMP-2 and NTT-MMP-2 transcripts. Statistical analysis revealed highly significant associations between FL-MMP-2 and NTT-MMP-2 transcript abundance and the extent of tubular injury, with NTT-MMP-2 having the strongest association. We conclude that two distinct MMP-2 isoforms are

  19. DNMT3B7, a truncated DNMT3B isoform expressed in human tumors, disrupts embryonic development and accelerates lymphomagenesis

    Science.gov (United States)

    Shah, Mrinal Y.; Vasanthakumar, Aparna; Barnes, Natalie Y.; Figueroa, Maria E.; Kamp, Anna; Hendrick, Christopher; Ostler, Kelly R.; Davis, Elizabeth M.; Lin, Shang; Anastasi, John; Le Beau, Michelle M.; Moskowitz, Ivan; Melnick, Ari; Pytel, Peter; Godley, Lucy A.

    2010-01-01

    Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the C-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic are bred with Eμ-Myc transgenic mice, which model aggressive B cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared to Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the re-distribution of DNA methylation characterizing virtually every human tumor. PMID:20587527

  20. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

    Directory of Open Access Journals (Sweden)

    Sandy Azzi

    2015-06-01

    Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

  1. Adamantyl ethanone pyridyl derivatives: potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase type 1.

    Science.gov (United States)

    Su, Xiangdong; Pradaux-Caggiano, Fabienne; Vicker, Nigel; Thomas, Mark P; Halem, Heather; Culler, Michael D; Potter, Barry V L

    2011-09-05

    Elevated levels of active glucocorticoids have been implicated in the development of several phenotypes of metabolic syndrome, such as type 2 diabetes and obesity. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular conversion of inactive cortisone to cortisol. Selective 11β-HSD1 inhibitors have shown beneficial effects in various conditions, including diabetes, dyslipidemia and obesity. A series of adamantyl ethanone pyridyl derivatives has been identified, providing potent and selective inhibitors of human 11β-HSD1. Lead compounds display low nanomolar inhibition against human and mouse 11β-HSD1 and are selective for this isoform, with no activity against 11β-HSD2 and 17β-HSD1. Structure-activity relationship studies reveal that an unsubstituted pyridine tethered to an adamantyl ethanone motif through an ether or sulfoxide linker provides a suitable pharmacophore for activity. The most potent inhibitors have IC₅₀ values around 34-48 nM against human 11β-HSD1, display reasonable metabolic stability in human liver microsomes, and weak inhibition of key human CYP450 enzymes.

  2. Adamantyl Ethanone Pyridyl Derivatives: Potent and Selective Inhibitors of Human 11β-Hydroxysteroid Dehydrogenase Type 1

    Science.gov (United States)

    Su, Xiangdong; Pradaux-Caggiano, Fabienne; Vicker, Nigel; Thomas, Mark P; Halem, Heather; Culler, Michael D; Potter, Barry V L

    2011-01-01

    Elevated levels of active glucocorticoids have been implicated in the development of several phenotypes of metabolic syndrome, such as type 2 diabetes and obesity. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular conversion of inactive cortisone to cortisol. Selective 11β-HSD1 inhibitors have shown beneficial effects in various conditions, including diabetes, dyslipidemia and obesity. A series of adamantyl ethanone pyridyl derivatives has been identified, providing potent and selective inhibitors of human 11β-HSD1. Lead compounds display low nanomolar inhibition against human and mouse 11β-HSD1 and are selective for this isoform, with no activity against 11β-HSD2 and 17β-HSD1. Structure–activity relationship studies reveal that an unsubstituted pyridine tethered to an adamantyl ethanone motif through an ether or sulfoxide linker provides a suitable pharmacophore for activity. The most potent inhibitors have IC50 values around 34–48 nm against human 11β-HSD1, display reasonable metabolic stability in human liver microsomes, and weak inhibition of key human CYP450 enzymes. PMID:21714097

  3. Binding and repressive activities of apolipoprotein E3 and E4 isoforms on the human ApoD promoter.

    Science.gov (United States)

    Levros, Louis-Charles; Labrie, Marilyne; Charfi, Cyndia; Rassart, Eric

    2013-12-01

    Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer's disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV-VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.

  4. Human heterochromatin protein 1 isoforms regulate androgen receptor signaling in prostate cancer.

    Science.gov (United States)

    Itsumi, Momoe; Shiota, Masaki; Yokomizo, Akira; Kashiwagi, Eiji; Takeuchi, Ario; Tatsugami, Katsunori; Inokuchi, Junichi; Song, Yoohyun; Uchiumi, Takeshi; Naito, Seiji

    2013-06-01

    Androgen receptor (AR) signaling is critical for the tumorigenesis and development of prostate cancer, as well as the progression to castration-resistant prostate cancer. We previously showed that the heterochromatin protein 1 (HP1) β isoform plays a critical role in transactivation of AR signaling as an AR coactivator that promotes prostate cancer cell proliferation. However, the roles of other HP1 isoforms, HP1α and HP1γ, in AR expression and prostate cancer remain unclear. Here, we found that knockdown of HP1γ, but not HP1α, reduced AR expression and cell proliferation by inducing cell cycle arrest at G1 phase in LNCaP cells. Conversely, overexpression of full-length HP1α and its C-terminal deletion mutant increased AR expression and cell growth, whereas overexpression of HP1γ had no effect. Similarly, HP1α overexpression promoted 22Rv1 cell growth, whereas HP1γ knockdown reduced the proliferation of CxR cells, a castration-resistant LNCaP derivative. Taken together, HP1 isoforms distinctly augment AR signaling and cell growth in prostate cancer. Therefore, silencing of HP1β and HP1γ may be a promising therapeutic strategy for treatment of prostate cancer.

  5. STIM and Orai isoform expression in pregnant human myometrium: a potential role in calcium signaling during pregnancy.

    Directory of Open Access Journals (Sweden)

    Evonne eChin-Smith

    2014-05-01

    Full Text Available Store-operated calcium (Ca2+ entry (SOCE can be mediated by two novel proteins, STIM/Orai. We have previously demonstrated that members of the TRPC family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labor associated stimuli IL-1β and mechanical stretch. Although STIM and Orai isoforms (1-3 have been reported in other smooth muscle cell types, there is little known about the expression or gestational regulation of STIM and Orai expression in human myometrium. Total RNA was isolated from lower segment human myometrial biopsies obtained at caesarean section from women at the time of preterm no labor (PTNL, preterm labor (PTL, term non-labor (TNL and term with labor (TL; primary cultured human uterine smooth muscle cells, and a human myometrial cell line (hTERT-HM. STIM1-2, and Orai1-3 mRNA expression was assessed by quantitative real-time PCR. All five genes were expressed in myometrial tissue and cultured cells. Orai2 was the most abundant Orai isoform in human myometrium. Expression of STIM1-2/Orai1-3 did not alter with the onset of labor. Orai1 mRNA expression in cultured cells was enhanced by IL-1β treatment. This novel report of STIM1-2 and Orai1-3 mRNA expression in pregnant human myometrium and Orai1 regulation by IL-1β indicates a potential role for these proteins in calcium signaling in human myometrium during pregnancy.

  6. Identification of the human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of the phytochemical ferulic acid.

    Science.gov (United States)

    Li, Xiaojun; Shang, Liang; Wu, Yaohua; Abbas, Suzanne; Li, Dong; Netter, Patrick; Ouzzine, Mohamed; Wang, Hui; Magdalou, Jacques

    2011-01-01

    Ferulic acid (FA), a member of the hydroxycinnamate family, is an abundant dietary antioxidant that may offer beneficial effects against cancer, cardiovascular disease, diabetes, osteoarthritis and Alzheimer's disease. In this study, evidence for sulfation and glucuronidation of FA was investigated upon incubation with human liver microsomes and cytosol. Two main glucuronides, M1 (ether O-glucuronide) and M2 (ester acylglucuronide), were formed with a similar affinity (apparent K(m) 3.53 and 5.15 mM, respectively). A phenol sulfoconjugate was also formed with a higher affinity (K(m) 0.53 mM). Identification of the UDP-glucuronosyltransferase (UGT) isoforms involved in FA glucuronidation was investigated with 12 human recombinant enzymes. FA was mainly glucuronidated by UGT1A isoforms and by UGT2B7. UGT1A4, 2B4, 2B15 and 2B17 failed to glucuronidate the substance. Examination of the kinetic constants revealed that FA was mainly glucuronidated by UGT1A1 at the two nucleophilic groups. UGT1A3 was able to glucuronidate these two positions with the same, but low, efficiency. UGT1A6 and 1A8 were involved in the formation of the ether glucuronide only, whereas UGT1A7, 1A10 and 2B7 preferentially glucuronidated the carboxyl group. Moreover, octyl gallate, a marker substrate of UGT1A1, competitively inhibited FA glucuronidation mediated by this isoform. Altogether, the results suggest that FA glucuronidation is primarily mediated by UGT1A1.

  7. Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

    DEFF Research Database (Denmark)

    Kononenko, Olga; Bazov, Igor; Watanabe, Hiroyuki

    2017-01-01

    of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of the signal peptide. ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted...... to the cell nuclei in a model cellular system. This may be driven by bipartite nuclear localization signal (NLS) that is cryptic in the full-length PDYN molecule and becomes functional when signal peptide is truncated. Nuclear PDYN isoform was identified by western blot and radioimmunoassay in neuronal nuclei...

  8. Expression of the WT1 gene -KTS domain isoforms suppresses the invasive ability of human lung squamous cell carcinoma cells.

    Science.gov (United States)

    Moriya, Shogo; Takiguchi, Masaki; Seki, Naohiko

    2008-02-01

    Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(Waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that p21(Waf1/Cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of p21(Waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.

  9. CD99 isoforms regulate CD1a expression in human monocyte-derived DCs through ATF-2/CREB-1 phosphorylation.

    Science.gov (United States)

    Mahiddine, Karim; Mallavialle, Aude; Bziouech, Hanen; Larbret, Frédéric; Bernard, Alain; Bernard, Ghislaine

    2016-06-01

    CD1a expression is considered one of the major characteristics qualifying in vitro human dendritic cells (DCs) during their generation process. Here, we report that CD1A transcription is regulated by a mechanism involving the long and short isoforms of CD99. Using a lentiviral construct encoding for a CD99 short hairpin RNA, we were able to inhibit CD99 expression in human primary DCs. In such cells, CD1a membrane expression increased and CD1A transcripts were much higher in abundance compared to cells expressing CD99 long form (CD99LF). We also show that CD1A transcription is accompanied by a switch in expression from CD99LF to expression at comparable levels of both CD99 isoforms during immature DCs generation in vitro. We demonstrate that CD99LF maintains a lower level of CD1A transcription by up-regulating the phosphorylated form of the ATF-2 transcription factor and that CD99 short form (SF) is required to counteract this regulatory mechanism. Elucidation of the molecular mechanisms related to CD99 alternative splicing will be very helpful to better understand the transcriptional regulatory mechanism of CD1a molecules during DCs differentiation and its involvement in the immune response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  11. Soluble human leukocyte antigen-G isoforms in maternal plasma in early and late pregnancy

    DEFF Research Database (Denmark)

    Rizzo, Roberta; Andersen, Anita Sylvest; Lassen, Michael Rud

    2009-01-01

    published studies that a high, detectable soluble HLA-G concentration in maternal plasma or serum is not mandatory for a successful pregnancy. However, complications during pregnancy, such as (severe) pre-eclampsia, spontaneous abortion, IUGR, and premature birth, are associated with a low or undetectable...... was to investigate the levels of different soluble HLA-G isoforms in maternal plasma in early and late pregnancy. METHOD OF STUDY: Soluble HLA-G (sHLA-G) can be detected in maternal blood, and in this study, two different isoforms of sHLA-G, namely sHLA-G1 generated by shedding of membrane-bound HLA-G1 and HLA......-G generated by specific HLA-G transcripts, have been investigated early [median of 16.4 weeks of gestation (GW)] and late (median: 38.9 GW) in pregnancy in an original cohort of 580 pregnant Caucasian women. RESULTS: Lower concentrations of sHLA-G1 were found late in pregnancy (>32 GW) in a group of women...

  12. The Effects of Formaldehyde on Cytochrome P450 Isoform Activity in Rats

    Directory of Open Access Journals (Sweden)

    Min Xu

    2017-01-01

    Full Text Available Formaldehyde (FA is an occupational and indoor pollutant. Long-term exposure to FA can irritate the respiratory mucosa, with potential carcinogenic effects on the airways. The effects of acute FA poisoning on the activities of CYP450 isoforms CYP1A2, CYP2C11, CYP2E1, and CYP3A2 were assessed by determining changes in the pharmacokinetic parameters of the probe drugs phenacetin, tolbutamide, chlorzoxazone, and testosterone, respectively. Rats were randomly divided into three groups: control, low FA dose (exposure to 110 ppm for 2 h for 3 days, and high FA dose (exposure to 220 ppm for 2 h for 3 days. A mixture of the four probe drugs was injected into rats and blood samples were taken at a series of time points. Plasma concentrations of the probe drugs were measured by HPLC. The pharmacokinetic parameters t1/2, AUC(0-t, and Cmax of tolbutamide, chlorzoxazone, and testosterone increased significantly in the high dose versus control group (P<0.05, whereas the CL of chlorzoxazone and testosterone decreased significantly (P<0.05. However, t1/2, AUC(0-t, and Cmax of phenacetin decreased significantly (P<0.05, whereas the CL of phenacetin increased significantly (P<0.05 compared to controls. Thus, acute FA poisoning suppressed the activities of CYP2C11, CYP2E1, and CYP3A2 and induced the activity of CYP1A2 in rats. And the change of CYP450 activity caused by acute FA poisoning may be associated with FA potential carcinogenic effects on the airways.

  13. The Effects of Formaldehyde on Cytochrome P450 Isoform Activity in Rats.

    Science.gov (United States)

    Xu, Min; Tang, Huaqiao; Rong, Qian; Zhang, Yuanli; Li, Yinglun; Zhao, Ling; Ye, Gang; Shi, Fei; Lv, Cheng

    2017-01-01

    Formaldehyde (FA) is an occupational and indoor pollutant. Long-term exposure to FA can irritate the respiratory mucosa, with potential carcinogenic effects on the airways. The effects of acute FA poisoning on the activities of CYP450 isoforms CYP1A2, CYP2C11, CYP2E1, and CYP3A2 were assessed by determining changes in the pharmacokinetic parameters of the probe drugs phenacetin, tolbutamide, chlorzoxazone, and testosterone, respectively. Rats were randomly divided into three groups: control, low FA dose (exposure to 110 ppm for 2 h for 3 days), and high FA dose (exposure to 220 ppm for 2 h for 3 days). A mixture of the four probe drugs was injected into rats and blood samples were taken at a series of time points. Plasma concentrations of the probe drugs were measured by HPLC. The pharmacokinetic parameters t1/2, AUC(0-t), and Cmax of tolbutamide, chlorzoxazone, and testosterone increased significantly in the high dose versus control group (P < 0.05), whereas the CL of chlorzoxazone and testosterone decreased significantly (P < 0.05). However, t1/2, AUC(0-t), and Cmax of phenacetin decreased significantly (P < 0.05), whereas the CL of phenacetin increased significantly (P < 0.05) compared to controls. Thus, acute FA poisoning suppressed the activities of CYP2C11, CYP2E1, and CYP3A2 and induced the activity of CYP1A2 in rats. And the change of CYP450 activity caused by acute FA poisoning may be associated with FA potential carcinogenic effects on the airways.

  14. E3B1/ABI-1 Isoforms Are Down-Regulated in Cancers of Human Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    Rafia A. Baba

    2012-01-01

    Full Text Available The expression of E3B1/ABI-1 protein and its role in cancer progression and prognosis are largely unknown in the majority of solid tumors. In this study, we examined the expression pattern of E3B1/ABI-1 protein in histologically confirmed cases of esophageal (squamous cell carcinoma and adenocarcinoma, gastro-esophageal junction, colorectal cancers and corresponding normal tissues freshly resected from a cohort of 135 patients, by Western Blotting and Immunofluorescence Staining. The protein is present in its phosphorylated form in cells and tissues. Depending on the extent of phosphorylation it is either present in hyper-phosphorylated (M. Wt. 72 kDa form or in hypo-phosphorylated form (M. Wt. 68 kDa and 65 kDa. A thorough analysis revealed that expression of E3B1/ABI-1 protein is significantly decreased in esophageal, gastro-esophageal junction and colorectal carcinomas irrespective of age, gender, dietary and smoking habits of the patients. The decrease in expression of E3B1/ABI-1 was consistently observed for all the three isoforms. However, the decrease in the expression of isoforms varied with different forms of cancers. Down-regulation of E3B1/ABI-1 expression in human carcinomas may play a critical role in tumor progression and in determining disease prognosis.

  15. The highly conserved human cytomegalovirus UL136 ORF generates multiple Golgi-localizing protein isoforms through differential translation initiation.

    Science.gov (United States)

    Liao, Huanan; Lee, Jung-Hyun; Kondo, Rikita; Katata, Marei; Imadome, Ken-Ichi; Miyado, Kenji; Inoue, Naoki; Fujiwara, Shigeyoshi; Nakamura, Hiroyuki

    2014-01-22

    The UL133-UL138 locus in the unique long b' (ULb') region of the human cytomegalovirus (HCMV) genome is considered to play certain roles in viral replication, dissemination and latency in a host cell type-dependent manner. Here we characterized the proteins encoded by UL136, one of the open reading frames (ORFs) in the locus. Comparative sequence analysis of UL136 among clinical isolates and laboratory strains indicates that its predicted amino-acid sequence is highly conserved. A polyclonal antibody against UL136 proteins (pUL136s) was raised against its carboxy-terminal region and this antibody specifically recognized at least five UL136-encoded protein isoforms of 29-17 kDa both in HCMV-infected cells and in cells transfected with a construct expressing pUL136. Immunofluorescence analysis with this antibody revealed localization of pUL136 in the Golgi apparatus. Analysis of several pUL136 mutants indicated that the putative transmembrane domain of pUL136 is required for its Golgi localization. Mutational analysis of multiple AUG codons in UL136 demonstrated that translation initiation from these AUG codons contributes in the generation of pUL136 isoforms.

  16. Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5'-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the “gt-ag” rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 17b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context.

  17. 探针药物法评价氟尼辛葡甲铵对猪肝CYP450酶的影响%Simultaneous evaluation of effect of flunixin meglumine on live cytochrome P450 enzymes in pigs by Cocktail probe drugs

    Institute of Scientific and Technical Information of China (English)

    胡屹屹; 杨海峰; 江善祥

    2008-01-01

    采用Cocktail探针药物法探讨氟尼辛葡甲铵对猪肝CYP450酶是否具有诱导或抑制作用.将12头健康猪随机均分为2组.公母各半.试验组每天肌肉注射氟尼辛葡甲铵1次(11 mg/kg),对照组给予相同体积的生理盐水,连续给药10 d.通过HPLC检测探针药物的代谢率,来评价各组CYP450水平.结果显示,试验组的氨苯砜代谢减慢,t1/2延长;而氯唑沙宗代谢加快,t1/2缩短.这说明氟尼辛葡甲铵对猪CYP3A4具有抑制作用,而对CYP2E1存在诱导效应.

  18. Expression profiles of muscle disease-associated genes and their isoforms during differentiation of cultured human skeletal muscle cells

    Directory of Open Access Journals (Sweden)

    Abdul-Hussein Saba

    2012-12-01

    Full Text Available Abstract Background The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins. Methods We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development. Results Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development. Conclusions In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.

  19. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

    Directory of Open Access Journals (Sweden)

    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  20. 14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

    Science.gov (United States)

    Noutsios, Georgios T; Ghattas, Paul; Bennett, Stephanie; Floros, Joanna

    2015-07-15

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

  1. Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.

    Science.gov (United States)

    Boros, Eszter; Szabó, András; Zboray, Katalin; Héja, Dávid; Pál, Gábor; Sahin-Tóth, Miklós

    2017-02-17

    Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

  2. Differential Expression of IGF Components and Insulin Receptor Isoforms in Human Seminoma Versus Normal Testicular Tissue

    Directory of Open Access Journals (Sweden)

    Tanja Pascale Neuvians

    2005-05-01

    Full Text Available Insulin-like growth factors (IGF have mitogenic and antiapoptotic functions, and may be involved in tumor growth. The purpose of the study was to investigate the role of IGF components in seminoma compared to normal testis. Normal testicular tissues from autopsy cases and seminoma from surgery cases were obtained for microarray and real-time reverse transcription polymerase chain reaction (RT-PCR analysis of IGF-1, IGF-2, IGF receptor type 1 (IGF-R1, IGF-R2, insulin receptor isoforms A (IR-A and B (IR-B, and IGF-binding proteins (IGFBP 1-6. IGF-2 was localized by immunohistochemistry. IGFBP-5 protein expression was evaluated by Western blot analysis. mRNA expression in microarray and real-time RT-PCR showed similar tendencies: IGF-1, IGF-R1, IGF-R2, IR-A, and IGFBP-2 were not different in both groups. IGF-2, IR-B, IGFBP-1, IGFBP-4, and IGFBP-6 mRNA were downregulated in seminoma. IGFBP-3 tended to be upregulated in pT1 seminoma, but downregulated in pT2 stages. IGFBP-5 and IGF-2 protein expression correlated with mRNA expression. In conclusion, downregulation of mainly inhibiting IGFBPs may allow a stimulated tumor growth. The downregulated IGF-2 does not seem to be involved in the growth regulation of seminoma. Constantly expressed genes (e.g., IGF-1, IGF-R1, IR-A, and IGFBP-2 may reflect an involvement in spermatogenesis, but may also play a major role in tumor growth as their expression is not downregulated despite the lack of spermatogenesis in tumor tissue.

  3. Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

    Science.gov (United States)

    de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

    2013-09-30

    We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test.

  4. Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4, and 6 in distinct membrane microdomains.

    Science.gov (United States)

    Bogard, Amy S; Xu, Congfeng; Ostrom, Rennolds S

    2011-04-01

    Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

  5. An evolutionary test of the isoform switching hypothesis of functional progesterone withdrawal for parturition: humans have a weaker repressive effect of PR-A than mice.

    Science.gov (United States)

    Wagner, Günter P; Tong, Yingchun; Emera, Deena; Romero, Roberto

    2012-03-27

    A decrease in maternal serum progesterone (P4) concentrations precedes the onset of labor in most placental mammals. Humans differ by maintaining high levels of P4 throughout birth. Parturition in humans probably includes mechanisms that undercut the pregnancy sustaining function of P4. One attractive hypothesis is the isoform switching hypothesis (ISH). ISH is supported by in vitro evidence that progesterone receptor isoform A (PR-A) inhibits PR-B and that the PR-A/PR-B ratio increases towards term. Here, we test the hypothesis that isoform switching is an adaptation to high levels of P4 at term, predicting that, in humans, PR-A mediated repression of PR-B is stronger than in mouse. We use reporter assays with human and mouse PRs to detect species differences in the repressive effects of PR-A. We found that human PR-B is less sensitive to repression by human PR-A than mouse PR-B, contrary to our prediction. The difference between human and mouse PR-B sensitivity is most pronounced at PR-A/PR-B ratios typical for the preterm myometrium. Our results are inconsistent with the ISH. We speculate that, instead, the lower sensitivity of human PR-B to PR-A may be relevant for the maintenance of pregnancy at high progesterone levels and increasing PR-A concentrations towards term.

  6. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili

    2015-04-01

    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  7. HPLC separation of human serum albumin isoforms based on their isoelectric points

    OpenAIRE

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2013-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the differ...

  8. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M; Kaul, A; Goke, M; Beil, W.; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  9. Expression of leptin and leptin receptor isoforms in the human stomach

    OpenAIRE

    Mix, H.; Widjaja, A; Jandl, O.; Cornberg, M.; Kaul, A.; GOKE, M; Beil, W; Kuske, M.; Brabant, G; Manns, M; Wagner, S.

    2000-01-01

    BACKGROUND—Leptin is an important regulator of food intake and energy expenditure. Initially it was thought to be expressed exclusively in and secreted by adipocytes. Recently, leptin expression was also noted in other tissues, including rat gastric mucosa. Information on leptin and leptin receptor expression in the human stomach is lacking.
AIM—To investigate expression of leptin and its corresponding receptors in human gastric epithelial cells.
METHODS—Fundic and antral gastric mucosal biop...

  10. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    Science.gov (United States)

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  11. Identification of cytochrome P450 isoforms involved in the metabolism of paroxetine and estimation of their importance for human paroxetine metabolism using a population-based simulator

    DEFF Research Database (Denmark)

    Jornil, Jakob; Jensen, Klaus Gjervig; Larsen, Frank

    2010-01-01

    the importance of the identified paroxetine-metabolizing P450 isoforms for human metabolism, taking mechanism-based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism-based inhibition) was also estimated by Simcyp. For extensive and poor metabolizers of CYP2D6......We identify here for the first time the low-affinity cytochrome P450 (P450) isoforms that metabolize paroxetine, using cDNA-expressed human P450s measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography-tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP......3A4, and CYP3A5 were identified as paroxetine-catechol-forming P450 isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing P450 isoforms by substrate depletion. Michaelis-Menten constants K(m) and V(max) were determined by product formation and substrate depletion. Using selective...

  12. Use of glucuronidation fingerprinting to describe and predict mono- and dihydroxyflavone metabolism by recombinant UGT isoforms and human intestinal and liver microsomes.

    Science.gov (United States)

    Tang, Lan; Ye, Ling; Singh, Rashim; Wu, Baojian; Lv, Chang; Zhao, Jie; Liu, Zhongqiu; Hu, Ming

    2010-06-07

    The present study aims to predict the regiospecific glucuronidation of three dihydroxyflavones and seven monohydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven monohydroxyflavones (or HFs), 2'-, 3'-, 4'-, 3-, 5-, 6-, and 7-hydroxyflavone, and three dihydroxyflavones (or diHFs), 3,7-dihydroxyflavone (3,7-diHF), 3,5-dihydroxyflavone (3,5-diHF), and 3,4'-dihydroxyflavone (3,4'-diHF), were chosen, and rates were measured at 2.5, 10, and 35 microM. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 are the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used, 3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intramolecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoform-specific metabolism rates generally correlated well with the rates of glucuronidation in human intestinal and liver microsomes at each of the three tested concentrations. In conclusion, the glucuronidation "fingerprint" of seven selected monohydroxyflavones was affected by UGT isoforms used, positions of the -OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.

  13. Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.

    Science.gov (United States)

    Kotnis, Smita; Bingham, Brendan; Vasilyev, Dmitry V; Miller, Scott W; Bai, Yuchen; Yeola, Sarita; Chanda, Pranab K; Bowlby, Mark R; Kaftan, Edward J; Samad, Tarek A; Whiteside, Garth T

    2010-06-01

    P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

  14. Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

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    McGuire John J

    2005-09-01

    Full Text Available Abstract Background The rTS gene (ENOSF1, first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.

  15. Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

    Science.gov (United States)

    Tan, Bo; Cai, Weimin; Zhang, Jinlian; Zhou, Ning; Ma, Guo; Yang, Ping; Zhu, Qing; Zhu, Yizhun

    2014-09-01

    Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

  16. Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1.

    Science.gov (United States)

    Root, L L; Shipley, G D

    2000-02-01

    Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which alpha FGF and beta FGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.

  17. Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

    OpenAIRE

    Giuliani, A.L.; Colognesi, D.; T. Ricco; Roncato, C.; Capece, M.; Amoroso, F; Wang, Q. G.; Marchi, E.; Gartland, A.; Di Virgilio, F; Adinolfi, E.

    2014-01-01

    The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tu...

  18. OPIOID PRECURSOR PROTEIN ISOFORM IS TARGETED TO THE CELL NUCLEI IN THE HUMAN BRAIN

    DEFF Research Database (Denmark)

    Kononenko, Olga; Bazov, Igor; Watanabe, Hiroyuki;

    2016-01-01

    Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. We here describe two novel splicing variants of human PDYN mRNA. Expression of one...

  19. Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues

    NARCIS (Netherlands)

    Coers, W; Timens, W; Kempinga, C; Klok, PA; Moshage, H

    1998-01-01

    Ten commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several an

  20. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characte

  1. ACC2 is expressed at high levels in human white adipose and has an isoform with a novel N-terminus [corrected].

    Directory of Open Access Journals (Sweden)

    John C Castle

    Full Text Available Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2 that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

  2. Ikaros isoforms in human pituitary tumors: distinct localization, histone acetylation, and activation of the 5' fibroblast growth factor receptor-4 promoter.

    Science.gov (United States)

    Ezzat, Shereen; Yu, Shunjiang; Asa, Sylvia L

    2003-09-01

    Targeted expression of a human pituitary tumor derived-fibroblast growth factor receptor-4 (FGFR4) recapitulates pituitary tumorigenesis. We have shown that FGFR4 is a target for Ikaros, a zinc finger-containing transcription factor that localizes to heterochromatin regions and participates in higher order chromatin complexes and control of gene expression. We report here the expression of Ikaros and functional differences between its alternatively spliced variants in human pituitary tumors. Ik1 expression was detected in human pituitary tumors and we also identified a truncated isoform consistent with the non-DNA-binding Ik6 isoform in a subset of adenomas by reverse transcriptase-polymerase chain reaction, sequencing, and Western immunoblotting. Transfection of Ik6 in GH4 pituitary cells resulted in predominantly cytoplasmic expression as compared to Ik1, which resulted in exclusively nuclear expression as determined by immunofluorescence and immunoblotting of fractionated protein. Immunohistochemistry of primary human pituitary adenomas localized Ikaros expression to the nuclear compartment but also in the cytoplasm, the latter consistent with Ik6. Expression of Ikaros and truncated non-DNA-binding isoforms was also suggested by electromobility shift assays using nuclear proteins from primary human pituitary adenomas. Ik6 resulted in reversal of the effects of Ik1 on wild-type 5' FGFR4 promoter activity, histone acetylation, and regulation of the endogenous gene. We conclude that dominant-negative Ik6 isoforms with their distinct localization and effects on Ik1 action may contribute to the altered expression of FGFR4 and possibly other target genes in human pituitary tumors.

  3. 维生素E对二(口恶)英染毒小鼠肝脏细胞色素P450酶基因转录的影响%Effect of Vitamin E on Transcription Activity of Liver CYP450 Gene in Mice Treated with TCDD

    Institute of Scientific and Technical Information of China (English)

    尹海萍; 徐建平; 周显青; 王迎

    2007-01-01

    目的 研究维生素E对2,3,7,8-四氯代二苯并二(口恶)英 (TCDD) 染毒小鼠肝脏细胞色素P4 50酶1A1、1A2、1B1基因转录的影响.方法 设计了5个实验组,将45只小鼠随机分为5组,5组动物给予TCDD和维生素E的水平依次为:TCDD 0 ng/kg和VE 0 mg/kg 、TCDD 100 ng/kg和VE 0 mg/kg、TCDD 100 ng/kg和VE 20 mg/kg、TCDD 100 ng/kg和VE 100 mg/kg、TCDD 100 ng/kg和VE 500 mg/kg.喂食4周后取其肝脏,用real-time PCR 法测小鼠细胞色素P450酶1A1、1A2、1B1基因mRNA水平.结果 TCDD染毒小鼠肝脏细胞色素P450酶1A1、1A2 mRNA水平与对照组相比有升高的趋势,而细胞色素P450酶1B1 mRNA水平明显降低.染毒并给予维生素E 20 mg/kg组,其肝脏CYP4501A1的mRNA水平有降低趋势,随着维生素E剂量的增加,YP450 1A1的mRNA水平反而有升高的趋势,当维生素E剂量为500 mg/kg时,其肝脏CYP450 1A1的mRNA 明显高于TCDD染毒组.CYP450 1A2的mRNA 水平随着维生素E剂量的增加有降低的趋势,在TCDD染毒并给予维生素E 500 mg/kg组,其肝脏CYP450 1A2的mRNA水平明显低于TCDD染毒组.而肝脏CYP450 1B1 的mRNA水平在染毒并给与维生素E 500 mg/kg组,与染毒组相比有上调趋势.结论 TCDD能影响或部分影响小鼠细胞色素P450酶1A1、1A2、1B1的基因转录,而维生素E能在一定程度上缓解TCDD的作用.

  4. Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1).

    Science.gov (United States)

    Qiao, Cheng; Jiang, Yajie; Deng, Cuilan; Huang, Zebo; Teng, Kaixuan; Chen, Lan; Liu, Xin

    2015-03-01

    TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA binding and C-terminal PRD and STY domains for transcriptional activation. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-11-66 and TEF3-1197-434 functioned as two independent activation domains, suggesting that N-terminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197-434 showed that only the peptides TEF3-1197-265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-11-66 and four β sheets in TEF3-1197-265. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber, cell proliferation, migration and targeted gene expression. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes.

  5. Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 Infection

    Directory of Open Access Journals (Sweden)

    Yu-Ping Chiang

    2014-12-01

    Full Text Available Individual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1 infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L, a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31 than their uninfected counterparts (n = 27 (p = 0.009. Fifty male homosexual subjects (20 of them are HIV-1 positive were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Bayesian multiple logistic regression revealed that playing a receptive role and increased levels of MRJ-L in macrophages were two risk factors for HIV-1 infection. A 1% rise in MRJ-L expression was associated with a 1.13 fold (95% CrI 1.06–1.29 increase in odds of contracting HIV-1 infection. Ex vivo experiments revealed that MRJ-L facilitated Vpr-dependent nuclear localization of virus. Infection of macrophage-tropic strain is a critical step in HIV-1 transmission. MRJ-L is a critical factor in this process; hence, subjects with higher macrophage MRJ-L levels are more vulnerable to HIV-1 infection.

  6. Hydrogen peroxide modifies both activity and isoforms of acetylcholinesterase in human neuroblastoma SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Alba Garcimartín

    2017-08-01

    Human neuroblastoma SH-SY5Y cells were treated with H2O2 (1–1000 µM for 24 h and AChE activity and AChE and cytochrome c levels were evaluated. AChE activity was strongly increased from 1 µM to 1000 µM of H2O2. The results of the kinetic study showed that H2O2 affected Vmax but not Km; and also that H2O2 changed the sigmoid kinetic observed in control samples to hyperbolic kinetic. Thus, results suggest that H2O2 acts as an allosteric activators. In addition, H2O2, (100–1000 µM reduced the total AChE content and modified its isoform profile (mainly 50-, 70-, and 132-kDa·H2O2 from 100 µM to 1000 µM induced cytochrome c release confirming cell death by apoptosis. All these results together suggest: a the involvement of oxidative stress in the imbalance of AChE; and b treatment with antioxidant agents may be a suitable strategy to protect cholinergic system alterations promoted by oxidative stress.

  7. Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins.

    Science.gov (United States)

    Torchetti, Enza M; Bonomi, Francesco; Galluccio, Michele; Gianazza, Elisabetta; Giancaspero, Teresa A; Iametti, Stefania; Indiveri, Cesare; Barile, Maria

    2011-11-01

    A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

  8. IL-13 Receptor Alpha 2 Membrane and Soluble Isoforms Differ in Human and Mouse1

    OpenAIRE

    Chen, Weiguo; Sivaprasad, Umasundari; Tabata, Yasuhiro; Gibson, Aaron M.; Stier, Matthew T.; Finkelman, Fred D.; Khurana Hershey, Gurjit K.

    2009-01-01

    Although mice have ng/ml serum levels of soluble (s) IL-13Rα2, humans lack sIL-13Rα2 in serum. Our data provide a mechanism for this biologic divergence. In mice, discrete transcripts encoding s and membrane (mem) forms of IL-13Rα2 are generated by alternative splicing. We utilized siRNA to specifically deplete the transcript encoding memIL-13Rα2 (full-length) or sIL-13Rα2 (ΔEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Rα2, but had no effect on the level of...

  9. The Expression of MTUS1/ATIP and Its Major Isoforms, ATIP1 and ATIP3, in Human Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Louis, Simon N.S., E-mail: simonnsl@unimelb.edu.au; Chow, Laurie T.C.; Varghayee, Naghmeh; Rezmann, Linda A.; Frauman, Albert G.; Louis, William J. [Clinical Pharmacology and Therapeutics Unit, Department of Medicine, University of Melbourne, Austin Health, Heidelberg 3084, Victoria (Australia)

    2011-10-11

    Angiotensin II (Ang II), the main effector of the renin angiotensin system, acts upon two distinct transmembrane receptors, the Ang II type 1 and the type 2 (AT{sub 2}-) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT{sub 2}-receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT{sub 2}-receptor interacting protein), inhibits the mitogenic effects of EGF in prostate cancer cell lines representing both early and late stage disease. This is the first report on the expression of ATIP in normal and malignant human prostatic biopsies. The expression of ATIP and its major isoforms, ATIP1 and ATIP3, in normal prostatic cells and three prostate cancer cell lines was examined using QPCR and immunohistochemistry. Human biopsies containing benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and well, moderately and poorly differentiated prostate cancer were also examined. Overall, ATIP1 and ATIP3 mRNA expression was increased in malignant compared to normal tissues and cell lines. ATIP immunostaining was low or absent in both the basal and columnar epithelial cell layers surrounding BPH acini; however, it was observed in high concentration in neoplastic epithelial cells of HGPIN and was clearly evident in cytoplasms of malignant cells in all prostate cancer grades. ATIP immunostaining was also identified in the cytoplasms of LNCaP and PC3 prostate cancer cells. As the AT{sub 2}-receptor/ATIP inhibitory signaling pathway exists in malignant cells in all grades of prostate cancer, enhancement of this pathway may be a therapeutic target even after the development of androgen-independence.

  10. Expression of differential nitric oxide synthase isoforms in human gastric mucosa infected with Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    屠振兴; 龚燕芳; 丁华; 许国铭; 李兆申; 满晓华

    2003-01-01

    Objective: To study the relationship between nitric oxide synthase (NOS) expression in human gastric mucosa and Helicobacter pylori (H.pylori) infection. Methods: Gastric mucosa samples were obtained from antrum of 33 patients received gastroendoscopy. H.pylori infection was confirmed by Giems staining and bacteria culture under microaerophilic conditions. Expression of iNOS, eNOS and nitrotyrosine were detected by immunohistochemistry. Results: (1) The positive rate of H. pylori infection was 66.7%(22/33). (2) iNOS positive staining in inflammatory cells was detected in 77.3%(17/22) of samples with H.pylori and 27.3%(3/11) without H.pylori infection (P0.05). (5) Moderate and severe infiltrations of inflammatory cells were found in 86.4%(19/22) of gastric biopsies with H. pylori and 9.1%(1/11) of samples without H. pylori infection (P<0.01). Conclusion: H.pylori infection might promote infiltration of mononuclear cells and macrophages in gastric mucosa and induce iNOS expression in these cells. The accumulated nitric oxide in local area may result in gastric mucosa damage.

  11. Crystal structure of the first WW domain of human YAP2 isoform.

    Science.gov (United States)

    Martinez-Rodriguez, Sergio; Bacarizo, Julio; Luque, Irene; Camara-Artigas, Ana

    2015-09-01

    The WW domains are the smallest modular domains known. The study of the structural basis of their stability is important to understand their physiological role. These domains are intrinsically flexible, which makes them difficult to crystallize. The first WW domain of the human Yes tyrosine kinase Associated Protein (YAP) has been crystallized and its structure has been solved by X-ray diffraction at 1.6 Å resolution. Crystals belong to the orthorhombic space group P21212 with unit cell parameters a=42.67, b=43.10 and c=21.30. The addition of proline and other small-molecule additives improves drastically the quality of the crystals. The interactions that stabilize this minimal modular domain have been analysed. This crystal structure reveals that, besides the stabilization of the hydrophobic core of the protein by the aromatic cluster formed by Trp177-Phe189-Pro202, some salt-bridges interactions might affect the stability of the domain. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.

    Science.gov (United States)

    Jones, M; Tussey, L; Athanasou, N; Jackson, D G

    2000-03-17

    The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ectodermal cells has recently been shown to regulate limb morphogenesis via presentation of fibroblast growth factor (FGF) 4/FGF 8 while expression on tumor cells was shown to sequester hepatocyte growth factor and promote tumor dissemination. To date, however, CD44 HSPG expression in tissue macrophages and lymphocytes has not been adequately investigated, despite the fact these cells actively synthesize growth factors and chemokines and indirect evidence that monocyte CD44 sequesters macrophage inflammatory protein-1beta. Here we show primary human monocytes rather than lymphocytes express CD44 HSPGs, but only following in vitro differentiation to macrophages or activation with the proinflammatory cytokine interleukin-1alpha or bacterial lipopolysaccharide. Furthermore, we show these isoforms are preferentially modified with heparan rather than chondroitin sulfate, bind the macrophage-derived growth factors FGF-2, vascular endothelial growth factor, and heparin-binding epidermal growth factor with varying affinities (K(d) 25-330 nM) and in the case of FGF-2, can stimulate productive binding to the high affinity tyrosine kinase FGF receptor 1 (FGFR1). In contrast, we find no evidence for significant binding to C-C chemokines. Last, we confirm by immunofluorescent antibody staining that inflamed synovial membrane macrophages express CD44 HSPGs and that expression is greatest in cells containing high FGF-2 levels. These results suggest a paracrine role for macrophage CD44 HSPG isoforms in the regulation of growth factor action during inflammation.

  13. Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

    Science.gov (United States)

    Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

    2016-11-01

    The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.

  14. The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2 upregulates galectin-7 expression in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Carole G Campion

    Full Text Available Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2, the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

  15. Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    Institute of Scientific and Technical Information of China (English)

    Guang-Jing Hu; Jia Chen; Xiao-Nan Zhao; Jia-Jia Xu; Dong-Qing Guo; Ming Lu; Ming Zhu

    2013-01-01

    Trans-splicing,a process involving the cleavage and joining of two separate transcripts,can expand the transcriptome and proteome in eukaryotes.Chimeric RNAs generated by trans-splicing are increasingly described in literatures.The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health.Certain antibiotic resistance genes,such as ampicillin resistance gene (Amp),are frequently used in recombinant plasmids.Until now,trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported.Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis.The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities.Here,we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA,which is presumably processed through a prior event of interchromosomal trans-splicing.Strikingly,DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells.Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production,reveal an exogenous-endogenous trans-splicing system,in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells,and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

  16. Identification of a novel splice variant of human PD-L1 Mrna encoding an isoform-lacking Igv-like domain

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2005-01-01

    Aim: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). Methods: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. Results: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon 2 encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intmcellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. Conclusion: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  17. Heterogeneous filament network formation by myosin light chain isoforms effects on contractile energy output of single cardiomyocytes derived from human induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Takeomi Mizutani

    2016-03-01

    Full Text Available Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs are expected to play an important role in heart therapies, in which hiPSC-CMs should generate sufficient contractile force to pump blood. However, recent studies have shown that the contractility of myocardial mimics composed of hiPSC-CMs is lower than that of adult human myocardium. To examine the mechanism by which contractile force output of hiPSC-CMs is weakened, we measured the contractile force of single hiPSC-CMs and observed the fibrous distribution of myosin II regulatory light chain (MRLC of cardiac (contributes to beating and non-cardiac (does not contribute to beating isoforms. Single hiPSC-CMs were cultured on an extracellular matrix gel, and the contractile force and strain energy exerted on the gel were measured. Strain energy was not uniform between cells and ranged from 0.2 to 5.8 pJ. The combination of contractile force measurement and immunofluorescent microscopy for MRLC isoforms showed that cells with higher strain energy expressed the weakened non-cardiac myosin II fibers compared to those of cells with lower strain energy. Observation of cardiac and non-cardiac MRLC showed that the MRLC isoforms formed heterogeneous filament networks. These results suggest that strain energy output from single hiPSC-CMs depends both cardiac and non-cardiac myosin fibers, which prevent deformation of the cell body.

  18. hMena+11a Isoform Serves as a Marker of Epithelial Phenotype and Sensitivity to EGFR Inhibition in Human Pancreatic Cancer Cell Lines

    Science.gov (United States)

    Pino, Maria S.; Balsamo, Michele; Di Modugno, Francesca; Mottolese, Marcella; Alessio, Massimo; Melucci, Elisa; Milella, Michele; McConkey, David J.; Philippar, Ulrike; Gertler, Frank B.; Natali, Pier Giorgio; Nistico, Paola

    2008-01-01

    Purpose hMena, member of the Ena/VASP protein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. Experimental design Human pancreatic ductal adenocarcinoma (PDAC) cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition (EMT), and expression of hMena and its isoform hMena+11a. The effects of EGF and erlotinib on hMena expression as well as the effect of hMena knock-down on cell proliferation were also evaluated. Results hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena+11a isoform was specifically associated with an epithelial phenotype, EGFR dependency and sensitivity to erlotinib. In epithelial BxPC3 cells EGF upregulated hMena/hMena+11a and erlotinib downregulated expression. hMena knock-down reduced cell proliferation and MAPK and AKT activation in BxPC3 cells and promoted the growth inhibitory effects of erlotinib. Conclusions Collectively, our data indicate that the hMena+11a isoform is associated with an epithelial phenotype and identifies EGFR dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena+11a specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients. PMID:18676769

  19. Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species.

    Science.gov (United States)

    Mascarello, Francesco; Toniolo, Luana; Cancellara, Pasqua; Reggiani, Carlo; Maccatrozzo, Lisa

    2016-09-01

    In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species. To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b).

  20. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Institute of Scientific and Technical Information of China (English)

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song

    2008-01-01

    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  1. Induction of the expression of genes encoding TGF-beta isoforms and their receptors by inositol hexaphosphate in human colon cancer cells.

    Science.gov (United States)

    Kapral, Małgorzata; Wawszczyk, Joanna; Hollek, Andrzej; Weglarz, Ludmiła

    2013-01-01

    Transforming growth factors-beta (TGF-beta) are multifunctional cytokines involved in the regulation of cell development, differentiation, survival and apoptosis. They are also potent anticancer agents that inhibit uncontrolled proliferation of cells. Incorrect TGF-beta regulation has been implicated in the pathogenesis of many diseases including inflammation and cancer. In humans, the TGF-beta family consists of three members (TGF-beta1, 2, 3) that show high similarity and homology. TGF-betas exert biological activities on various cell types including neoplastic cells via their specific receptors. Inositol hexaphosphate (phytic acid, IP6), a phytochemical has been reported to possess various health benefits. The aim of this study was to examine the effect of IP6 on the expression of genes encoding TGF-beta1, TGF-beta2, TGF-beta3 isoforms and their receptors TbetaRI, TbetaRII, TbetaRIII in human colorectal cancer cell line Caco-2. The cells were treated with 0.5, 1 and 2.5 mM IP6 for 3, 6 and 12 h. The untreated Caco-2 cells were used as the control. Quantification of genes expression was performed by real time QRT-PCR technique with a SYBR Green I chemistry. The experimental data revealed that the TGF-beta1 mRNA was the predominant isoform in Caco-2 cells and that IP6 enhanced transcriptional activity of genes of all three TGF-beta isoforms and their receptors TbetaRI, TbetaRII TbetaRIII in these cells. At concentrations up to 1 mM, IP6 over-expressed the genes in 6 h lasting cultures, and its higher dose (2.5 mM) caused successively increasing transcript level of TGF-beta isoforms and receptors with the duration of experiment up to 12 h. The findings of this study indicate that one of anti-cancer abilities of IP6 can be realized by enhancing the gene expression of TGF-beta isoforms and their receptors at the transcriptional level.

  2. High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS.

    Science.gov (United States)

    Lee, Miran; Park, Jeonghyeon; Lim, Mi-sun; Seong, Sook Jin; Lee, Joomi; Seo, Jeong Ju; Park, Yong-Ki; Lee, Hae Won; Yoon, Young-Ran

    2012-01-01

    Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

  3. Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

    OpenAIRE

    Ostler, Kelly R.; Yang, Qiwei; Looney, Timothy J.; Li ZHANG; Vasanthakumar, Aparna; Tian, Yufeng; Kocherginsky, Masha; Stacey L. Raimondi; DeMaio, Jessica G.; Salwen, Helen R.; Gu, Song; Chlenski, Alexandre; Naranjo, Arlene; Gill, Amy; Peddinti, Radhika

    2012-01-01

    Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7 which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tum...

  4. Tissue- and Condition-Specific Isoforms of Mammalian Cytochrome c Oxidase Subunits: From Function to Human Disease

    Directory of Open Access Journals (Sweden)

    Christopher A. Sinkler

    2017-01-01

    Full Text Available Cytochrome c oxidase (COX is the terminal enzyme of the electron transport chain and catalyzes the transfer of electrons from cytochrome c to oxygen. COX consists of 14 subunits, three and eleven encoded, respectively, by the mitochondrial and nuclear DNA. Tissue- and condition-specific isoforms have only been reported for COX but not for the other oxidative phosphorylation complexes, suggesting a fundamental requirement to fine-tune and regulate the essentially irreversible reaction catalyzed by COX. This article briefly discusses the assembly of COX in mammals and then reviews the functions of the six nuclear-encoded COX subunits that are expressed as isoforms in specialized tissues including those of the liver, heart and skeletal muscle, lung, and testes: COX IV-1, COX IV-2, NDUFA4, NDUFA4L2, COX VIaL, COX VIaH, COX VIb-1, COX VIb-2, COX VIIaH, COX VIIaL, COX VIIaR, COX VIIIH/L, and COX VIII-3. We propose a model in which the isoforms mediate the interconnected regulation of COX by (1 adjusting basal enzyme activity to mitochondrial capacity of a given tissue; (2 allosteric regulation to adjust energy production to need; (3 altering proton pumping efficiency under certain conditions, contributing to thermogenesis; (4 providing a platform for tissue-specific signaling; (5 stabilizing the COX dimer; and (6 modulating supercomplex formation.

  5. Isoforms of human C4b-binding protein. II. Differential modulation of the C4BPA and C4BPB genes by acute phase cytokines

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, O.C.; Sanchez-Corral, P.; Rodriguez de Cordoba, S. [Center for Biological Investigations (CSIC), Velazquez, Madrid (Spain)

    1995-10-15

    Human C4b-binding protein (C4BP) controls activation of the complement system and inactivates the anticoagulant vitamin K-dependent protein S using two distinct polypeptides known as C4BP{alpha} and C4BP{beta}, respectively. C4BP presents three isoforms, {alpha}7{beta}1, {alpha}7{beta}0, and {alpha}6{beta}1, the proportion of which depends on the relative levels of C4BP{alpha} and C4BP{beta}. To better understand the regulation of C4BP during the acute phase response we analyzed the C4BP isoforms in 23 serial samples of acute phase patients and characterized the effect of various acute phase cytokines on the expression of the C4BPA and C4BPB genes using Hep3B cells. We show that the elevation of C4BP during acute phase response leads to changes in the proportion of the C4BP isoforms. However, there are striking differences among acute phase individuals. Some of them present a pattern of induction that primarily affects the {alpha}7{beta}0 isoform, whereas others present the opposite situation, increasing the C4BP{beta}-containing isoforms. In vitro studies demonstrate that IL-6, IL-1{beta}, and INF-{gamma} increase the levels of both C4BP{alpha} and C4BP{beta}-mRNAs, whereas TNF-{alpha} down-regulates these mRNAs. INF-{gamma} shows, in addition, a differential effect on the C4BP{alpha} and C4BP{beta}-mRNAs. Differential modulation of the C4BPA and C4BPB genes has been postulated as an efficient mechanism to maintain steady concentrations of C4BP{beta} when C4BP is induced. A synergistic 10-fold induction of C4BP{alpha}-mRNA, but a marginal increase of C4BP{beta}-mRNA, was observed when INF-{gamma} was used together with TNF-{alpha}, suggesting that association of these cytokines is critical to avoid elevation of C4BP{beta} during the acute phase induction of C4BP. 33 refs., 6 figs.

  6. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome.

    Science.gov (United States)

    Puisac, Beatriz; Teresa-Rodrigo, María-Esperanza; Hernández-Marcos, María; Baquero-Montoya, Carolina; Gil-Rodríguez, María-Concepción; Visnes, Torkild; Bot, Christopher; Gómez-Puertas, Paulino; Kaiser, Frank J; Ramos, Feliciano J; Ström, Lena; Pié, Juan

    2017-02-23

    Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

  7. mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

    Directory of Open Access Journals (Sweden)

    Beatriz Puisac

    2017-02-01

    Full Text Available Cornelia de Lange syndrome (CdLS is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction. Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys, showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

  8. Leptin receptor isoform 219.1: an example of protein evolution by LINE-1-mediated human-specific retrotransposition of a coding SVA element.

    Science.gov (United States)

    Damert, Annette; Löwer, Johannes; Löwer, Roswitha

    2004-04-01

    Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

  9. The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

    Science.gov (United States)

    Donzelli, Massimiliano; Derungs, Adrian; Serratore, Maria-Giovanna; Noppen, Christoph; Nezic, Lana; Krähenbühl, Stephan; Haschke, Manuel

    2014-03-01

    Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the

  10. Single-fiber expression and fiber-specific adaptability to short-term intense exercise training of Na+-K+-ATPase α- and β-isoforms in human skeletal muscle.

    Science.gov (United States)

    Wyckelsma, V L; McKenna, M J; Serpiello, F R; Lamboley, C R; Aughey, R J; Stepto, N K; Bishop, D J; Murphy, R M

    2015-03-15

    The Na(+)-K(+)-ATPase (NKA) plays a key role in muscle excitability, but little is known in human skeletal muscle about fiber-type-specific differences in NKA isoform expression or adaptability. A vastus lateralis muscle biopsy was taken in 17 healthy young adults to contrast NKA isoform protein relative abundance between type I and IIa fibers. We further investigated muscle fiber-type-specific NKA adaptability in eight of these adults following 4-wk repeated-sprint exercise (RSE) training, comprising three sets of 5 × 4-s sprints, 3 days/wk. Single fibers were separated, and myosin heavy chain (I and IIa) and NKA (α1-3 and β1-3) isoform abundance were determined via Western blotting. All six NKA isoforms were expressed in both type I and IIa fibers. No differences between fiber types were found for α1-, α2-, α3-, β1-, or β3-isoform abundances. The NKA β2-isoform was 27% more abundant in type IIa than type I fibers (P training increased β1 in type IIa fibers (pretraining 0.70 ± 0.25, posttraining 0.84 ± 0.24 arbitrary units, 42%, P training effects were found for other NKA isoforms. Thus human skeletal muscle expresses all six NKA isoforms and not in a fiber-type-specific manner; this points to their different functional roles in skeletal muscle cells. Detection of elevated NKA β1 after RSE training demonstrates the sensitivity of the single-fiber Western blotting technique for fiber-type-specific intervention effects.

  11. Disulfiram Metabolite S-Methyl-N, N-Diethylthiocarbamate Quantitation in Human Plasma with Reverse Phase Ultra Performance Liquid Chromatography and Mass Spectrometry

    Science.gov (United States)

    Hochreiter, Jill; McCance-Katz, Elinore F.; Lapham, Jill; Ma, Qing; Morse, Gene D.

    2012-01-01

    Disulfiram has been used extensively for alcohol abuse and may have a role in treatment for cocaine addiction. Recent data suggest that disulfiram may also reactivate latent HIV in reservoirs. Disulfiram has complex pharmacokinetics with rapid metabolism to active metabolites, including S-Methyl-N, N-Diethylthiocarbamate (DET-Me) which is formed from cytochrome P450 (CYP450). Assessing disulfiram in HIV-infected individuals with a CYP450 inducing drug (e.g., efavirenz) or a CYP450 inhibiting drug (e.g., HIV-1 protease inhibitors) requires an assay that can measure a metabolite that is formed directly via CYP450 oxidation. Therefore, an assay to measure concentrations of DET-Me in human plasma was validated. DET-Me and the internal standard, S-ethyldipropylthiocarbamate (EPTC) were separated by isocratic ultra performance liquid chromatography using a Waters Acquity HSS T3 column (2.1×100mm, 1.8μm) and detection via electrospray coupled to a triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used with DET-Me at 148/100 and the internal standard at 190/128 with a linear range of 0.500 to 50.0 ng/mL with a five minute run time. Human plasma (500 μL) was extracted using a solid phase procedure. The interassay variation ranged from 1.86 to 7.74% while the intra assay variation ranged from 3.38 to 5.94% over three days. Representative results are provided from samples collected from subjects receiving daily doses of disulfiram 62.5 mg or 250 mg. PMID:22534656

  12. Human CD72 splicing isoform responsible for resistance to systemic lupus erythematosus regulates serum immunoglobulin level and is localized in endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Hitomi Yuki

    2012-12-01

    Full Text Available Abstract Background CD72 is an inhibitory co-receptor expressed on B cells. We previously demonstrated significant association of the polymorphism of the CD72 gene with susceptibility to human systemic lupus erythematosus (SLE in individuals carrying a SLE-susceptible FCGR2B genotype (FCGR2B-232Thr/Thr. The human CD72 locus generates a splicing isoform that lacks exon 8 (CD72Δex8 as well as full-length CD72 (CD72fl, and the CD72 polymorphism regulates exon 8 skipping. Results Here we demonstrated that individuals carrying the disease-protective CD72 genotype exhibit significantly lower serum immunoglobulin levels than do individuals carrying other CD72 genotypes (P CD72 genotype, the protein level of CD72Δex8 was increased in individuals carrying the disease-protective CD72 genotype, suggesting a crucial role of CD72Δex8 in regulation of antibody production. By expressing these human CD72 isoforms in mouse cell lines, we further demonstrated that CD72Δex8 is accumulated in endoplasmic reticulum (ER and fails to regulate BCR signaling whereas human CD72fl is efficiently transported to the cell surface and inhibits signaling through the B cell antigen receptor (BCR, as is the case for mouse CD72. Conclusion Human CD72 polymorphism appears to regulate antibody production as well as susceptibility to SLE by regulating expression of ER-localizing CD72Δex8.

  13. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram; Finerty, Jr., Patrick J.; MacKenzie, Farrell; Dimov, Svetoslav; Vedadi, Masoud; Park, Hee-Won (Toronto)

    2010-07-06

    Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

  14. Multiple isoforms of the tumor protein p73 are expressed in the adult human telencephalon and choroid plexus and present in the cerebrospinal fluid.

    Science.gov (United States)

    Cabrera-Socorro, Alfredo; Pueyo Morlans, Mercedes; Suarez Sola, Maria Luisa; Gonzalez Delgado, Francisco J; Castañeyra-Perdomo, Agustin; Marin, Maria C; Meyer, Gundela

    2006-04-01

    p73, a homolog of the p53 tumor suppressor, codes for full-length transactivating (TA) and N-terminally truncated (DeltaN) isoforms, with pro- and anti-apoptotic activities, respectively. We examined the expression of the main p73 isoforms in adult human and mouse telencephalon and choroid plexus by immunohistochemistry on paraffin sections, and immunoblotting (IB) of tissue extracts and cerebrospinal fluid (CSF), using antibodies against different protein domains. Cortical neurons expressed TAp73 predominantly in the cytoplasm and DeltaNp73 mainly in the nucleus, with partial overlap in the cytoplasm. Highest expression was found in the hippocampus. IB showed an array of TAp73 variants in adult human cortex and hippocampus. IB of human choroid plexus and CSF using TAp73-specific antibodies revealed the presence of a approximately 90-kDa protein whose molecular weight was reduced after N-deglycosylation, suggesting that glycosylated TAp73 is exported into the CSF. In the mouse, high expression of TAp73 was also detected in the subcommissural organ (SCO), an ependymal gland absent in adult humans. TAp73 colocalized with anti-fibra-Reissner-antibody (AFRU), which is a marker of Reissner's fiber, the secreted SCO product. p73-deficient mice had generalized cortical hypoplasia and hydrocephalus; in addition, we observed a dramatic size reduction of the choroid plexus. However, the SCOs were apparently unaltered and continued to secrete Reissner's fiber. Our findings point to complex and widespread p73 activities in the maintenance of adult cortical neurons and in brain homeostasis. TAp73 in the CSF may play important roles in the maintenance of the adult ventricular wall as well as in the development of the proliferating neuroepithelium.

  15. Carbonic anhydrase inhibitors: synthesis and inhibition of the human carbonic anhydrase isoforms I, II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties.

    Science.gov (United States)

    Sethi, Kalyan K; Verma, Saurabh M; Tanç, Muhammet; Purper, Gaultier; Calafato, Gaetan; Carta, Fabrizio; Supuran, Claudiu T

    2014-03-01

    A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (Kis in the range of 295-10,000 nM), but were more effective hCA II inhibitors (Kis of 1.7-887 nM). The tumor-associated hCA IX was also inhibited, with Kis in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90-3,746 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. 氟甲喹对异育银鲫细胞色素CYP450主要药酶的影响%Effects of flumequine on cytochrome P450 enzymes in allogynogenetic silver crucian carp,Carassius auratus gibelio

    Institute of Scientific and Technical Information of China (English)

    胡晓; 房文红; 汪开毓; 孙贝贝; 胡琳琳; 周帅; 周俊芳

    2011-01-01

    activity(P<0.01). However, the other P450 monooxygenases, such as erythromycin N-demethylase (ERND) [177.98 pmol/(mg.min)], aminopyrine N-demethylase [934.40 pmol/(mg.min)] and ethoxycoumarin O-deethylase (ECOD) [9.84pmol/ (mg.min)]were not significantly affected by flumequine, comparing with these activities in control [140.90 pmol/(mg·min), 850.71 pmol/(mg.min) and 8.93 pmol/(mg.min)], respectively. Except that the ERND activity in kidney tissue was higher than that in liver, the activities ofAPD, EROD and ECOD were the highest in liver tissue. An immunoblot analysis with rabbit anti-rat P4501A polyclonal antibody showed that CYP 1A protein expression in flumequine-treated carps was markedly higher than that in control carps, which was consistent with the trend in EROD activities. These findings suggest that P450 lA isoform, constitutively expressed in crucian carp, was particularly susceptible to activation by flumequine. Semiquantitative RT-PCR indicated that CYP 1A mRNA was not affected by flumequine. In vitro assay, microsomes incubating with various concentrations of flumequine demonstrated that this fluoroquinolone antibiotic had no induction or inhibition of CYP1A. On the basis of the above, liver CYP 1A induction of flumequine on carp occurs at the post-translation level, which may enhance protein stability.

  17. Loss of the SxxSS motif in a human T-cell factor-4 isoform confers hypoxia resistance to liver cancer: an oncogenic switch in Wnt signaling.

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    Hironori Koga

    Full Text Available PURPOSE: Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. METHODS: We investigated the TCF-4J and K isoform pair characterized by the presence (K or absence (J of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells and K (K cells to grow as solid tumors in nude mice was explored. RESULTS: TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells. The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. CONCLUSIONS: Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.

  18. Human adult chondrocytes express hepatocyte growth factor (HGF) isoforms but not HgF: potential implication of osteoblasts on the presence of HGF in cartilage.

    Science.gov (United States)

    Guévremont, Melanie; Martel-Pelletier, Johanne; Massicotte, Frédéric; Tardif, Ginette; Pelletier, Jean-Pierre; Ranger, Pierre; Lajeunesse, Daniel; Reboul, Pascal

    2003-06-01

    HGF is increased in human OA cartilage, possibly from Ob's. RT-PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross-talk between subchondral bone and cartilage may occur during OA. Recently, hepatocyte growth factor (HGF) has been identified by immunohistochemistry in cartilage and more particularly in the deep zone of human osteoarthritic (OA) cartilage. By investigating HGF expression in cartilage, we found that chondrocytes did not express HGF; however, they expressed the two truncated isoforms, namely HGF/NK1 and HGF/NK2. Because the only other cells localized near the deep zone are osteoblasts from the subchondral bone plate, we hypothesized that they were expressing HGF. Indeed, we found that HGF was synthesized by osteoblasts from the subchondral bone plate. Moreover, OA osteoblasts produced five times more HGF than normal osteoblasts and almost no HGF/NK1, unlike normal osteoblasts. Because prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 are involved in OA progression, we investigated whether these factors impact HGF produced by normal osteoblasts. PGE2 was the only factor tested that was able to stimulate HGF synthesis. However, the addition of NS398, a selective inhibitor of cyclo-oxygenase-2 (COX-2) had no effect on HGF produced by OA osteoblasts. HGF/NK2 had a moderate stimulating effect on HGF production by normal osteoblasts, whereas osteocalcin was not modulated by either HGF or HGF/NK2. When investigating signaling routes that might be implicated in OA osteoblast-produced HGF, we found that protein kinase A was at least partially involved. In summary, this study raises the hypothesis that the HGF found in articular cartilage is produced by osteoblasts, diffuses into the cartilage, and may be implicated in the OA process.

  19. Expression of the human isoform of glutamate dehydrogenase, hGDH2, augments TCA cycle capacity and oxidative metabolism of glutamate during glucose deprivation in astrocytes.

    Science.gov (United States)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw; Stridh, Malin H; Zaganas, Ioannis; Skytt, Dorte M; Schousboe, Arne; Bak, Lasse K; Enard, Wolfgang; Pääbo, Svante; Waagepetersen, Helle S

    2017-03-01

    A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [(3) H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of (13) C and (14) C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.

  20. The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme.

    Science.gov (United States)

    Cao, Zhenbo; van Lith, Marcel; Mitchell, Lorna J; Pringle, Marie Anne; Inaba, Kenji; Bulleid, Neil J

    2016-04-01

    The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.

  1. Glucuronidation of zearalenone, zeranol and four metabolites in vitro: formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms.

    Science.gov (United States)

    Pfeiffer, Erika; Hildebrand, Andreas; Mikula, Hannes; Metzler, Manfred

    2010-10-01

    Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

  2. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  3. Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5′-diphospho-glucuronosyltransferase isoforms

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    Munirah Haron

    2015-01-01

    Full Text Available Background: Glucuronidation catalyzed by uridine 5′- diphospho-glucuronosyltransferase (UGT is a major phase II drug metabolism reaction which facilitates drug elimination. Inhibition of UGT activity can cause drug-drug interaction. Therefore, it is important to determine the inhibitory potentials of drugs on glucuronidation. Objective: The objective was to evaluate the inhibitory potentials of mitragynine, 7-hydroxymitragynine, ketamine and buprenorphine, respectively on 4-methylumbelliferone (4-MU glucuronidation in rat liver microsomes, human liver microsomes and recombinant human UGT1A1 and UGT2B7 isoforms. Materials and Methods: The effects of the above four compounds on the formation of 4-MU glucuronide from 4-MU by rat liver microsomes, human liver microsomes, recombinant human UGT1A1 and UGT2B7 isoforms were determined using high-performance liquid chromatography with ultraviolet detection. Results: For rat liver microsomes, ketamine strongly inhibited 4-MU glucuronidation with an IC 50 value of 6.21 ± 1.51 mM followed by buprenorphine with an IC 50 value of 73.22 ± 1.63 mM. For human liver microsomes, buprenorphine strongly inhibited 4-MU glucuronidation with an IC 50 value of 6.32 ± 1.39 mM. For human UGT1A1 isoform, 7-hydroxymitragynine strongly inhibited 4-MU glucuronidation with an IC 50 value of 7.13 ± 1.16 mM. For human UGT2B7 isoform, buprenorphine strongly inhibited 4-MU glucuronidation followed by 7-hydroxymitragynine and ketamine with respective IC 50 values of 5.14 ± 1.30, 26.44 ± 1.31, and 27.28 ± 1.18 mM. Conclusions: These data indicate the possibility of drug-drug interaction if 7-hydroxymitragynine, ketamine, and buprenorphine are co-administered with drugs that are UGT2B7 substrates since these three compounds showed significant inhibition on UGT2B7 activity. In addition, if 7-hydroxymitragynine is to be taken with other drugs that are highly metabolized by UGT1A1, there is a possibility of drug

  4. Inhibitory Effect of Selaginellins from Selaginella tamariscina (Beauv. Spring against Cytochrome P450 and Uridine 5′-Diphosphoglucuronosyltransferase Isoforms on Human Liver Microsomes

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    Jae-Kyung Heo

    2017-09-01

    Full Text Available Selaginella tamariscina (Beauv. has been used for traditional herbal medicine for treatment of cancer, hepatitis, and diabetes in the Orient. Numerous bioactive compounds including alkaloids, flavonoids, lignans, and selaginellins have been identified in this medicinal plant. Among them, selaginellins having a quinone methide unit and an alkylphenol moiety have been known to possess anticancer, antidiabetic, and neuroprotective activity. Although there have been studies on the biological activities of selaginellins, their modulatory potential of cytochrome P450 (P450 and uridine 5′-diphosphoglucuronosyltransferase (UGT activities have not been previously evaluated. In this study, we investigated the drug interaction potential of two selaginellins on ten P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and 3A and six UGT isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7 using human liver microsomes and liquid chromatography-tandem mass spectrometry. Selaginellin and selaginellin M had high inhibitory potential for CYP2C8-mediated amodiaquine O-demethylation with IC50 values of 0.5 and 0.9 μM, respectively. Selaginellin and selaginellin M also showed medium inhibitory potential against CYP2C9, CYP2J2, UGT1A1, and UGT1A3 (1 μM < IC50 < 5 μM. These two selaginellins had low inhibitory potential against CYP1A2, CYP2A6, CYP2E1, and UGT1A6 (IC50 > 25 μM. This information might be helpful to predict possible drug interaction potential of between selaginellins and co-administered drugs.

  5. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    Science.gov (United States)

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  6. E6^E7, a novel splice isoform protein of human papillomavirus 16, stabilizes viral E6 and E7 oncoproteins via HSP90 and GRP78.

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    Ajiro, Masahiko; Zheng, Zhi-Ming

    2015-02-17

    Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy. HPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E

  7. 刺五加注射液对大鼠肝微粒体四种CYP450亚型酶活性的影响%Effects of Ciwujia Injection on four cytochrome P450 isoforms in rat liver microsomes in vitro

    Institute of Scientific and Technical Information of China (English)

    曾超; 刘艳; 刘高峰; 李想

    2012-01-01

    AIM: To investigate the effects of Ciwujia Injection on CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in rat liver microsomes in vitro, providing references for clinical rational use of drug combination therapy. METHODS: Probe drugs of tolbutamide(TB), dextromethor-phan(DM), chlorzoxazone(CLZ) and testoster-one(TS) as well as Ciwujia Injection of different concentrations were added to incubation systems of rat liver microsomes in vitro, the concentrations of metabolites of the probe drugs in blank control, low, medium and high dose of Ciwujia Injection groups were determined by HPLC after incubation, and the differences of metabolic rates were compared to evaluate the effects of Ciwujia Injection on the activities of the four subtypes. The strength of inhibition (IC50 and K, values) was further determined if the activity in the medium dose group significantly1 decreased. RESULTS: Compared with the blank control group, the effects on CYP3A4 activity were statistically significant in low, medium and high dose groups of Ciwujia Injection (P0. 05); the activities of CYP2C9, CYP2E1 were not statistically significant in low dose groups (P>0. 05), meanwhile the inhibitory effects on the two subtype activities in medium and high dose groups were statistically significant (P<0.05), but the rates of inhibition were less than 8. 50% in the medium dose groups and less than 12.00% in the high dose groups. CONCLUSION: Ciwujia Injection exhibited inhibition against the activity of CYP3A4 in rat liver microsomes in vitro, with mixed inhibition model, and weak inhibition on CYP2C9, CYP2E1, but no effect on CYP2D6.%目的:研究刺五加注射液在大鼠体外肝微粒体中对CYP2C9、CYP2D6、CYP2E1和CYP3A4活性的影响,为临床合理联合用药提供参考.方法:在大鼠体外肝微粒体中分别加入四种亚型酶的探针药物甲苯磺丁脲(TB)、右美沙芬(DM)、氯唑沙宗(CLZ)、睾酮(TS)和低、中、高剂量的刺五加注射液,温孵后用HPLC法测定各空白对照组和不同剂量刺五加注射液给药组中各探针药物代谢产物的浓度并比较代谢率的差异,以评价刺五加注射液对各亚型酶活性的影响;中剂量组活性显著降低的亚型酶进一步考察抑制作用的强弱(即IC50和Ki值).结果:与空白对照组比较,刺五加注射液低、中、高剂量给药组对CYP3A4活性的影响均有统计学意义(P<0.01),抑制率分别为 10.22%、19.00%、30.29%,其IC50和K1值分别为3.96%和2.74%(V/V);低、中、高剂量给药组对CYP2D6活性的影响均无统计学差异(P>0.05);低剂量给药组对CYP2C9、CYP2E1活性的影响无统计学差异(P>0.05),中、高剂量给药组对两个亚型的抑制作用有统计学差异(P<0.05),但中剂量给药组抑制率均小于8.50%,高剂量给药组抑制率均小于12.00%.结论:刺五加注射液对大鼠体外肝微粒体CYP3A4有抑制作用,且符合混合型抑制模型;对CYP2C9、CYP2E1抑制作用较弱;对CYP2D6活性无影响.

  8. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    OpenAIRE

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Jing-quan LI; Chu, Rui-Ai; Zhang, Wei; Wang, Hui

    2013-01-01

    Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using q...

  9. In vitro metabolic clearance of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms

    Science.gov (United States)

    Species differences in the intrinsic clearance (CLint) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, β-cyfluthrin, cypermethrin, cis-per...

  10. Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

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    Reesha R Patel

    Full Text Available Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs. Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have

  11. Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human.

    Science.gov (United States)

    Monk, D; Sanches, R; Arnaud, P; Apostolidou, S; Hills, F A; Abu-Amero, S; Murrell, A; Friess, H; Reik, W; Stanier, P; Constância, M; Moore, G E

    2006-04-15

    Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

  12. Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

    Science.gov (United States)

    Wu, Jing-Jing; Ge, Guang-Bo; He, Yu-Qi; Wang, Ping; Dai, Zi-Ru; Ning, Jing; Hu, Liang-Hai; Yang, Ling

    2016-01-01

    Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

  13. Antagonistic effects of vitamin E on transcription activity of CYP450 and antioxidant enzyme genes in the liver of mice treated acutely with TCDD%维生素E拮抗急性二(噁)英对小鼠肝脏细胞色素P450酶和抗氧化物酶基因转录的影响

    Institute of Scientific and Technical Information of China (English)

    尹海萍; 徐建平; 杨绍华; 周显青

    2008-01-01

    为了探讨维生素E对急性2,3,7,8-四氯代二苯并二(噁)英(TCDD)染毒小鼠肝脏细胞色素P450酶(CYP450)基因和抗氧化物酶基因转录的影响,我们设计了对照组、TCDD(30 μg/kg)染毒组和染毒同时给予100mg/kg维生素E组(实验组)3组实验.用Real-Time CR法检测小鼠细胞色素P450酶1A1、1A2、1B1、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)基因表达的变化.结果显示:1)急性TCDD染毒小鼠肝脏细胞色素P450酶1A1、1A2、1B1 mRNA水平与对照组相比明显升高,100 mg/kg的维生素E具有部分拮抗肝脏细胞色素P450酶1A1、1A2、1B1 RNA水平升高的作用;2)急性TCDD染毒小鼠肝脏SOD和GSH-PX mRNA水平呈现降低的趋势,CAT的mRNA水平明显降低,染毒并给予100 mg/kg维生素E,SOD和CATmRNA水平与染毒组相比明显升高,GSH-Px mRNA水平也有升高的趋势.上述结果表明:TCDD能影响或部分影响小鼠肝脏细胞色素P450酶1A1、1A2、1B1、SOD、CAT、GSH-Px的基因转录,而维生素E能在一定程度上缓解TCDD的作用[动物学报54(6):1038-1043,2008].

  14. Endogenous retrovirus drives hitherto unknown proapoptotic p63 isoforms in the male germ line of humans and great apes.

    Science.gov (United States)

    Beyer, Ulrike; Moll-Rocek, Julian; Moll, Ute M; Dobbelstein, Matthias

    2011-03-01

    TAp63, but not its homolog p53, eliminates oocytes that suffered DNA damage. An equivalent gene for guarding the male germ line is currently not known. Here we identify hitherto unknown human p63 transcripts with unique 5'-ends derived from incorporated exons upstream of the currently mapped TP63 gene. These unique p63 transcripts are highly and specifically expressed in testis. Their most upstream region corresponds to a LTR of the human endogenous retrovirus 9 (ERV9). The insertion of this LTR upstream of the TP63 locus occurred only recently in evolution and is unique to humans and great apes (Hominidae). A corresponding p63 protein is the sole p63 species in healthy human testis, and is strongly expressed in spermatogenic precursors but not in mature spermatozoa. In response to DNA damage, this human male germ-cell-encoded TAp63 protein (designated GTAp63) is activated by caspase cleavage near its carboxyterminal domain and induces apoptosis. Human testicular cancer tissues and cell lines largely lost p63 expression. However, pharmacological inhibition of histone deacetylases completely restores p63 expression in testicular cancer cells (>3,000-fold increase). Our data support a model whereby testis-specific GTAp63 protects the genomic integrity of the male germ line and acts as a tumor suppressor. In Hominidae, this guardian function was greatly enhanced by integration of an endogenous retrovirus upstream of the TP63 locus that occurred 15 million years ago. By providing increased germ-line stability, this event may have contributed to the evolution of hominids and enabled their long reproductive periods.

  15. The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer.

    Science.gov (United States)

    Cotter, Kristina; Liberman, Rachel; Sun-Wada, GeHong; Wada, Yoh; Sgroi, Dennis; Naber, Stephen; Brown, Dennis; Breton, Sylvie; Forgac, Michael

    2016-07-19

    The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

  16. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.

    Science.gov (United States)

    Phanstiel, Doug; Brumbaugh, Justin; Berggren, W Travis; Conard, Kevin; Feng, Xuezhu; Levenstein, Mark E; McAlister, Graeme C; Thomson, James A; Coon, Joshua J

    2008-03-18

    Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

  17. Isoform selectivity and kinetics of morphine 3- and 6-glucuronidation by human udp-glucuronosyltransferases: evidence for atypical glucuronidation kinetics by UGT2B7.

    Science.gov (United States)

    Stone, Andrew N; Mackenzie, Peter I; Galetin, Aleksandra; Houston, J Brian; Miners, John O

    2003-09-01

    Morphine elimination involves UDP-glucuronosyltransferase (UGT) catalyzed conjugation with glucuronic acid to form morphine 3- and 6-glucuronides (M3G and M6G, respectively). It has been proposed that UGT2B7 is the major enzyme involved in these reactions, but there is evidence to suggest that other isoforms also catalyze morphine glucuronidation in man. Thus, we have characterized the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs. UGT 1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed M3G formation, but only UGT2B7 formed M6G. The kinetics of M3G formation by the UGT1A family isoforms was consistent with a single enzyme Michaelis-Menten model, with apparent Km values ranging from 2.6 to 37.4 mM. In contrast, M3G and M6G formation by UGT2B7 exhibited atypical kinetics. The atypical kinetics may be described by a model with high- and low-affinity Km values (0.42 and 8.3 mM for M3G, and 0.97 and 7.4 mM for M6G) from fitting to a biphasic Michaelis-Menten model. However, a multisite model with an interaction between two identical binding sites in a negative cooperative manner provides a more realistic approach to modeling these data. According to this model, the respective binding affinities (Ks) for M3G and M6G were 1.76 and 1.41 mM, respectively. These data suggest that M6G formation may be used as a selective probe for UGT2B7 activity, and morphine glucuronidation by UGT2B7 appears to involve the simultaneous binding of two substrate molecules, highlighting the need for careful analysis of morphine glucuronidation kinetics in vitro.

  18. Enzyme kinetic study of a new cardioprotective agent, KR-32570 using human liver microsomes and recombinant CYP isoforms.

    Science.gov (United States)

    Kim, Hyojin; Seo, Kyung-Ah; Kim, Hyunmi; Lee, Hye Suk; Lee, Choong-Hwan; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2007-04-01

    KR-32570 (5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. Human liver microsomal incubation of KR-32570 in the presence of NADPH resulted in the formation of two metabolites, hydroxy-KR-32570 and O-desmethyl-KR-32570. In this study, a kinetic analysis of the metabolism of two metabolites from KR-32570 was performed in human liver microsomes, and recombinant CYP1A2, and CYP3A4. The metabolism for hydroxy- and O-desmethyl-KR-32570 formation from KR-32570 by human liver microsomes was best described by a Michaelis-Menten equation and a Hill equation, respectively. The Cl(int) values of hydroxy- and O-desmethyl-KR-32570 formation were similar to each other (0.03 vs 0.04 microL/min/pmol CYP, respectively). CYP3A4 mediated the formation of hydroxy-KR-32570 from KR-32570 with Cl(int) = 0.24 microL/min/pmol CYP3A4. The intrinsic clearance for O-desmethyl-KR-32570 formation by CYP1A2 was 0.83 AL/min/pmol CYP1A2. These findings suggest that CYP3A4 and CYP1A2 enzymes are major enzymes contributing to the metabolism of KR-32570.

  19. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B;

    1999-01-01

    -AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change...... in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...

  20. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Science.gov (United States)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  1. Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

    Science.gov (United States)

    Pinsonneault, Julia K; Frater, John T; Kompa, Benjamin; Mascarenhas, Roshan; Wang, Danxin; Sadee, Wolfgang

    2017-01-01

    Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

  2. An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

    Science.gov (United States)

    Mühmer, Mario; Ditthardt, Denise; Jäkel, Jörg; Wischmann, Viktoria; Moll, Roland; Schmidt, Ansgar

    2014-01-01

    The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

  3. Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

    Directory of Open Access Journals (Sweden)

    Ali Mobasheri

    2012-04-01

    Full Text Available Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons. Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4 using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes.

  4. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

    Directory of Open Access Journals (Sweden)

    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  5. Effects of 4-nonylphenol on hepatic gene expression of peroxisome proliferator-activated receptors and cytochrome P450 isoforms (CYP1A1 and CYP3A4) in juvenile sole (Solea solea).

    Science.gov (United States)

    Cocci, Paolo; Mosconi, Gilberto; Palermo, Francesco Alessandro

    2013-10-01

    The objective of the present study was to investigate the modulatory effects of the xenoestrogen 4-nonylphenol (4-NP) on hepatic peroxisome proliferator-activated receptor (PPAR) α and β gene expression patterns in relation to the detoxification pathways mediated by cytochrome P450 isoforms (CYP1A1 and CYP3A4). Waterborne 4-NP-induced effects were compared with those of 10(-8)M 17β-estradiol (E2) by using in vivo dose-response experiments carried out with juvenile sole (Solea solea). Compared to the controls, significantly higher levels of PPARα mRNAs were found in fish treated with E2 or 4-NP (10(-6)M) 3 d after exposure; the highest dose of 4-NP also caused up-regulation of retinoid X receptor α (RXRα) transcript levels. On the contrary, PPARβ gene expression was not modulated by E2 or 4-NP. Our data show that 4-NP-induced PPARα mRNA levels coincide with suppression of CYP1A1 and CYP3A4 expression similarly to E2. The results from these in vivo studies suggest the presence of cross-talk between nuclear receptor-mediated signaling pathways and PPARα that may result in modulation of CYP450 isoforms expression following 4-NP treatment in sole liver. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

    Science.gov (United States)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte; Pedersen, Bente K; Saltin, Bengt; Pilegaard, Henriette

    2006-09-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

  7. Progesterone receptor (PR) isoforms PRA and PRB differentially regulate expression of the breast cancer resistance protein in human placental choriocarcinoma BeWo cells.

    Science.gov (United States)

    Wang, Honggang; Lee, Eun-Woo; Zhou, Lin; Leung, Peter C K; Ross, Douglas D; Unadkat, Jashvant D; Mao, Qingcheng

    2008-03-01

    Breast cancer resistance protein (BCRP) plays a significant role in drug disposition and in conferring multidrug resistance in cancer cells. Previous studies have shown that steroid hormones such as 17beta-estradiol and progesterone can affect BCRP expression in cancer cells. In this study, we investigated the molecular mechanism by which BCRP expression in human placental choriocarcinoma BeWo cells is regulated by progesterone. Transfection of the progesterone receptor (PR) isoforms PRA and PRB resulted in a similarly increased expression of PRA and PRB, respectively. However, progesterone significantly increased BCRP expression and activity only in PRB-transfected cells. This stimulatory effect of progesterone was abrogated by the PR antagonist mifepristone (RU-486). Consistently, transcriptional activity of the BCRP promoter was induced 2- to 6-fold by 10(-8) to 10(-5) M progesterone in PRB-transfected cells. Progesterone had little effect on BCRP expression and activity and transcriptional activity of the BCRP promoter in PRA-transfected cells; however, cotransfection of PRA and PRB significantly decreased the progesterone-response compared with that in cells transfected with only PRB. Mutations in a novel progesterone response element (PRE) identified between -243 to -115 bp of the BCRP promoter region significantly attenuated the progesterone-response in PRB-transfected cells, and deletion of the PRE nearly completely abrogated the progesterone effect. Specific binding of both PRA and PRB to the BCRP promoter through the identified PRE was confirmed using the electrophoretic mobility shift assay. Collectively, progesterone induces BCRP expression in BeWo cells via PRB but not PRA. PRA represses the PRB activity. Thus, PRA and PRB differentially regulate BCRP expression in BeWo cells.

  8. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

    Science.gov (United States)

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-12-01

    The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains

    Directory of Open Access Journals (Sweden)

    Kuypers Frans A

    2006-07-01

    Full Text Available Abstract Background The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL genes have been identified with as many as 3 different transcript variants for each. Results Acyl-CoA Synthetase Long-chain member 6 (ACSL6 is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe or tyrosine (Tyr. Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs. Conclusion We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes.

  10. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    Energy Technology Data Exchange (ETDEWEB)

    Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  11. 鼻咽癌抑瘤基因NOR1第2外显子选择性剪切异构体在组织和细胞中的分布规律%Distribution and expression of alternative splice isoforms of NORI in human tissues and cell lines

    Institute of Scientific and Technical Information of China (English)

    向波; 王卫; 易梅; 李文娟; 周鸣; 李小玲; 李桂源

    2011-01-01

    Objective To analyze the distribution and subcellular localization of NORI alternative splice isoforms in human tissues and cell lines. Methods NORI open reading frame(ORF)was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector. The level of NORI mRNA in human tissues was determined by real-time RT-PCR. Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced. The expression plasmids of NORI were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NORI protein isoforms in human cells. Results The expression of NORI mRNA was high in human adult testis, moderate in human fetus nasopharynx, trachea,brain, and kidney tissues, and weak or undetectable in other tissues. Two splice variants of human NORI gene resulted from alternative splicing at exon 2 were identified. Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain. Isoform 2 was the sole isoform in other tissues but brain. Only isoform 2 mRNA was detected in cell lines used in this study, though no genomic deletion of exon 2 could be found in all these cell lines. Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm. Conclusion Alternative splice isoforms of tumor suppressor gene NORI are identified. NORI isoform 1 and isoform 2 are both detected in fetus brain. NORI isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain. The exon 2 encoded peptide does not affect the subcellular location of NORI protein.%目的:研究NOR1基因全长及第2外显子选择性剪切异构体在人细胞系和组织中的分布规律以及NOR1蛋白全长及剪切异构体的亚细胞定位模式.方法:以人胎脑cDNA文库质粒为模板,PCR扩增NOR1基因开放阅读框(open reading frame,ORF),酶切后连接pCMV/myc载体.以pCMV/myc-NOR1(isoform 1)质粒为标准品,采用Real-time RT-PCR方法检测NOR1 m

  12. Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

    Science.gov (United States)

    Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S; Hernandez-Andrade, Edgar; Than, Nandor Gabor

    2015-01-01

    Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One

  13. Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms: relevance to excitotoxic neurodegeneration.

    Science.gov (United States)

    Li, Yuju; Peer, Justin; Zhao, Runze; Xu, Yinghua; Wu, Beiqing; Wang, Yi; Tian, Changhai; Huang, Yunlong; Zheng, Jialin

    2017-01-01

    Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis, which converts glutamine into glutamate. Glutamate is the major neurotransmitter of excitatory synapses, executing important physiological functions in the central nervous system. There are two isoforms of glutaminase 1, KGA and GAC, both of which are generated through alternative splicing from the same gene. KGA and GAC both transcribe 1-14 exons in the N-terminal, but each has its unique C-terminal in the coding sequence. We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection. Furthermore, glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and hepatic encephalopathy. Core enzyme structure of KGA and GAC has been published recently. However, how other coding sequences affect their functional enzyme activity remains unclear. We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids (AAs). Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate. When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal, KGA and GAC retained enzyme activity comparable to the full length proteins. In contrast, deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC. Consistently, when both N- and C-terminal of the KGA and GAC were removed, creating a truncated protein that expressed the central 219 AA - 550 AA, the protein retained enzyme activity. Furthermore, expression of the core 219 AA - 550 AA coding

  14. Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

    Directory of Open Access Journals (Sweden)

    Gabor Szalai

    Full Text Available Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1 not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1 develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a in preeclampsia; 2 determine blood pressures in non-stressed conditions; and 3 develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP monitoring.Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11 or green fluorescent protein (GFP; n = 9 on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18. Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2; GD18. Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2. Placental and fetal weights did not differ between the groups

  15. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

    Full Text Available Objective. To investigate expression of vascular endothelial growth factor (VEGF antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

  16. ApoE isoform-dependent changes in hippocampal synaptic function

    Directory of Open Access Journals (Sweden)

    Sullivan Patrick M

    2009-05-01

    Full Text Available Abstract The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.

  17. Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit.

    Science.gov (United States)

    Maltsev, Victor A; Kyle, John W; Undrovinas, Albertas

    2009-05-01

    Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I(NaL)) for the function of both normal and, especially, failing myocardium, in which I(NaL) is reportedly increased. While recent molecular studies identified the cardiac Na(+) channel (NaCh) alpha subunit isoform (Na(v)1.5) as a major contributor to I (NaL), the molecular mechanisms underlying alterations of I(NaL) in heart failure (HF) are still unknown. Here we tested the hypothesis that I(NaL) is modulated by the NaCh auxiliary beta subunits. tsA201 cells were transfected simultaneously with human Na(v)1.5 (former hH1a) and cardiac beta(1) or beta(2) subunits, and whole-cell patch-clamp experiments were performed. We found that I(NaL) decay kinetics were significantly slower in cells expressing alpha + beta(1) (time constant tau = 0.73 +/- 0.16 s, n = 14, mean +/- SEM, P < 0.05) but remained unchanged in cells expressing alpha + beta(2) (tau = 0.52 +/- 0.09 s, n = 5), compared with cells expressing Na(v)1.5 alone (tau = 0.54 +/- 0.09 s, n = 20). Also, beta(1), but not beta(2), dramatically increased I(NaL) relative to the maximum peak current, I(NaT) (2.3 +/- 0.48%, n = 14 vs. 0.48 +/- 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary beta(1) subunit modulates I(NaL), produced by the human cardiac Na(+) channel Na(v)1.5 by slowing its decay and increasing I(NaL) amplitude relative to I(NaT). Because expression of Na(v)1.5 reportedly decreases but beta(1) remains unchanged in chronic HF, the relatively higher expression of beta(1) may contribute to the known I(NaL) increase in HF via the modulation mechanism found in this study.

  18. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

    Laura; A; Perez-Casellas; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

  19. High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22(Phos) → Phe variant

    Science.gov (United States)

    Iavarone, Federica; D’Alessandro, Alfredo; Tian, Na; Cabras, Tiziana; Messana, Irene; Helmerhorst, Eva J.; Oppenheim, Frank G.; Castagnola, Massimo

    2015-01-01

    During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ~22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22(phos)→Phe. PMID:24771659

  20. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors

    DEFF Research Database (Denmark)

    Khan, N.; Jeffers, M.; Kumar, S.;

    2008-01-01

    ) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rh...

  1. Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Hans Prochnow

    Full Text Available Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU, which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.

  2. Glucuronidation of edaravone by human liver and kidney microsomes: biphasic kinetics and identification of UGT1A9 as the major UDP-glucuronosyltransferase isoform.

    Science.gov (United States)

    Ma, Liping; Sun, Jianguo; Peng, Ying; Zhang, Rong; Shao, Feng; Hu, Xiaoling; Zhu, Jianping; Wang, Xiaojin; Cheng, Xuefang; Zhu, Yinci; Wan, Ping; Feng, Dong; Wu, Hui; Wang, Guangji

    2012-04-01

    Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL(int1)) and low-affinity phase (CL(int2)) were 8.4 ± 3.3 and 1.3 ± 0.2 μl · min(-1) · mg(-1), respectively, for HLM and were 45.3 ± 8.2 and 1.8 ± 0.1 μl · min(-1) · mg(-1), respectively, for HKM. However, in microsomal incubations contained with 2% bovine serum albumin, CL(int1) and CL(int2) were 16.4 ± 1.2 and 3.7 ± 0.3 μl · min(-1) · mg(-1), respectively, for HLM and were 78.5 ± 3.9 and 3.6 ± 0.5 μl · min(-1) · mg(-1), respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL(int1) = 42.4 ± 9.5 μl · min(-1) · mg(-1)), followed by UGT2B17 (CL(int) = 3.3 ± 0.4 μl · min(-1) · mg(-1)) and UGT1A7 (CL(int) = 1.7 ± 0.2 μl · min(-1) · mg(-1)). Inhibition study found that inhibitor of UGT1A9 (propofol) attenuated edaravone glucuronidation in HLM and HKM. In addition, edaravone glucuronidation in a panel of seven HLM was significantly correlated (r = 0.9340, p = 0.0021) with propofol glucuronidation. Results indicated that UGT1A9 was the main UGT isoform

  3. Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

    DEFF Research Database (Denmark)

    Kleiveland, Charlotte Ramstad; Kassem, Moustapha; Lea, Tor

    2008-01-01

    with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G0/G1 phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem....... Furthermore, PGE2 treatment leads to enhanced nuclear translocation of beta-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II......The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects...

  4. Evidence for leptin receptor isoforms heteromerization at the cell surface.

    Science.gov (United States)

    Bacart, Johan; Leloire, Audrey; Levoye, Angélique; Froguel, Philippe; Jockers, Ralf; Couturier, Cyril

    2010-06-01

    Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

  5. The Functional Role of MnSOD as a Biomarker of Human Diseases and Therapeutic Potential of a New Isoform of a Human Recombinant MnSOD

    Directory of Open Access Journals (Sweden)

    Antonella Borrelli

    2014-01-01

    Full Text Available Reactive oxygen species (ROS are generated as a consequence of metabolic reactions in the mitochondria of eukaryotic cells. This work describes the role of the manganese superoxide dismutase (MnSOD as a biomarker of different human diseases and proposes a new therapeutic application for the prevention of cancer and its treatment. The paper also describes how a new form of human MnSOD was discovered, its initial application, and its clinical potentials. The MnSOD isolated from a human liposarcoma cell line (LSA was able to kill cancer cells expressing estrogen receptors, but it did not have cytotoxic effects on normal cells. Together with its oncotoxic activity, the recombinant MnSOD (rMnSOD exerts a radioprotective effect on normal cells irradiated with X-rays. The rMnSOD is characterized by the presence of a leader peptide, which allows the protein to enter cells: this unique property can be used in the radiodiagnosis of cancer or chemotherapy, conjugating radioactive substances or chemotherapic drugs to the leader peptide of the MnSOD. Compared to traditional chemotherapic agents, the drugs conjugated with the leader peptide of MnSOD can selectively reach and enter cancer cells, thus reducing the side effects of traditional treatments.

  6. Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

    Science.gov (United States)

    Hu, Yiding; Kupfer, David

    2002-12-01

    Methoxychlor, a currently used pesticide that in mammals elicits proestrogenic/estrogenic activity and reproductive toxicity, has been classified as a prototype endocrine disruptor. Methoxychlor is prochiral, and its metabolites 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M); 1,1,1-trichloro- 2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M); and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M) are chiral; whereas 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M) is achiral. These metabolites are formed during methoxychlor incubation with liver microsomes or recombinant cytochrome p450s (rp450s). Since methoxychlor-metabolite enantiomers may have different estrogenic/antiestrogenic/antiandrogenic activities than corresponding racemates, the possibility that p450s preferentially generate or use R or S enantiomers, was examined. Indeed, rCYP1A2 and r2A6 mono-demethylated methoxychlor primarily into (R)-mono-OH-M at 91 and 75%, respectively, whereas rCYP1A1, 2B6, 2C8, 2C9, 2C19, and 2D6 formed the (S)-enantiomer at 69, 66, 75, 95, 96, and 80%, respectively. However, rCYP3A4, 3A5, and 2B1(rat) weakly demethylated methoxychlor without enantioselectivity. Human liver microsomes generated (S)-mono-OH-M (77-87%), suggesting that CYP1A2 and 2A6 display only minor catalytic contribution. P450 inhibitors demonstrated that CYP2C9 and possibly 2C19 are major hepatic catalysts forming (S)-mono-OH-M, and CYP1A2 is primarily involved in forming the (R)-mono-OH-M. Demethylation rate of (S)-mono-OH-M versus (R)-mono-OH-M forming achiral bis-OH-M by rCYP1A2 was 97/3, compared with 15/85 and 17/83 for rCYP2C9 and 2C19, respectively, indicating opposite substrate enantioselectivity of rCYP1A2 versus 2C9 and 2C19. Also, rCYP1A2 preferentially O-demethylated (R)-catechol-M into (R)-tris-OH-M (at 80%), contrasting r2C9 and r2C19 that yielded (S)-tris-OH-M at 80 and 77%, respectively. Ortho-hydroxylation of

  7. Adiponectin isoforms: a potential therapeutic target in rheumatoid arthritis?

    Science.gov (United States)

    Frommer, Klaus W; Schäffler, Andreas; Büchler, Christa; Steinmeyer, Jürgen; Rickert, Markus; Rehart, Stefan; Brentano, Fabia; Gay, Steffen; Müller-Ladner, Ulf; Neumann, Elena

    2012-10-01

    Several clinical studies have suggested the adipocytokine adiponectin is involved in the progression of rheumatoid arthritis (RA). From this point of view, adiponectin might present a new therapeutic target. However, as adiponectin also exerts beneficial effects in the human organism, a strategy that would allow its detrimental effects to be abolished while maintaining the positive effects would be highly favourable. To elucidate such a strategy, the authors analysed whether the different adiponectin isoforms induce diverging effects, especially with regard to rheumatoid arthritis synovial fibroblasts (RASF), a central cell type in RA pathogenesis capable of invading into and destroying cartilage. Affymetrix microarrays were used to screen for changes in gene expression of RASF. Messenger RNA levels were quantified by real-time PCR, protein levels by immunoassay. The migration of RASF and primary human lymphocytes was analysed using a two-chamber migration assay. In RASF, the individual adiponectin isoforms induced numerous genes/proteins relevant in RA pathogenesis to clearly different extents. In general, the most potent isoforms were the high molecular weight/middle molecular weight isoforms and the globular isoform, while the least potent isoform was the adiponectin trimer. The chemokines secreted by RASF upon adiponectin stimulation resulted in an increased migration of RASF and lymphocytes. The results clearly suggest a pro-inflammatory and joint-destructive role of all adiponectin isoforms in RA pathophysiology, indicating that in chronic inflammatory joint diseases the detrimental effects outweigh the beneficial effects of adiponectin.

  8. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    Science.gov (United States)

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

  9. Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data

    DEFF Research Database (Denmark)

    Tay, Aidan P; Pang, Chi Nam Ignatius; Twine, Natalie a;

    2015-01-01

    data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against...... the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1...

  10. Effects of Polygonum multiflorum on the activity of cytochrome P450 isoforms in rats.

    Science.gov (United States)

    Zhang, Yuan; Ding, Ting; Diao, Tingting; Deng, Mengjiao; Chen, Suhong

    2015-01-01

    Polygonum multiflorum is a traditional Chinese medicinal herb used in clinical medicine to nourish the liver and kidney. However, in recent years, there have been increased reports of clinical adverse reactions associated with Polygonum multiflorum preparations, especially due to liver injury. The cocktail method can be used to assess the influence of Polygonum multiflorum on the activity of cytochrome P450 (CYP450) isoforms CYP2B6, CYP2C19, CYP2C9, CYP1A2, CYP3A4, and CYP2D6, which were reflected by changes in pharmacokinetic parameters in six specific probe drugs: bupropion, omeprazole, tolbutamide, phenacetin, midazolam, and metoprolol. Comprised the experimental rats were randomly divided into five groups: control group, alcohol extraction A group, alcohol extraction B group, water extraction A group, and water extraction B group. Each group five male rats and five female rats. Each of the groups received treatments by gavage as follows: control group was given normal saline, alcohol extraction A group was given 15 g/kg alcohol extract of Polygonum multiflorum (E15), alcohol extraction B group was given with 30 g/kg alcohol extract (E30), water extraction A group was given 15 g/kg water extract (W15), and water extraction B group was given 30 g/kg water extract (W30). The extract solution was orally administered once a day for 28 consecutive days. The mixture of six probe drugs was given by gavage, and blood samples were collected through the tail vein at different time points. Probe drug concentration in rat plasma was measured by liquid chromatography-mass spectrometry (LC-MS). In the treatment and control groups, Polygonum multiflorum alcoholic extract inhibited the activity of CYP2C19 and CYP2C9 and induced the activity of CYP1A2. Polygonum multiflorum aquous extract inhibited the activity of CYP2B6, CYP2C19, CYP2C9, CYP1A2, and CYP2D6. Pathological sections showed that in the alcohol extract group the liver was degenerated inconspicuously, and in the water

  11. Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

    Science.gov (United States)

    Zhou, Momei; Yu, Qin; Wechsler, Anya

    2013-01-01

    Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes. PMID:23804643

  12. Engineering of insulin receptor isoform-selective insulin analogues.

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    Tine Glendorf

    Full Text Available BACKGROUND: The insulin receptor (IR exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.

  13. Therapeutic Targeting the Cell Division Cycle 25 (CDC25 Phosphatases in Human Acute Myeloid Leukemia — The Possibility to Target Several Kinases through Inhibition of the Various CDC25 Isoforms

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    Annette K. Brenner

    2014-11-01

    Full Text Available The cell division cycle 25 (CDC25 phosphatases include CDC25A, CDC25B and CDC25C. These three molecules are important regulators of several steps in the cell cycle, including the activation of various cyclin-dependent kinases (CDKs. CDC25s seem to have a role in the development of several human malignancies, including acute myeloid leukemia (AML; and CDC25 inhibition is therefore considered as a possible anticancer strategy. Firstly, upregulation of CDC25A can enhance cell proliferation and the expression seems to be controlled through PI3K-Akt-mTOR signaling, a pathway possibly mediating chemoresistance in human AML. Loss of CDC25A is also important for the cell cycle arrest caused by differentiation induction of malignant hematopoietic cells. Secondly, high CDC25B expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors in primary human AML cells, and inhibition of this isoform seems to reduce AML cell line proliferation through effects on NFκB and p300. Finally, CDC25C seems important for the phenotype of AML cells at least for a subset of patients. Many of the identified CDC25 inhibitors show cross-reactivity among the three CDC25 isoforms. Thus, by using such cross-reactive inhibitors it may become possible to inhibit several molecular events in the regulation of cell cycle progression and even cytoplasmic signaling, including activation of several CDKs, through the use of a single drug. Such combined strategies will probably be an advantage in human cancer treatment.

  14. Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT: analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

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    Gaudernack Gustav

    2006-08-01

    Full Text Available Abstract Background Human telomerase reverse transcriptase (hTERT is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. Results By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. Conclusion The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

  15. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-11-17

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

  16. Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human.

    Science.gov (United States)

    Wang, Pei; Zhao, Yunli; Zhu, Yingdong; Sun, Jianbo; Yerke, Aaron; Sang, Shengmin; Yu, Zhiguo

    2016-02-05

    Dictamnine, a furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae), is reported to have a wide range of pharmacological activities. In this study, the in vitro metabolic profiles of dictamnine in mouse, rat, dog, monkey, and human liver microsomes were investigated and compared. Dictamnine was incubated with liver microsomes in the presence of an NADPH-regenerating system, resulting in the formation of eight metabolites (M1-M8). M1 is an O-desmethyl metabolite. M5 and M6 are formed by a mono-hydroxylation of the benzene ring of dictamnine. M8 was tentatively identified as an N-oxide metabolite. The predominant metabolic pathway of dictamnine occurs through the epoxidation of the 2,3-olefinic to yield a 2,3-epoxide metabolite (M7), followed by the ring of the epoxide opening to give M4. Likewise, cleavage of the furan ring forms M2 and M3. Slight differences were observed in the in vitro metabolic profiles of dictamnine among the five species tested. A chemical inhibition study with a broad and five specific CYP450 inhibitors revealed that most of the dictamnine metabolites in liver microsomes are mediated by CYP450, with CYP3A4 as the predominant enzyme involved in the formation of M7, the major metabolite. These findings provide vital information to better understand the metabolic processes of dictamnine among various species.

  17. Cytochrome P450 genetic polymorphism in neonatal drug metabolism: role and practical consequences towards a new drug culture in neonatology.

    Science.gov (United States)

    Fanni, D; Ambu, R; Gerosa, C; Nemolato, S; Castagnola, M; Van Eyken, P; Faa, G; Fanos, V

    2014-01-01

    The cytochrome P450 superfamily (CYP450) in humans is formed by 57 functional monooxygenases critical for the metabolism of numerous endogenous and exogenous compounds. The superfamily is organized into 18 families and 44 subfamilies. CYP nomenclature is based on the identity of amino acids. The most important functions of the CYP450 are related to metabolism of endogenous compounds, detoxification of exogenous xenobiotics and decomposition of the vast majority of currently used drugs. The expression of CYP450 enzymes in the human body is characterized by a marked substrate and tissue specificity, the most important being localized in the liver, but also present in kidney, lung, brain, breast, prostate and in the small intestine. The human cytochrome P450 3A gene family (CYP3A) accounts for the largest portion of CYP450 proteins in human liver and includes 4 genes: CYP3A4, CYP3A5, CYP3A7, CYP3A43. Multiple and complex genetic variations, marked interindividual, interethnic and gender variability have been reported regarding CYP3A isoform expression and activity. Multiple factors may affect CYP3A expression and activity, such as inducers like rifampicin, phenobarbital, 3-methylcholantrene, beta-naphtoflavone, and dexamethasone. The maturation of organ systems, paralleled by ontogeny of drug-metabolizing enzymes during fetal life and in the first months of postnatal life, surely exerts profound effects on drug disposition, probably being the predominant factor accounting for age-associated changes in drug clearance. In fact, drug dosage in the perinatal period represents a continuous challenge for neonatologists. The purpose of this article is to provide a brief review of the pharmacokinetic differences between neonates and adults, showing the peculiarities of liver CYP450-related drug metabolism in the perinatal period and at birth, and to report the toxic mechanisms of liver injury in neonates, due to the most frequently utilized drugs in NICU centers.

  18. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  19. A common role for various human truncated adenomatous polyposis coli isoforms in the control of beta-catenin activity and cell proliferation.

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    Shree Harsha Vijaya Chandra

    Full Text Available The tumour suppressor gene adenomatous polyposis coli (APC is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR. The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5 with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the "just right" level.

  20. Histamine H3-receptor isoforms.

    Science.gov (United States)

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  1. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

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    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  2. Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

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    French Frank S

    2004-08-01

    Full Text Available Abstract Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed

  3. Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS

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    Xianbao Shi

    2016-01-01

    Full Text Available Morusin, the important active component of a traditional Chinese medicine, Morus alba L., has been shown to exhibit many vital pharmacological activities. In this study, six recombinant CYP450 supersomes and liver microsomes were used to perform metabolic studies. Chemical inhibition studies and screening assays with recombinant human cytochrome P450s were also used to characterize the CYP450 isoforms involved in morusin metabolism. The morusin metabolites identified varied greatly among different species. Eight metabolites of morusin were detected in the liver microsomes from pigs (PLMs, rats (RLMs, and monkeys (MLMs by LC-MS/MS and six metabolites were detected in the liver microsomes from humans (HLMs, rabbits (RAMs, and dogs (DLMs. Four metabolites (M1, M2, M5, and M7 were found in all species and hydroxylation was the major metabolic transformation. CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19 contributed differently to the metabolism of morusin. Compared to other CYP450 isoforms, CYP3A4 played the most significant role in the metabolism of morusin in human liver microsomes. These results are significant to better understand the metabolic behaviors of morusin among various species.

  4. Insulin receptor isoforms : an integrated view focused on gestational diabetes mellitus

    NARCIS (Netherlands)

    Westermeier, F.; Sáez, T.; Arroyo, P.; Toledo, F.; Gutierrez, J.; Sanhueza, C.; Pardo, F.; Leiva, A.; Sobrevia, L.

    2016-01-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms r

  5. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

    DEFF Research Database (Denmark)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T

    2014-01-01

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms...

  6. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation.

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    Ansgar Zoch

    Full Text Available The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

  7. Chemical origins of isoform selectivity in histone deacetylase inhibitors.

    Science.gov (United States)

    Butler, Kyle V; Kozikowski, Alan P

    2008-01-01

    Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.

  8. p53 isoform profiling in glioblastoma and injured brain.

    Science.gov (United States)

    Takahashi, R; Giannini, C; Sarkaria, J N; Schroeder, M; Rogers, J; Mastroeni, D; Scrable, H

    2013-06-27

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10 to 70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, some of which can modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry and reverse transcription-PCR. We found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared with tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions.

  9. STEADY-STATE TRANSCRIPT LEVELS OF CYTOCHROME-C-OXIDASE GENES DURING HUMAN MYOGENESIS INDICATE SUBUNIT SWITCHING OF SUBUNIT VIA AND COEXPRESSION OF SUBUNIT VIIA ISOFORMS

    NARCIS (Netherlands)

    TAANMAN, JW; HERZBERG, NH; DEVRIES, H; BOLHUIS, PA; VANDENBOGERT, C

    1992-01-01

    Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the beta-subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so

  10. Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

    Science.gov (United States)

    Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A; Larsson, Lars; Ge, Ying

    2016-04-01

    Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases.

  11. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  12. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

    Directory of Open Access Journals (Sweden)

    Martin Krøyer Rasmussen

    2014-09-01

    Full Text Available Cytochrome P450 (CYP450 is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole, one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint.

  13. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

    Science.gov (United States)

    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

  14. Marked Differences in C9orf72 Methylation Status and Isoform Expression between C9/ALS Human Embryonic and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yaara Cohen-Hadad

    2016-11-01

    Full Text Available We established two human embryonic stem cell (hESC lines with a GGGGCC expansion in the C9orf72 gene (C9, and compared them with haploidentical and unrelated C9 induced pluripotent stem cells (iPSCs. We found a marked difference in C9 methylation between the cells. hESCs and parental fibroblasts are entirely unmethylated while the iPSCs are hypermethylated. In addition, we show that the expansion alters promoter usage and interferes with the proper splicing of intron 1, eventually leading to the accumulation of repeat-containing mRNA following neural differentiation. These changes are attenuated in C9 iPSCs, presumably owing to hypermethylation. Altogether, this study highlights the importance of neural differentiation in the pathogenesis of disease and points to the potential role of hypermethylation as a neuroprotective mechanism against pathogenic mRNAs, envisaging a milder phenotype in C9 iPSCs.

  15. Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms I and II with (reduced) Schiff's bases incorporating sulfonamide, carboxylate and carboxymethyl moieties.

    Science.gov (United States)

    Nasr, Gihane; Cristian, Alina; Barboiu, Mihail; Vullo, Daniella; Winum, Jean-Yves; Supuran, Claudiu T

    2014-05-15

    A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn(2+)-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The Ki values of the Schiff bases were in the range of 7.0-21,400nM against hCA II and of 52-8600nM against hCA I, respectively. The corresponding amines showed Ki values in the range of 8.6nM-5.3μM against hCA II, and of 18.7-251nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications.

  16. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  17. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  18. RNA-Seq of Human Breast Ductal Carcinoma In Situ Models Reveals Aldehyde Dehydrogenase Isoform 5A1 as a Novel Potential Target

    Science.gov (United States)

    Kaur, Hitchintan; Mao, Shihong; Li, Quanwen; Sameni, Mansoureh; Krawetz, Stephen A.; Sloane, Bonnie F.; Mattingly, Raymond R.

    2012-01-01

    Breast ductal carcinoma in situ (DCIS) is being found in great numbers of women due to the widespread use of mammography. To increase knowledge of DCIS, we determined the expression changes that are common among three DCIS models (MCF10.DCIS, SUM102 and SUM225) compared to the MCF10A model of non-tumorigenic mammary epithelial cells in three dimensional (3D) overlay culture with reconstituted basement membrane (rBM). Extracted mRNA was subjected to 76 cycles of deep sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of RNA-Seq results showed 295 consistently differentially expressed transcripts in the DCIS models. These differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFβ signaling, show association with cell-cell signaling, cell-cell adhesion and cell proliferation, and have a notable bias toward localization in the extracellular and plasma membrane compartments. RNA-Seq data was validated by quantitative real-time PCR of selected differentially expressed genes. Aldehyde dehydrogenase 5A1 (ALDH5A1) which is an enzyme that is involved in mitochondrial glutamate metabolism, was over-expressed in all three DCIS models at both the mRNA and protein levels. Disulfiram and valproic acid are known to inhibit ALDH5A1 and are safe for chronic use in humans for other disorders. Both of these drugs significantly inhibited net proliferation of the DCIS 3D rBM overlay models, but had minimal effect on MCF10A 3D rBM overlay models. These results suggest that ALDH5A1 may play an important role in DCIS and potentially serve as a novel molecular therapeutic target. PMID:23236365

  19. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease.

    Science.gov (United States)

    Kamphuis, Willem; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-03-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Receptor-isoform-selective insulin analogues give tissue-preferential effects

    DEFF Research Database (Denmark)

    Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi

    2011-01-01

    The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

  1. Lipoprotein lipase isoelectric point isoforms in humans

    DEFF Research Database (Denmark)

    Badia-Villanueva, M.; Carulla, P.; Carrascal, M.

    2014-01-01

    Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-hepari...

  2. CCAAT/enhancer binding protein β (C/EBPβ isoforms as transcriptional regulators of the pro-invasive CDH3/P-cadherin gene in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    André Albergaria

    Full Text Available P-cadherin is a cell-cell adhesion molecule codified by the CDH3 gene, which expression is highly associated with undifferentiated cells in normal adult epithelial tissues, as well as with poorly differentiated carcinomas. In breast cancer, P-cadherin is frequently overexpressed in high-grade tumours and is a well-established indicator of aggressive tumour behaviour and poor patient prognosis. However, till now, the mechanisms controlling CDH3 gene activation have been poorly explored. Since we recently described the existence of several CCAAT/Enhancer Binding Protein β (C/EBPβ transcription factor binding sites at the CDH3 promoter, the aim of this study was to assess if the distinct C/EBPβ isoforms were directly involved in the transcriptional activation of the CDH3 gene in breast cancer cells. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed. We demonstrated that C/EBPβ is co-expressed with P-cadherin in breast cancer cells and all the three isoforms function as transcriptional regulators of the CDH3 gene, directly interacting with specific regions of its promoter. Interestingly, this transcriptional activation was only reflected at the P-cadherin protein level concerning the LIP isoform. Taken together, our data show that CDH3 is a newly defined transcriptional target gene of C/EBPβ isoforms in breast cancer, and we also identified the binding sites that are relevant for this activation.

  3. Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

    Science.gov (United States)

    Ziak, M; Meier, M; Etter, K S; Roth, J

    2001-01-12

    We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

  4. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  5. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel

    2011-01-01

    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  6. Effect of high sucrose diet on liver enzyme content and activity and aflatoxin B1-induced mutagenesis.

    Science.gov (United States)

    Peters, Leandra P; Teel, Robert W

    2003-01-01

    Aflatoxin B1 (AFB1) is a fungal toxin and contaminant that has been implicated in human liver carcinogenesis. In this study we evaluated the effect of a 65% of total calories from sucrose diet (HSD) for 90 days on hepatic cytochrome P450 (CYP450) and glutathione-S-transferase (GST) activity compared to rats maintained on standard lab chow (0% sucrose). There was a statistically significant increase in the number of S. typhimurium His+ revertants (p < 0.001) generated from the incubation of AFB1 with hepatic microsomes from rats fed a HSD. The HSD did not affect the total microsomal CYP450 content nor content of CYP450 1A2, 2B1, 2 isoforms which activate AFB1. Alkoxyresorufin O-dealkylase activity (MROD, PROD) of microsomes from animals fed HSD was decreased by 73% and 49%, respectively. MROD activity is linked to CYP 1A2 activity while PROD is linked to CYP 2B1,2 activity. Although the amount of CYP 3A was significantly decreased in rats fed a HSD, its activity, determined by the presence of the fluorometric metabolite 7-hydroxyquinidine, was unchanged. GST activity was significantly lower in the rats fed HSD.

  7. Metabolism-mediated interaction potential of standardized extract of Tinospora cordifolia through rat and human liver microsomes

    Directory of Open Access Journals (Sweden)

    Shiv Bahadur

    2016-01-01

    Full Text Available Objective: Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. Materials and Methods: Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC 50 values were determined. Results: The content of tinosporaside was found to be 1.64% (w/w in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC 50 (μg/ml value was 136.45 (CYP3A4, 144.37 (CYP2D6, 127.55 (CYP2C9, and 141.82 (CYP1A2. Tinosporaside and extract showed higher IC 50 (μg/ml value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. Conclusions: Plant extract showed significantly higher IC 50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics.

  8. Metabolism-mediated interaction potential of standardized extract of Tinospora cordifolia through rat and human liver microsomes

    Science.gov (United States)

    Bahadur, Shiv; Mukherjee, Pulok K.; Milan Ahmmed, S. K.; Kar, Amit; Harwansh, Ranjit K.; Pandit, Subrata

    2016-01-01

    Objective: Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. Materials and Methods: Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC50 values were determined. Results: The content of tinosporaside was found to be 1.64% (w/w) in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC50 (μg/ml) value was 136.45 (CYP3A4), 144.37 (CYP2D6), 127.55 (CYP2C9), and 141.82 (CYP1A2). Tinosporaside and extract showed higher IC50 (μg/ml) value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. Conclusions: Plant extract showed significantly higher IC50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics. PMID:27721546

  9. Disulfide isoforms of recombinant glia maturation factor beta.

    Science.gov (United States)

    Zaheer, A; Lim, R

    1990-09-14

    Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.

  10. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity.

    Science.gov (United States)

    Gardella, Kacie A; Muro, Israel; Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W

    2016-03-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies.

  11. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin

    Science.gov (United States)

    Fornasa, Giulia; Tsilingiri, Katerina; Caprioli, Flavio; Botti, Fiorenzo; Mapelli, Marina; Meller, Stephan; Kislat, Andreas; Homey, Bernhard; Di Sabatino, Antonio; Sonzogni, Angelica; Viale, Giuseppe; Diaferia, Giuseppe; Gori, Alessandro; Longhi, Renato; Penna, Giuseppe; Rescigno, Maria

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. Objective In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. Methods We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. Results We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. Conclusions We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform. PMID:26014813

  12. CYP450 polymorphisms as risk factors for early-onset lung cancer: gender-specific differences.

    Science.gov (United States)

    Timofeeva, Maria N; Kropp, Silke; Sauter, Wiebke; Beckmann, Lars; Rosenberger, Albert; Illig, Thomas; Jäger, Birgit; Mittelstrass, Kirstin; Dienemann, Hendrik; Bartsch, Helmut; Bickeböller, Heike; Chang-Claude, Jenny C; Risch, Angela; Wichmann, Heinz-Erich

    2009-07-01

    Cytochrome P450 (CYP) enzymes, involved in metabolism of tobacco carcinogens, are also involved in estrogen metabolism and many are regulated by estrogens. These genes may thus be of relevance to gender-specific differences in lung cancer risk, particularly in early-onset lung cancer, where a high proportion of women is observed. We conducted a case-control study to investigate genetic polymorphisms in cytochromes that might modify the risk of developing early-onset lung cancer. In total, 638 Caucasian patients under the age of 51 with primary lung cancer and 1300 cancer-free control individuals, matched by age and sex, were included in this analysis. Thirteen polymorphisms in the CYP1A1, CYP1B1, CYP2A13, CYP3A4 and CYP3A5 genes were analyzed. No significant association was found for any of the analyzed polymorphisms and lung cancer risk overall. However, among women, a significantly increased risk of early-onset lung cancer was observed for carriers of the minor allele of CYP1B1 SNP rs1056836 [odds ratio (OR) 1.97; 95% confidence interval (CI) 1.32-2.94; P lung cancer risk was observed in the group of women carriers of the minor allele of CYP2A13 SNP rs1709084 (OR 1.64; 95% CI 1.00-2.70; P = 0.05). The effect of these two polymorphisms was shown to be modified by smoking. Haplotype analysis was performed for CYP1B1 and CYP2A13. No differences between cases and controls were observed for both genes (P = 0.63 and P = 0.42 for CYP1B1 and CYP2A13, respectively). Our results suggest that the CYP1B1 and the CYP2A13 genotypes may contribute to individual susceptibility to early-onset lung cancer in women.

  13. Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery

    OpenAIRE

    Sánchez-Sánchez, Lorena; Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Patterson, Dustin P; Cadena-Nava, Ruben D.; Douglas, Trevor; Vazquez-Duhalt, Rafael

    2015-01-01

    Background The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. Results We have designed a nano-bioreactor based on the encapsu...

  14. FSH isoform pattern in classic galactosemia

    OpenAIRE

    Gubbels, C.S.; Thomas, C. M.; Wodzig, K.W.H.; Olthaar, A. J.; Jaeken, J.; Sweep, F.C.; Rubio-Gozalbo, M.E.

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  15. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    Science.gov (United States)

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  17. [Clinical relevance of myosin isoforms in the diaphragm].

    Science.gov (United States)

    Gayan-Ramirez, G; Decramer, M

    2000-06-01

    The diaphragm as a striated muscle is characterized by the repetition of a single element arranged in series: the sarcomere containing two kinds of myofilaments: a thick one constituted by the myosin, and a thin one primarily composed of actin. The myosin molecule consists of two heads where two myosin heavy chains (MHC) are fixed, a flexible hinge with two light (MLC) chains, and long rod-shaped tails. The diaphragm contains 4 MHC isoforms (MHC-slow, MHC-2A, MHC-2B, MHC-2X) and 6 MLC isoforms (MLC-1f, MLC-3f, MLC-1sa, MLC-1sb, MLC-2f, MLC-2s/v). In humans, the diaphragm contains mainly fibers expressing the isoforms MHC-slow, MHC-2A, and MLC-2f, MLC-2s et MLC-1f. For the mechanical properties of the different isoforms, there is a gradient from the MHC-slow to the MHC-2A, MHC-2B and MHC-2X/2B. According to the circumstances, the diaphragm will adapt towards a slow profile (COPD, cardiac failure and in animals: Duchenne muscular dystrophy, denervation-1 week, age-female, corticosteroids, chronic stimulation), or a fast profile (in animals: chronic hypoxia, denervation-2 weeks, age-males) or a more oxidative profile (in animals: cachexia, obesity). The reasons why the diaphragm adapts towards a slower or a faster muscle are not known. In fact, for a given pathological situation, several factors are able to influence the fiber composition of the diaphragm. Therefore, the net result of the influence of these different factors in terms of MHC and MLC diaphragm adaptation is difficult to predict.

  18. Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease.

    Science.gov (United States)

    Boczonadi, Veronika; Giunta, Michele; Lane, Maria; Tulinius, Mar; Schara, Ulrike; Horvath, Rita

    2015-06-01

    Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues. We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  19. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    Science.gov (United States)

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  20. Role of ROCK Isoforms in Regulation of Stiffness Induced Myofibroblast Differentiation in Lung Fibrosis.

    Science.gov (United States)

    Htwe, Su S; Cha, Byung H; Yue, Kan; Khademhosseini, Ali; Knox, Alan J; Ghaemmaghami, Amir M

    2017-02-22

    Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

  1. Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells.

    Science.gov (United States)

    Cappelletti, Vera; Miodini, Patrizia; Di Fronzo, Giovanni; Daidone, Maria Grazia

    2006-05-01

    High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

  2. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  3. The FU gene and its possible protein isoforms

    Directory of Open Access Journals (Sweden)

    Nöthen Markus M

    2004-07-01

    Full Text Available Abstract Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci. Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1 maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing

  4. The role of wild type RAS isoforms in cancer.

    Science.gov (United States)

    Zhou, Bingying; Der, Channing J; Cox, Adrienne D

    2016-10-01

    Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.

  5. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  6. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  7. Alterations of Lymphoid Enhancer Factor-1 Isoform Expression in Solid Tumors and Acute Leukemias

    Institute of Scientific and Technical Information of China (English)

    Wenbing WANG; Carsten M(U)LLER-TIDOW; Ping JI; Bj(o)rn STEFFEN; Ralf METZGER; Paul M. SCHNEIDER; Hartmut HALFTER; Mark SCHRADER; Wolfgang E. BERDEL; Hubert SERVE

    2005-01-01

    Two major transcripts of lymphoid enhancer factor-1 (LEF-1) have been described. The long isoform with β-catenin binding domain functions as a transcriptional enhancer factor. The short isoform derives from an intronic promoter and exhibits dominant negative activity. Recently, alterations of LEF- 1isoforms distribution have been described in colon cancer. In the current study we employed a quantitative real-time reverse transcription PCR method (TaqMan) to analyze expression of LEF-1 isoforms in a large cohort of human tumor (n=304) and tumor-free control samples (n=56). The highest expression level of LEF-1 was found in carcinoma samples whereas brain cancer samples expressed little. Expression of LEF1 was different in distinct cancer types. For example, the mRNA level of LEF-1 was lower in testicular tumor samples compared with tumor-free control samples. Besides epithelial cancers, significant LEF-1expression was also found in hematopoietic cells. In hematological malignancies, overall LEF-1 level was higher in lymphocytic leukemias compared with myeloid leukemias and normal hematopoiesis. However,acute myeloid leukemia and acute lymphocytic leukemia showed a significantly increased fraction of the oncogenic LEF-1 compared with chronic lymphocytic leukemia and chronic myeloid leukemia. Taken together,these data suggest that LEF-1 is abundantly expressed in human tumors and the ratio of the oncogenic and the dominant negative short isoform altered not only in carcinomas but also in leukemia.

  8. Differential expression of the interleukin 5 receptor alpha isoforms in blood and tissue eosinophils of nasal polyp patients

    OpenAIRE

    Gevaert, Philippe; Hellman, C.; Lundblad, L.; J. Lundahl; Holtappels, Gabriële; Van Cauwenberge, Paul; Tavernier, Jan; Bachert, Claus

    2009-01-01

    Given the key role of interleukin-5 (IL-5) in eosinophil function, we investigated the regulated expression of the membrane-anchored (TM-IL-5R alpha) isoform, or a secreted (SOL IL-5R alpha) isoform, on both protein and transcript level in vitro and in vivo. A real-time PCR, FACS and ELISA were established to determine IL-5R alpha isoform expression in peripheral blood and nasal tissue from control subjects and nasal polyp (NP) patients with or without asthma. Human peripheral blood eosino...

  9. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  10. p53 isoforms change p53 paradigm

    OpenAIRE

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  11. Isoforms of receptors of fibroblast growth factors.

    Science.gov (United States)

    Gong, Siew-Ging

    2014-12-01

    The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

  12. Absolute quantitation of protein posttranslational modification isoform.

    Science.gov (United States)

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

  13. Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a ...

    African Journals Online (AJOL)

    Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a) Levels In Nigerian Subjects With and Without Coronary Heart Disease. ... Plasma lipoprotein (a) Concentrations and apo(a) isoforms were determined in 101 ... Article Metrics.

  14. Identification of alternatively translated Tetherin isoforms with differing antiviral and signaling activities.

    Directory of Open Access Journals (Sweden)

    Luis J Cocka

    2012-09-01

    Full Text Available Tetherin (BST-2/CD317/HM1.24 is an IFN induced transmembrane protein that restricts release of a broad range of enveloped viruses. Important features required for Tetherin activity and regulation reside within the cytoplasmic domain. Here we demonstrate that two isoforms, derived by alternative translation initiation from highly conserved methionine residues in the cytoplasmic domain, are produced in both cultured human cell lines and primary cells. These two isoforms have distinct biological properties. The short isoform (s-Tetherin, which lacks 12 residues present in the long isoform (l-Tetherin, is significantly more resistant to HIV-1 Vpu-mediated downregulation and consequently more effectively restricts HIV-1 viral budding in the presence of Vpu. s-Tetherin Vpu resistance can be accounted for by the loss of serine-threonine and tyrosine motifs present in the long isoform. By contrast, the l-Tetherin isoform was found to be an activator of nuclear factor-kappa B (NF-κB signaling whereas s-Tetherin does not activate NF-κB. Activation of NF-κB requires a tyrosine-based motif found within the cytoplasmic tail of the longer species and may entail formation of l-Tetherin homodimers since co-expression of s-Tetherin impairs the ability of the longer isoform to activate NF-κB. These results demonstrate a novel mechanism for control of Tetherin antiviral and signaling function and provide insight into Tetherin function both in the presence and absence of infection.

  15. Identification of an alternative splicing isoform of chicken Lmbr1.

    Science.gov (United States)

    Huang, Yanqun; Chen, Wen; Li, Ning; Deng, Xuemei; Kang, Xiangtao; Liu, Xiaojun

    2011-10-01

    Lmbr1 is the key candidate gene for limb development. Until now, at least five and four alternative splicing isoforms of Lmbr1 gene have been found in human and mouse, respectively. However, only two alternative splicing isoforms of this homologous gene have been reported in chicken. In the present study, one novel chicken Lmbr1 transcript variant (designated Lmbr1-1) was identified by 5' RACE and RT-PCR. Chicken Lmbr1-1 possesses one novel transcription start site different from Lmbr1-N, and was predicted to encode one 192 amino acid protein with length variation in comparison with chicken LMBR1-N protein, which was produced by 5' spliced site variation of chicken Lmbr1-N exon 10. Comparing with Lmbr1-N transcript, chicken Lmbr1-1 exhibited restricted tissue distribution of the expression. Comparative sequence analysis revealed a highly conservative intron element between chicken and mammalians from the intron 9 of chicken Lmbr1-N, indicating their possible importance as intronic elements in the regulation of alternative splicing of Lmbr1 in vertebrates. By direct PCR sequencing the exon 10 and its flanking sequences in chicken Lmbr1-N, four variation sites/haplotypes were identified from six chicken breeds. One 797A/G nonsynonymous mutation (266Arg/Gln) locating in exon 10 of chicken Lmbr1-N was predicted to affect the exon splice enhancer motif for serine/arginine-rich protein recognition. These data demonstrated that chicken Lmbr1 was alternatively spliced to generate multiple splice forms, as was the case in mammals and each of the alternative splicing isoforms might function differentially.

  16. Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2

    DEFF Research Database (Denmark)

    Lee, Yun-Han; Seo, Daekwan; Choi, Kyung-Ju

    2014-01-01

    Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit H...

  17. INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

    Science.gov (United States)

    Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

  18. The relationship between gene isoform multiplicity, number of exons and protein divergence.

    Directory of Open Access Journals (Sweden)

    Jordi Morata

    Full Text Available At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly. In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences.

  19. Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers.

    Science.gov (United States)

    Sellin, Mikael E; Stenmark, Sonja; Gullberg, Martin

    2012-11-01

    Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform-specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks-a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.

  20. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

    Directory of Open Access Journals (Sweden)

    Jean-Simon eFortin

    2013-12-01

    Full Text Available The peptide repertoire (peptidome associated with MHC class II molecules (MHCIIs is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  1. Exposing the Specific Roles of the Invariant Chain Isoforms in Shaping the MHC Class II Peptidome.

    Science.gov (United States)

    Fortin, Jean-Simon; Cloutier, Maryse; Thibodeau, Jacques

    2013-12-13

    The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  2. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

    Science.gov (United States)

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J; Vacic, Vladimir; Calderwood, Michael A; Roth, Frederick P; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M

    2014-04-11

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

  3. Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

    Science.gov (United States)

    Bridges, Hannah R; Mohammed, Khairunnisa; Harbour, Michael E; Hirst, Judy

    2017-03-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

  4. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  5. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have u...... synthesized cohorts of pumps from the Golgi apparatus to the plasma membrane....

  6. FSH isoform pattern in classic galactosemia.

    Science.gov (United States)

    Gubbels, Cynthia S; Thomas, Chris M G; Wodzig, Will K W H; Olthaar, André J; Jaeken, Jaak; Sweep, Fred C G J; Rubio-Gozalbo, M Estela

    2011-04-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

  7. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.

    Science.gov (United States)

    Gilbreth, Ryan N; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B; Li, Nan-Sheng; Piccirilli, Joseph A; Chen, Yuan; Koide, Shohei

    2011-05-10

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  8. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

    Energy Technology Data Exchange (ETDEWEB)

    Gilbreth, Ryan N.; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B.; Li, Nan-Sheng; Piccirilli, Joseph A.; Chen, Yuan; Koide, Shohei (UC); (CHNMC)

    2011-07-25

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a 'monobody,' a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K{sub d} values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  9. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

    Directory of Open Access Journals (Sweden)

    Siliakus Melvin

    2010-04-01

    Full Text Available Abstract Background Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6. Results Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms. Conclusion The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.

  10. Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

    Science.gov (United States)

    Pires, Susana S; Hughes, Steven; Turton, Michael; Melyan, Zare; Peirson, Stuart N; Zheng, Lei; Kosmaoglou, Maria; Bellingham, James; Cheetham, Michael E; Lucas, Robert J; Foster, Russell G; Hankins, Mark W; Halford, Stephanie

    2009-09-30

    Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

  11. Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein

    Science.gov (United States)

    Minervini, Giovanni; Mazzotta, Gabriella M.; Masiero, Alessandro; Sartori, Elena; Corrà, Samantha; Potenza, Emilio; Costa, Rodolfo; Tosatto, Silvio C. E.

    2015-01-01

    Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization. PMID:26211615

  12. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  13. Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure.

    Science.gov (United States)

    Bousfield, George R; Butnev, Vladimir Y; Bidart, Jean-Michel; Dalpathado, Dilusha; Irungu, Janet; Desaire, Heather

    2008-02-12

    Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the

  14. Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms.

    Science.gov (United States)

    Rouleau, Agnès; Héron, Anne; Cochois, Véronique; Pillot, Catherine; Schwartz, Jean-Charles; Arrang, Jean-Michel

    2004-09-01

    The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.

  15. Cadmium effects on p38/MAPK isoforms in MDA-MB231 breast cancer cells.

    Science.gov (United States)

    Casano, Caterina; Agnello, Maria; Sirchia, Rosalia; Luparello, Claudio

    2010-02-01

    Emerging evidence seems to indicate that the heavy metal cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in normal and pathological eukaryotic cells, also affecting intracellular signalization events. Human p38 is a family of mitogen-activated protein kinases consisting of four isoforms (alpha, beta, gamma and delta) which mediate signal transduction cascades controlling several aspects of cell physiology. In this study we examined whether exposure of MDA-MB231 tumor cells from the human breast to Cd may exert some effect on p38 isoform expression and accumulation, as well as on p38 activation. Employing a combination of proliferation tests, conventional and semiquantitative multiplex (SM)-polymerase chain reaction (PCR) and Western blot assays, we report that the treatment of breast cancer cells with 5 microM CdCl(2) induces a diversified modulation of the transcription patterns of p38 isoform genes and of the accumulation of the related protein products, which are, on the other hand, also affected by alpha and beta isoform functional inactivation induced by SB203580. Our findings suggest the existence of so far unexplored mechanisms of gene regulation in our model system and validate that MDA-MB231 cell line is a suitable in vitro model for further and more detailed studies on the intracellular mechanisms underlying the control of p38 expression, synthesis and activation in mammary tumor cells exposed to different stresses.

  16. Expression of Contractile Protein Isoforms in Microgravity

    Science.gov (United States)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  17. Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

    Science.gov (United States)

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

  18. The HER4 isoform JM-a/CYT2 relates to improved survival in bladder cancer patients but only if the estrogen receptor α is not expressed

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque; Poulsen, Steen S

    2013-01-01

    Abstract Bladder cancer tumors expressing human epidermal growth factor receptor 4 (HER4) demonstrate improved patient survival. HER4 isoforms and estrogen receptor alpha (ER-α) can form chaperone complexes causing cell-proliferation. We wanted to explore if HER4 isoforms and ER-α could correlate...... to poor prognosis in bladder cancers. We developed mRNA assays for HER4 isoforms (JM-a, JM-b, CYT1, and CYT2) and for ER-α. Expression was analyzed in tumors from 85 bladder cancer patients and compared to overall survival (median follow-up of 5.1 years). ER-α was expressed in 38% (n = 32) of tumors...... but did not correlate to survival (p = 0.4698). HER4 was expressed in 42% (n = 36) and in all cases as the ER-α binding isoform JM-a. The JM-a isoform can be alternatively spliced to either a CYT1 isoform (JM-a/CYT1) or a CYT2 isoform (JM-a/CYT2). All HER4 expressing tumors expressed the JM-a/CYT2 isoform...

  19. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity.

    Science.gov (United States)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T; Clausen, Henrik; Linstedt, Adam D

    2014-10-31

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

  20. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    Science.gov (United States)

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

  1. Progesterone receptor isoforms in the mammary gland of cats and dogs.

    Science.gov (United States)

    Gracanin, A; de Gier, J; Zegers, K; Bominaar, M; Rutteman, G R; Schaefers-Okkens, A C; Kooistra, H S; Mol, J A

    2012-12-01

    Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

  2. Evaluation of cytochrome P450 activity in vitro, using dermal and hepatic microsomes from four species and two keratinocyte cell lines in culture.

    Science.gov (United States)

    Rolsted, Kamilla; Kissmeyer, Anne-Marie; Rist, Gerda Marie; Hansen, Steen Honoré

    2008-01-01

    The Cytochrome P450 (CYP450) enzymes are expressed in the skin, and despite a low activity, as compared to the hepatic counterpart, a role during transdermal delivery of a drug cannot be excluded. Additionally, the enzymes may play a role in local toxicity, and further knowledge of dermal CYP450 activity can contribute to elucidate this issue. To achieve this, a cocktail of six selective CYP450 probe substrates were incubated with dermal and hepatic microsomes isolated from mouse, rat, minipig and man. Different species were used to evaluate if a reliable substitute for human tissue was possible. Further, the hepatic microsomes were included in this study, to estimate if the hepatic CYP450 activity is predictive of dermal CYP450 activity. The CYP450 activity was determined in two keratinocyte cell lines as well, as this in vitro model is desirable due to the ease in handling, among other factors. Overall, the metabolism found in the dermal microsomes was very low, and major differences were observed between species. When comparing the activities in dermal and hepatic microsomes, the qualitative pattern was to some extent similar within species, but also a number of differences were observed. The CYP450 metabolic activity in the two keratinocyte cell lines was not comparable to metabolism in the human dermal microsomes.

  3. Regulation of estrogen, progesterone and mifepristone on the expression of two human progesterone receptor isoforms in endometrial carcinoma cells%雌孕激素及米非司酮对人子宫内膜癌细胞孕激素受体亚型调控的研究

    Institute of Scientific and Technical Information of China (English)

    李大鹏; 盛修贵; 魏萍; 魏玲; 宋宝

    2011-01-01

    目的:通过体外实验探讨雌激素、孕激素及米非司酮对人子宫内膜癌细胞孕激素受体亚型表达的调节.方法:体外培养表达不同孕激素受体亚型的人子宫内膜癌细胞HECCA-A(PRA+)、HECCA-B(PRB+)及HECCA-AB(PRA+和PRB+),分别加入雌激素、孕激素和米非司酮,作用24、48h后采用蛋白质印迹法测定各组细胞中2种孕激素受体亚型蛋白的表达.结果:雌激素作用48 h后,HECCA-A、HECCA-AB细胞的PRA表达水平明显升高,PRA/PRB比值升高,P<0.05;孕激素及米非司酮作用48 h后,HECCA-B、HECCA-AB细胞的PRB表达水平明显降低,PRA/PRB比值升高,P<0.05.结论:雌激素上调子宫内膜癌细胞PRA的蛋白表达水平,孕激素及米非司酮下调PRB的蛋白表达水平.%OBJECTIVE: To investigate the regulatory effects of strogen, progesterone and mifepristone on the expression of two human progesterone receptor isoforms in endometrial carcinoma cells in vitro. METHODS: Three endometrial carcinoma cell lines were cultured in vitro, HECCA-A (PRA + ), HECCA-B (PRB + ) and HECCA-AB (PRA+,PRB+). The cells were incubated with estrogen, progesterone and mifepristone for 24 hours and 48 hours individually. The expressions of two progesterone receptor isoforms were detected by Western Blot. RESULTS: When stimulated by estrogen for 48 hours, the expression of PRA and the ratio of PRA/PRB in HECCA-A and HECCA-AB cells raised significantly (P<0. 05), while after being stimulated by progesterone or mifepristone, the expression of PRB in HECCA-B and HECCAAB cells decreased, and the ratio of PRA/PRB raised significiantly (P<0. 05). CONCLUSION: Estrogen can up-regulate the expression of PRA at protein level, progesterone or mifepristone can decrease the protein expression of PRB.

  4. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    Science.gov (United States)

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  5. Tocopherol isoforms in parenteral lipid emulsions and neutrophil activation.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Beunk, J.; Naber, A.H.J.; Swinkels, D.W.

    2002-01-01

    BACKGROUND AND AIMS: Tocopherol is a lipid-soluble anti-oxidant that exists in several isoforms. Patients on total parenteral nutrition depend on lipid emulsions for their tocopherol intake. In the present study, we analysed the content of tocopherol isoforms in various lipid emulsions. We also

  6. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

    OpenAIRE

    Jean-Simon eFortin; Maryse eCloutier; Jacques eThibodeau

    2013-01-01

    The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common...

  7. A Naturally Occurring Isoform Inhibits Parathyroid Hormone Receptor Trafficking and Signaling

    OpenAIRE

    Alonso, Ver?nica; Ardura, Juan A; WANG Bin; Sneddon, W Bruce; Peter A Friedman

    2010-01-01

    Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (?e14-PTHR) that encodes transmembrane domain 7. ?e14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the ?e14-PTHR is extracellular, and cell surface expression...

  8. Muscle Specific Fragile X Related Protein 1 Isoforms are Sequestered in the Nucleus of Undifferentiated Myoblast

    Directory of Open Access Journals (Sweden)

    Khandjian Edouard W

    2000-12-01

    Full Text Available Abstract Background The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied. Results Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism. Conclusions The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

  9. Bronchial Epithelial Cells from Asthmatic Patients Display Less Functional HLA-G Isoform Expression.

    Science.gov (United States)

    Carlini, Federico; Picard, Christophe; Garulli, Céline; Piquemal, David; Roubertoux, Pierre; Chiaroni, Jacques; Chanez, Pascal; Gras, Delphine; Di Cristofaro, Julie

    2017-01-01

    Not all asthmatic patients adequately respond to current available treatments, such as inhaled corticosteroids or omalizumab(®). New treatments will aim to target the bronchial epithelium-immune response interaction using different pathways. HLA-G is involved in immunomodulation and may promote epithelial cell differentiation and proliferation. HLA-G protein has several isoforms generated by alternative splicing that might have differential functionalities. HLA-G protein expression and genetic polymorphisms have been reported to be associated with asthma. Our hypothesis is that bronchial epithelium from asthmatic patients displays less functional HLA-G isoforms. HLA-G transcriptional isoforms were quantified by real-time PCR in human bronchial epithelium cells (HBEC) grown in air-liquid interface culture obtained from five healthy controls (HC), seven patients with mild asthma (MA), and seven patients with severe asthma (SA). They were re-differentiated, and IL-13 exposure was used as a proxy for a pro-inflammatory cytokine. HLA-G protein expression was assessed by western blot analysis. HLA-G allele was typed by direct sequencing. Our results showed that both MA and SA display less functional HLA-G isoforms than HC (p G*01:06 frequency in MA and SA was significantly higher than in the healthy population (p = 0.03 and p G expression. Our results support that an impaired expression of HLA-G isoforms in asthmatic patients could contribute to the loss of inflammation control and epithelium structural remodeling. Therefore, HLA-G might be an interesting alternative target for asthmatic patients not adequately responding to current drugs.

  10. ADAPTIVE CHANGES OF MYOSIN ISOFORMS IN RESPONSE TO LONG-TERM STRENGTH AND POWER TRAINING IN MIDDLE-AGED MEN

    Directory of Open Access Journals (Sweden)

    Raivo Puhke

    2006-06-01

    Full Text Available The purpose of the study was to examine the adaptive changes in myosin heavy chain (MHC and light chain (MLC isoforms in human vastus lateralis muscle caused by long-term strength and power training (54 weeks, approximately 3 times a week in untrained middle- aged men (16 in the training and 6 in the control group. Muscular MHC and MLC isoforms were determined by means of SDS-PAGE gel electrophoresis. During the training period, maximal anaerobic cycling power increased by 64 W (p < 0.001 and the maximal jumping height by 1.5 cm (p < 0. 05 in the training group, but no significant changes were found in the control group. However, the group by time effect was not significant. In the training group, the increase of the maximal jumping height correlated with the number of strength and power training sessions (r = 0.56; p < 0.05. The change of the proportion of MHC IIa isoform from 52.6 ± 12.2% to 59.4 ± 11.6% did not reach statistical significance (p = 0.070 for group by time; within training group p = 0.061 and neither did the change of the proportion of MHC IIx isoform from 18.1 ± 11.4% to 11.1 ± 9.1% (p = 0.104 for group by time; within training group p=0.032. The degree of change of MHC IIx isoform correlated with the amount of earlier recreational sports activity (r = 0.61; p < 0.05. In the training group, the changes of MLC1s isoform correlated negatively with the changes of MLC1f isoform (r = -0. 79; p < 0.05 as well as with the changes in maximal anaerobic cycling power (r = -0.81; p < 0.05, and positively with those of MHC I isoform (r = 0.81; p < 0.05. In conclusion, the long- term strength and power training ~3 times a week seemed to have only slight effects on fast MHC isoforms in the vastus lateralis muscle of untrained middle-aged men; the proportion of MHC IIa tended to increase and that of MHC IIx tended to decrease. No changes in MLC isoform profile could be shown

  11. Estimation of bisphenol A-Human toxicity by 3D cell culture arrays, high throughput alternatives to animal tests.

    Science.gov (United States)

    Lee, Dong Woo; Oh, Woo-Yeon; Yi, Sang Hyun; Ku, Bosung; Lee, Moo-Yeal; Cho, Yoon Hee; Yang, Mihi

    2016-09-30

    Bisphenol A (BPA) has been widely used for manufacturing polycarbonate plastics and epoxy resins and has been extensively tested in animals to predict human toxicity. In order to reduce the use of animals for toxicity assessment and provide further accurate information on BPA toxicity in humans, we encapsulated Hep3B human hepatoma cells in alginate and cultured them in three dimensions (3D) on a micropillar chip coupled to a panel of metabolic enzymes on a microwell chip. As a result, we were able to assess the toxicity of BPA under various metabolic enzyme conditions using a high-throughput and micro assay; sample volumes were nearly 2,000 times less than that required for a 96-well plate. We applied a total of 28 different enzymes to each chip, including 10 cytochrome P450s (CYP450s), 10 UDP-glycosyltransferases (UGTs), 3 sulfotransferases (SULTs), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase 2 (ALDH2). Phase I enzyme mixtures, phase II enzyme mixtures, and a combination of phase I and phase II enzymes were also applied to the chip. BPA toxicity was higher in samples containing CYP2E1 than controls, which contained no enzymes (IC50, 184±16μM and 270±25.8μM, respectively, panimal testing for estimating BPA toxicity via human metabolic systems. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. The synthesis of novel pyrazole-3,4-dicarboxamides bearing 5-amino-1,3,4-thiadiazole-2-sulfonamide moiety with effective inhibitory activity against the isoforms of human cytosolic carbonic anhydrase I and II.

    Science.gov (United States)

    Mert, Samet; Alım, Zuhal; İşgör, Mehmet Mustafa; Beydemir, Şükrü; Kasımoğulları, Rahmi

    2016-10-01

    A series of 1-(3-substituted-phenyl)-5-phenyl-N(3),N(4)-bis(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3,4-dicarboxamides (4-15) were synthesized. The structures of these pyrazole-sulfonamides were confirmed by FT-IR, (1)H NMR, (13)C NMR and elemental analysis methods. Human cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes (hCA I and II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of newly synthesized derivatives (4-15) were investigated in vitro on esterase activities of these isozymes. The Ki values were determined as 0.119-3.999μM for hCA I and 0.084-0.878μM for hCA II. The results showed that the compound 6 for hCA I and the compound 11 for hCA II had the highest inhibitory effect. Beside that, the compound 8 had the lowest inhibition effect on both isozymes.

  13. Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture.

    Science.gov (United States)

    Gutiérrez-Zúñiga, Gabriela Guadalupe; Hernández-López, José Luis

    2016-01-01

    A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule ((Btn)Ab-AgPSA-(HRP)Ab), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population.

  14. Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1.

    Directory of Open Access Journals (Sweden)

    Shinobu Tsuzuki

    2007-05-01

    Full Text Available AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.These data demonstrate that the "a" isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood

  15. Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1

    Science.gov (United States)

    Tsuzuki, Shinobu; Hong, Dengli; Gupta, Rajeev; Matsuo, Keitaro; Seto, Masao; Enver, Tariq

    2007-01-01

    Background AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo. Methods and Findings The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo. Conclusions These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of

  16. CYP-450 isoenzymes catalyze the generation of hazardous aromatic amines after reaction with the azo dye Sudan III

    OpenAIRE

    Zanoni, Thalita Boldrin; Lizier, Thiago M. [UNESP; Assis,Marilda das Dores; Zanoni, Maria Valnice Boldrin; de Oliveira, Danielle Palma

    2013-01-01

    This work describes the mutagenic response of Sudan III, an adulterant food dye, using Salmonella typhimurium assay and the generation of hazardous aromatic amines after different oxidation methods of this azo dye. For that, we used metabolic activation by S9, catalytic oxidation by ironporphyrin and electrochemistry oxidation in order to simulate endogenous oxidation conditions. The oxidation reactions promoted discoloration from 65% to 95% of Sudan III at 1×10-4molL-1 and generation of 7.6×...

  17. CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine

    Directory of Open Access Journals (Sweden)

    Pybus Brandon S

    2012-08-01

    Full Text Available Abstract Background The 8-aminoquinoline (8AQ drug primaquine (PQ is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ’s haemotoxic and anti-malarial properties are not fully understood. Methods In the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP and mono-amine oxidase (MAO families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements. Results Relative activity factor (RAF-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species. Conclusions As a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

  18. Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

    Science.gov (United States)

    Bahadoor, Adilah; Schneiderman, Danielle; Gemmill, Larissa; Bosnich, Whynn; Blackwell, Barbara; Melanson, Jeremy E; McRae, Garnet; Harris, Linda J

    2016-01-22

    A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography.

  19. Nonmuscle myosin II isoforms coassemble in living cells.

    Science.gov (United States)

    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

  20. Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases.

    Science.gov (United States)

    Rouabhia, Mahmoud; Schaller, Martin; Corbucci, Cristina; Vecchiarelli, Anna; Prill, Stephan K-H; Giasson, Luc; Ernst, Joachim F

    2005-08-01

    The PMT gene family in Candida albicans encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of pmt mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All pmt mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The pmt5 mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the pmt4 and pmt6 mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of C. albicans and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.

  1. eIF5A isoforms and cancer: two brothers for two functions?

    Science.gov (United States)

    Caraglia, M; Park, M H; Wolff, E C; Marra, M; Abbruzzese, A

    2013-01-01

    Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. The role of hypusine formation in the eIF5A protein in the regulation of cell proliferation and apoptosis is addressed in the present review. Moreover, vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. However, the biological functions of these two isoforms may be significantly different. In fact, eIF5A-1 is demonstrable in most cells of different histogenesis, whereas eIF5A-2 protein is detectable only in certain human cancer cells or tissues, suggesting its role as a potential oncogene. In this review we focus our attention on the involvement of eIF5A-1 in the triggering of an apoptotic program and in the regulation of cell proliferation. In addition, the potential oncogenic role and prognostic significance of eIF5A-2 in the prediction of the survival of cancer patients is described. eIF5A-1 and/or the eIF5A-2 isoform may serve as a new molecular diagnostic or prognostic marker or as a molecular target for anti-cancer therapy.

  2. A crustin isoform from black tiger shrimp, Penaeus monodon exhibits broad spectrum anti-bacterial activity

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    Debashis Banerjee

    2015-11-01

    Full Text Available Crustaceans have a powerful non-specific immune mechanism that responds to pathogen invasion and together with cellular responses, generates powerful humoral factors such as antimicrobial peptides. Crustins are a diverse class of antimicrobial peptides that are expressed by the circulating haemocytes of crustaceans. Several isoforms of this molecule are reported and in this study, one isoform from the black tiger shrimp, Penaeus monodon was cloned and expressed in Escherichia coli SG 13009. The purified recombinant crustin peptide had a molecular weight of 22 kDa and exhibited potent anti-bacterial activity in vitro against several Gram positive and Gram negative bacteria that included pathogens of aquatic animals and humans. The recombinant crustin showed a minimal inhibitory concentration of 0.5 μg ml−1 against the vibrio pathogens of shrimp, which suggests its promise for application in aquaculture.

  3. Numerous isoforms of Fgf8 reflect its multiple roles in the developing brain.

    Science.gov (United States)

    Sunmonu, N Abimbola; Li, Kairong; Li, James Y H

    2011-07-01

    Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination, and morphogenesis during development of multicellular organisms. Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces eight splice variants, which encode isoform proteins with different N-termini and distinct receptor-binding affinity and biological activity. In this article, we review the roles of Fgf8 in vertebrate development and summarize the recent findings on the in vivo function of different Fgf8 splice variants. We propose that multiple Fgf8 isoform proteins act in concert to regulate the overall function of Fgf8 and account for the diverse and essential role of Fgf8 during vertebrate development.

  4. Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

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    Linda V. Hjørnevik

    2015-12-01

    Full Text Available Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s. A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

  5. GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease.

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    Willem Kamphuis

    Full Text Available Glial fibrillary acidic protein (GFAP is the main astrocytic intermediate filament (IF. GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ, whereas GFAP(+1 is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-β, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS, neurogenic astrocytes of the subgranular zone (SGZ, and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP(+1 staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain.

  6. Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

    Science.gov (United States)

    Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

    2002-01-01

    Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

  7. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

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    Francesca Rappa

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

  8. 人类成纤维细胞生长因子受体1的Ⅲb亚型在胰腺癌细胞中的作用%The role of human fibroblast growth factor receptor 1-Ⅲb isoform in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    刘占兵; 杨尹默; 乔岐禄; 赵建勋; 黄莚庭; Marko Kornmann

    2007-01-01

    Objective To study the role of Ⅲb isoform of human fibroblast growth factor receptor 1 (FGFR1-Ⅲb) in PANC-1 pancreatic cancer cells. Methods The plasmid of human full-length FGFR1-Ⅲb isoform,pSVK4/FGFR1-Ⅲb, was stable transfected into cultured PNAC-1 pancreatic cancer cell lines facilitated by lipofectamine. The function of FGFR1-Ⅲb in transfected pancreatic cancer cells were examined by MTT assay, soft agar assay, cell migration assay, single cell movement assay, In vivo tumorigenicity assay. Results The basal anchorage-dependent and -independent cell growth was significantly inhibited. Additionally, FGFR1-Ⅲb expression inhibited single cell movement and in vitro invasion as determined by time-lapse microscopy and boyden chamber assay as well as in vivo tumor formation and growth in nude mice. Microscopic analysis of the xenograft tumors revealed a reduced Ki-67 labelling, lower amount of tumor necrosis and higher grade of differentiation in FGFR1-Ⅲb expressing tumors. Conclusion We identified a functional human FGFR mRNA splice variant that inhibits the transforming potential of pancreatic cancer cells.%目的 探讨人类成纤维细胞生长因子受体1的Ⅲb亚型(FGFR1-Ⅲb)在胰腺癌细胞中的作用. 方法 以脂质体法将人类全长FGFR1-Ⅲb表达质粒pSVK4/FGFR1-Ⅲb稳定转染入PANC-1胰腺癌细胞,通过MTT试验、软琼脂试验、细胞迁移试验、单个细胞运动试验和裸鼠移植瘤试验检测FGFR1-Ⅲb在转染的胰腺癌细胞中的功能. 结果 转染FGFR1-Ⅲb受体的胰腺癌细胞,在细胞生长、细胞迁移、细胞运动、移植瘤形成及生长方面均受抑,且移植瘤Ki-67表达减低、肿瘤组织中坏死灶减少. 结论 人类FGFR1-Ⅲb受体为胰腺癌细胞的功能性受体,对胰腺癌细胞具有抑制作用.

  9. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Fu, J.

    2002-03-01

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding

  10. Role of nuclear progesterone receptor isoforms in uterine pathophysiology.

    Science.gov (United States)

    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    required for growth. In contrast, progesterone via PR activation appears to increase leiomyoma growth. The exact role of PRs in cervical cancer is unclear. PRs regulate implantation and therefore aberrant PR function may be implicated in recurrent pregnancy loss (RPL). PRs likely regulate key immunogenic factors involved in RPL. However, the exact role of PRs in the pathophysiology of RPL and the use of progesterone for therapeutic benefit remains uncertain. PRs are key mediators of progesterone action in uterine tissues and are essential for normal uterine function. Aberrant PR function (due to abnormal expression and/or function) is a major cause of uterine pathophysiology. Further investigation of the underlying mechanisms of PR isoform action in the uterus is required, as this knowledge will afford the opportunity to create progestin/PR-based therapeutics to treat various uterine pathologies. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Isoform-specific targeting of ROCK proteins in immune cells

    OpenAIRE

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform ...

  12. Neuronal profilin isoforms are addressed by different signalling pathways.

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    Kai Murk

    Full Text Available Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF, on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

  13. Yeast ADP/ATP Carrier Isoform 2

    Science.gov (United States)

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  14. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

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    Kubo Takeo

    2010-02-01

    Full Text Available Abstract Background The ecdysone receptor (EcR regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. Results The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Conclusions Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional

  15. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.

    Science.gov (United States)

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram; Madsen, Thomas Daugbjerg; Gerken, Thomas A; Vester-Christensen, Malene B; Wandall, Hans H; Bennett, Eric Paul; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

  16. TrkB Isoforms Differentially Affect AICD Production through Their Intracellular Functional Domains

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    Sara Ansaloni

    2011-01-01

    Full Text Available We report that NTRK2, the gene encoding for the TrkB receptor, can regulate APP metabolism, specifically AICD levels. Using the human neuroblastoma cell line SH-SY5Y, we characterized the effect of three TrkB isoforms (FL, SHC, T on APP metabolism by knockdown and overexpression. We found that TrkB FL increases AICD-mediated transcription and APP levels while it decreases sAPP levels. These effects were mainly mediated by the tyrosine kinase activity of the receptor and partially by the PLC-- and SHC-binding sites. The TrkB T truncated isoform did not have significant effects on APP metabolism when transfected by itself, while the TrkB SHC decreased AICD-mediated transcription. TrkB T abolished TrkB FL effects on APP metabolism when cotransfected with it while TrkB SHC cotransfected with TrkB FL still showed increased APP levels. In conclusion, we demonstrated that TrkB isoforms have differential effects on APP metabolism.

  17. Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder.

    Science.gov (United States)

    Lopez, A Y; Wang, X; Xu, M; Maheshwari, A; Curry, D; Lam, S; Adesina, A M; Noebels, J L; Sun, Q-Q; Cooper, E C

    2016-12-13

    ANK3, encoding the adaptor protein Ankyrin-G (AnkG), has been implicated in bipolar disorder by genome-wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce the expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin (PV) interneurons and principal cells differentially express ANK3 first exon subtypes. PV interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone or both 1e and 1b. In transgenic mice deficient for exon 1b, PV interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy and sudden death. Thus ANK3's important association with human bipolar susceptibility may arise from imbalance between AnkG function in interneurons and principal cells and resultant excessive circuit sensitivity and output. AnkG isoform imbalance is a novel molecular endophenotype and potential therapeutic target.Molecular Psychiatry advance online publication, 13 December 2016; doi:10.1038/mp.2016.233.

  18. The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands.

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    Fabian E Saenz

    Full Text Available Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54 sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies.

  19. Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1.

    Science.gov (United States)

    Inserra, Marco C; Israel, Mathilde R; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R; Harrington, Andrea M; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y; Zimmermann, Katharina; Lewis, Richard J; Brierley, Stuart M; Vetter, Irina

    2017-02-22

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

  20. Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1

    Science.gov (United States)

    Inserra, Marco C.; Israel, Mathilde R.; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R.; Harrington, Andrea M.; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y.; Zimmermann, Katharina; Lewis, Richard J.; Brierley, Stuart M.; Vetter, Irina

    2017-01-01

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation. PMID:28225079

  1. Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

    Science.gov (United States)

    Wong, Marty Kwok-Shing; Pipil, Supriya; Kato, Akira; Takei, Yoshio

    2016-09-01

    Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other