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Sample records for human cyp450 isoforms

  1. Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism.

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    Veronique Michaud

    2010-12-01

    Full Text Available Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68 of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7, samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

  2. The effects of Andrographis paniculata (Burm.f.) Nees extract and diterpenoids on the CYP450 isoforms' activities, a review of possible herb-drug interaction risks.

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    Tan, Mei Lan; Lim, Lin Ee

    2015-01-01

    Andrographis paniculata (Burm.f.) Nees is a popular medicinal plant and its components are used in various traditional product preparations. However, its herb-drug interactions risks remain unclear. This review specifically discusses the various published studies carried out to evaluate the effects of Andrographis paniculata (Burm.f.) Nees plant extracts and diterpenoids on the CYP450 metabolic enzyme and if the plant components pose a possible herb-drug interaction risk. Unfortunately, the current data are insufficient to indicate if the extracts or diterpenoids can be labeled as in vitro CYP1A2, CYP2C9 or CYP3A4 inhibitors. A complete CYP inhibition assay utilizing human liver microsomes and the derivation of relevant parameters to predict herb-drug interaction risks may be necessary for these isoforms. However, based on the current studies, none of the extracts and diterpenoids exhibited CYP450 induction activity in human hepatocytes or human-derived cell lines. It is crucial that a well-defined experimental design is needed to make a meaningful herb-drug interaction prediction.

  3. Effect of acute paraquat poisoning on CYP450 isoforms activity in rats by cocktail method

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    Wang, Shuanghu; Wang, Zhiyi; Chen, Dongxin; Chen, Mengchun; Lin, Yingying; Liu, Zezheng; Zhang, Lijing; Wen, Congcong; Wang, Xianqin; Ma, Jianshe

    2015-01-01

    Paraquat is a highly effective contact herbicide that is marketed worldwide as a fantastical, non-selective compound for broadleaf weed control. As compared to most pesticides, paraquat is extremely toxic to humans and the lack of strategies to manage paraquat poisoning has resulted in high fatality rates. The rats were randomly divided into acute paraquat poisoning group and control group. The paraquat group rats were given 36 mg/kg paraquat by intragastric administration. The influence of a...

  4. Relative contributions of the major human CYP450 to the metabolism of icotinib and its implication in prediction of drug-drug interaction between icotinib and CYP3A4 inhibitors/inducers using physiologically based pharmacokinetic modeling.

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    Chen, Jia; Liu, Dongyang; Zheng, Xin; Zhao, Qian; Jiang, Ji; Hu, Pei

    2015-06-01

    Icotinib is an anticancer drug, but relative contributions of CYP450 have not been identified. This study was carried out to identify the contribution percentage of CYP450 to icotinib and use the results to develop a physiologically based pharmacokinetic (PBPK) model, which can help to predict drug-drug interaction (DDI). Human liver microsome (HLM) and supersome using relative activity factor (RAF) were employed to determine the relative contributions of the major human P450 to the net hepatic metabolism of icotinib. These values were introduced to develop a PBPK model using SimCYP. The model was validated by the observed data in a Phase I clinical trial in Chinese healthy subjects. Finally, the model was used to simulate the DDI with ketoconazole or rifampin. Final contribution of CYP450 isoforms determined by HLM showed that CYP3A4 provided major contributions to the metabolism of icotinib. The percentage contributions of the P450 to the net hepatic metabolism of icotinib were determined by HLM inhibition assay and RAF. The AUC ratio under concomitant use of ketoconazole and rifampin was 3.22 and 0.55, respectively. Percentage of contribution of CYP450 to icotinib metabolism was calculated by RAF. The model has been proven to fit the observed data and is used in predicting icotinib-ketoconazole/rifampin interaction.

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

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    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.

  6. Acetaldehyde and parkinsonism: role of CYP450 2E1

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    Francesca eVaglini

    2013-06-01

    Full Text Available The present review update the relationship between acetaldehyde and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly.We have indicated that acetaldehyde is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine neurons of rodent Substantia Nigra, but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the Substantia Nigra induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. Acetaldehyde is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson’s Disease patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and

  7. Assaying Oxidative Coupling Activity of CYP450 Enzymes.

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    Agarwal, Vinayak

    2018-01-01

    Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products. © 2018 Elsevier Inc. All rights reserved.

  8. The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes.

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    Shi, Xianbao; Yang, Shuman; Zhang, Gang; Song, Yonggui; Su, Dan; Liu, Yali; Guo, Feng; Shan, Lina; Cai, Jiqun

    2016-01-01

    1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes. 2. 100 μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 μM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 μM, respectively. 3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 μM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively. 4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

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

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    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.

  10. On the deduction and analysis of singlet and two-state gating-models from the static structures of mammalian CYP450.

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    Zawaira, Alexander; Coulson, Lauren; Gallotta, Marco; Karimanzira, Owen; Blackburn, Jonathan

    2011-02-01

    Differential tunnel-opening patterns were established in static structures of mammalian CYP450 isoforms and subsequently applied to identify tunnel-intersecting residues. The identified tunnel-intersecting residues permitted the subsequent construction of gating models via the identification of intra-protein interactions. We define 28 two-state gating models and 37 singlet gating-residue models. Our results reveal the preponderance of aromatic gating residues in CYP3A4 and CYP2A6, whereas we find a preponderance of polar/charged residues in CYP2C5. In CYP2C8 there is balanced presence of polar/charged and hydrophobic aliphatic residues in gating models, whilst in CYP2C9 there is balanced presence of all residue-types. These patterns suggest fast evolutionary dynamics for gating residues and we find that the average rate of evolution of gating residues in CYP2C5, CYP2C8, CYP2C9 and CYP2A6 is significantly faster than the average rate of evolution of the entire sequence. Our study identifies 67% of calculable gating models identified in the literature by molecular dynamics approaches and 92% of residues appearing in literature models appear in our models. However, only 6% of the models identified in this work had been previously-described in the literature. This suggests that our study has defined the most comprehensive list yet of tunnel-gating models in mammalian CYP450 and in doing so have created a benchmark for molecular dynamics approaches to the ligand-tunnelling problem in CYP450. Copyright © 2010 Elsevier Inc. All rights reserved.

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

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    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

  12. Lipoprotein lipase isoelectric point isoforms in humans

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    Badia-Villanueva, M.; Carulla, P.; Carrascal, M.

    2014-01-01

    -heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation...

  13. Effects of Quercetin on CYP450 and Cytokines in Aroclor 1254 Injured Endometrial Cells of the Pregnant Rats

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    Lina Xu

    2014-01-01

    Full Text Available Polychlorinated biphenyls (PCBs are widespread persistent residual environmental pollutants, which affect seriously the growth and reproductive alterations in humans and animals. Aroclor 1254 is a commercial mixture of PCBs. Quercetin is a flavonoid, which acts on estrogen receptors and causes the development of estrogen-related diseases. In this paper, the primary cultured endometrial cells in the pregnant rats were isolated and Aroclor 1254 was used to induce the injured endometrial cells model. The cells were treated with gradient quercetin, the viability of the endometrial cells, the expressions of CYP450, the contents of TNF-α, IL-6, estradiol (E2, and progesterone (P4 were measured. It showed that the viability of the cultured endometrial cells, the expression of CYP1A1 and CYP2B1, and the contents of TNF-α, E2, and IL-6 in the injured endometrial cells increased with the treatment of quercetin. It shows that quercetin has protective effect on the injured endometrial cells in the pregnant rats, this provide a basis on herbal medicine protection for animal reproductive diseases caused by environmental endocrine disruptors.

  14. GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

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    Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic, Marko D; Guillet, Fabrice; Bouaita, Belkacem; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, Mouhssin

    2018-01-01

    In a screening of natural products for allosteric modulators of GABA A receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABA A and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Influences of Oldenlandia diffusa on the CYP450 Activities in Rats Using a Cocktail Method by UHPLC-MS/MS

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    Yiping Lin

    2018-01-01

    Full Text Available Oldenlandia diffusa has been used to treat various cancers. Cytochrome P450, a drug metabolic enzyme, might be influenced by herbal medicine. Currently, the problem that remains is the effective treatment in drug-drug interaction situation. Potential influences of Oldenlandia diffusa were elucidated on the CYP450 activities in rats using a cocktail method. Blood samples were precipitated by acetonitrile. Quantitative determination of target test object was done by ultra-performance liquid chromatography tandem mass spectrometry detection. Influences of oldenlandia diffusa on the activities of five CYP450 subtypes in rats were evaluated by five specific probe drugs (phenacetin for CYP1A2, omeprazole for CYP2C19, tolbutamide for CYP2C9, metoprolol for CYP2D6, and midazolam for CYP3A4 according to the pharmacokinetic parameters changes. No statistically significant difference (P>0.05 in pharmacokinetic behaviors can be observed in the five probe drugs. There is a potential guidance on clinical drug combination with Oldenlandia diffusa. Oldenlandia diffusa in compound preparation showed well security.

  16. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

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    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.

  17. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

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    Gates, Simon; Miners, John O

    1999-01-01

    Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

  18. [Gene polymorphism of CYP450 2C9 and VKORC1 in Chinese population and their relationships to the maintaining dosage of warfarin].

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    Zhang, Ya-nan; Cui, Wei; Han, Mei; Zheng, Bin; Liu, Fan; Xie, Rui-qin; Yang, Xiao-hong; Gu, Guo-qiang; Zheng, Hong-mei; Wen, Jin-kun

    2010-02-01

    To investigate the distribution of gene polymorphism of CYP450 2C9 and VKORC1-1639A/G in the Chinese population as well as the difference of genetic polymorphism between Chinese Han population and other ethnic populations. Contribution of CYP2C9 and VKORC1 genotype to the maintenance doses on warfarin was also studied. The genotype and allele frequencies were calculated and compared with those in other populations. One hundred and one patients with stable anticoagulation with warfarin under a target international normalized ratio (INR) of 2.0 to 3.0 were enrolled for studying the relationship between the CYP2C9 and VKORC1 gene polymorphism and the warfarin maintaining dosage. CYP450 2C9*3 + 1075C/A allele frequencies were:AA in 449 cases (92.2%), AC in 36 cases (7.4%) and CC in 2 cases (0.4%), respectively. VKORC1 -1639A/G allele frequencies were AA in 415 cases (85.2%), GA in 72 cases (14.8%), but GG in no case (0.0%), respectively. When linear stepwise regression analysis was used to identify factors contributing to warfarin stable dose, the final equation was: ln (D) = 0.346 + 0.017 (weight) - 0.376 (CYP450 2C9*3 + 1075C/A) + 0.148 (VKORC1-1639A/G) - 0.002 (age) (r = 0.827, P = 0.02). There existed significant gene polymorphism CYP450 2C9*3 + 1075C/A and VKORC1-1639A/G in the Chinese Han population. Both Gene polymorphisms of CYP450 2C9*3 + 1075C/A and VKORC1-1639A/G were significantly affecting the maintaining dose of warfarin in the Chinese population.

  19. Expression of two isoforms of CD44 in human endometrium.

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    Behzad, F; Seif, M W; Campbell, S; Aplin, J D

    1994-10-01

    The distribution of the cell-surface adhesion glycoprotein CD44 in human endometrium was examined by immunofluorescence using six monoclonal antibodies to epitopes common to all forms of the molecule, and by reverse transcription-polymerase chain reaction (RT-PCR). Immunoreactivity was observed throughout the menstrual cycle in stroma, vessels, glandular, and luminal epithelium. Variations in staining intensity were observed, especially in the epithelial compartment. CD44 was also expressed strongly by decidualized stromal cells of first-trimester pregnancy. No systematic variation of immunoreactivity was observed with stages of the normal cycle, but a fraction (25%) of the specimens lacked reactivity in the epithelium. To determine the molecular size of the epithelial isoform, an immunoprecipitation technique was developed using surface-radioiodinated, detergent-extracted glands. This indicated the presence at the cell surface of a single dominant CD44E species with an approximate molecular mass of 130 kDa. RT-PCR was used to investigate the isoforms present in whole endometrial tissue, isolated gland fragments, and Ishikawa endometrial carcinoma cells. Complementary DNA produced from total endometrial mRNA was PCR-amplified across the splice junction between exons 5 and 15. Transcripts corresponding to the hyaluronate receptor CD44H as well as a larger isoform were identified. CD44H was absent, or very scarce, in cDNA from purified gland epithelium. In contrast, Ishikawa cells expressed this form abundantly. The glands and Ishikawa cells also expressed CD44E containing sequences encoded by exons 12, 13, and 14. These data demonstrate the presence of CD44 in human endometrium and decidua, and show that different isoforms of CD44 are associated with tissue compartments in which different functional roles can be anticipated.

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

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    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 distinct...... receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known...... revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor...

  1. A proteomic method for analysis of CYP450s protein expression changes in carbon tetrachloride induced male rat liver microsomes

    International Nuclear Information System (INIS)

    Jia Nuan; Liu Xin; Wen Jun; Qian Linyi; Qian Xiaohong; Wu Yutian; Fan Guorong

    2007-01-01

    Carbon tetrachloride (CCl 4 ) is a well-known model compound for producing chemical hepatic injury. Cytochrome P450 is an important monooxygenase in biology. We investigated the CYP450 protein expression in the in vivo hepatotoxicity of rats induced by CCl 4 . In this experiment, CCl 4 were administered to male rats, and their livers at 24 h post-dosing were applied to the proteomic analysis. Blood biochemistry and histopathology were examined to identify specific changes. At the same time, a novel acetylation stable isotopic labeling method coupled with LTQ-FTICR mass spectrometry was applied to disclose the changes of cytochrome P450 expression amounts. The quantitative proteomics method demonstrated its correlation coefficient was 0.9998 in a 100-fold dynamic range and the average ratio of the labeled peptides was 1.04, which was very close to the theoretical ratio of 1.00 and the standard deviation (S.D.) of 0.21. With this approach, 17 cytochrome P450 proteins were identified and quantified with high confidence. Among them, the expression amount of 2C11, 3A2, and 2 E1 were down-regulated, while that of 2C6, 2B2, and 2B1 were up-regulated

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

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    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.

  3. 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.

  4. [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.

  5. NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

    Science.gov (United States)

    Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

    2016-11-16

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H 2 O 2 . Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. 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...

  7. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

    Energy Technology Data Exchange (ETDEWEB)

    Spaggiari, Dany, E-mail: dany.spaggiari@unige.ch [School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland); Daali, Youssef, E-mail: youssef.daali@hcuge.ch [Clinical Pharmacology and Toxicology Service, Geneva University Hospitals, Rue Gabrielle Perret-Gentil, 1211 Genève 14 (Switzerland); Rudaz, Serge, E-mail: serge.rudaz@unige.ch [School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland); Swiss Centre for Applied Human Toxicology, University of Geneva, Boulevard d' Yvoy 20, 1211 Geneva 4 (Switzerland)

    2016-07-01

    Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC{sub 50} values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values generated 10

  8. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

    International Nuclear Information System (INIS)

    Spaggiari, Dany; Daali, Youssef; Rudaz, Serge

    2016-01-01

    Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC 50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC 50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC 50 values generated 10-fold faster

  9. Interaction between Red Yeast Rice and CYP450 Enzymes/P-Glycoprotein and Its Implication for the Clinical Pharmacokinetics of Lovastatin

    Directory of Open Access Journals (Sweden)

    Chia-Hao Chen

    2012-01-01

    Full Text Available Red yeast rice (RYR can reduce cholesterol through its active component, lovastatin. This study was to investigate the pharmacokinetic properties of lovastatin in RYR products and potential RYR-drug interactions. Extracts of three registered RYR products (LipoCol Forte, Cholestin, and Xuezhikang were more effective than pure lovastatin in inhibiting the activities of cytochrome P450 enzymes and P-glycoprotein. Among CYP450 enzymes, RYR showed the highest inhibition on CYP1A2 and CYP2C19, with comparable inhibitory potencies to the corresponding typical inhibitors. In healthy volunteers taking the RYR product LipoCol Forte, the pharmacokinetic properties of lovastatin and lovastatin acid were linear in the dose range of 1 to 4 capsules taken as a single dose and no significant accumulation was observed after multiple dosing. Concomitant use of one LipoCol Forte capsule with nifedipine did not change the pharmacokinetics of nifedipine. Yet, concomitant use of gemfibrozil with LipoCol Forte resulted in a significant increase in the plasma concentration of lovastatin acid. These findings suggest that the use of RYR products may not have effects on the pharmacokinetics of concomitant comedications despite their effects to inhibit the activities of CYP450 enzymes and P-gp, whereas gemfibrozil affects the pharmacokinetics of lovastatin acid when used concomitantly with RYR products.

  10. 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.

  11. 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.

  12. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    Science.gov (United States)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  13. The landscape of isoform switches in human cancers

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

    2017-01-01

    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...

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Sang Soo Kim

    Full Text Available 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.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.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.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 the treatment of diabetic renal

  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......-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...

  17. 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.

  18. Expression of a novel cardiac-specific tropomyosin isoform in humans

    International Nuclear Information System (INIS)

    Denz, Christopher R.; Narshi, Aruna; Zajdel, Robert W.; Dube, Dipak K.

    2004-01-01

    Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3 ' end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1α, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1κ and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1κ fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin

  19. 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.

  20. Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

    Science.gov (United States)

    Meyer-Roxlau, Stefanie; Lämmle, Simon; Opitz, Annett; Künzel, Stephan; Joos, Julius P; Neef, Stefan; Sekeres, Karolina; Sossalla, Samuel; Schöndube, Friedrich; Alexiou, Konstantin; Maier, Lars S; Dobrev, Dobromir; Guan, Kaomei; Weber, Silvio; El-Armouche, Ali

    2017-07-01

    Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

  1. Differential protein expression and localization of CYP450 enzymes in three species of earthworm; is this a reflection of environmental adaptation?

    Science.gov (United States)

    Lu, Xiaoxu; Li, Yinsheng; Thunders, Michelle; Cavanagh, Jo; Matthew, Cory; Wang, Xiuhong; Zhou, Xinchu; Qiu, Jiangping

    2017-03-01

    Cytochrome P450 (CYP450) is a hemoprotein superfamily, among which CYP1, CYP2 and CYP3 play a major role in the metabolism of vast array of xenobiotics and endobiotics. This paper reports on three CYP enzyme variants (CYP1A2, CYP2E1 and CYP3A4) in three species of earthworm (Eisenia fetida, Metaphire guillelmi and Amynthas carnosus). The relative expression levels and localization of the three associated proteins were investigated at three life-cycle points (juvenile, sub-adult and adult), through comparison of anterior and posterior body tissue and between specific organs (body wall, intestine and reproductive tissues) using western blot analysis. This study confirmed the presence of CYP3A4, CYP1A2 and CYP2E1 in all three species of earthworm tested. The levels of expression varied with earthworm species, age, and body location. These differences in occurrence of the three CYP enzymes appeared to reflect the ecological niche (the spatial and temporal location and functional relationship of each individual or population in populations or communities), and the likelihood of contact with soil contaminants of the respective species. These results may help to explain why earthworms are capable of adapting to very different and extensively polluted soil environments and provide important data for subsequent ecotoxicology and ecological adaptability studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    International Nuclear Information System (INIS)

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    2011-01-01

    Highlights: → A novel epsilon isoform of nesprin-2 has been discovered. → This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. → It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. → Like other nesprins, it is located at the nuclear envelope. → We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  3. 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.

  4. Isoforms of purified methyltransferase from human blood platelets ...

    African Journals Online (AJOL)

    ... purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation ...

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

    Science.gov (United States)

    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 PMID:24316526

  6. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    International Nuclear Information System (INIS)

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria

    2006-01-01

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl 2 , as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

  7. 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.

  8. 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...... the number of bands in IEF to two indicating the existence of two types of polypeptide chains....

  9. 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......, 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...... fragments from decomposed erythrocytes and granulocytes. The soluble FBPs may exert bacteriostatic effects and protect folates in plasma from biological degradation, whereas FRs on the surface of blood cells could be involved in intracellular folate uptake or serve as signal proteins. The latter receptors...

  10. Identification of nuclear τ isoforms in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I.

    1990-01-01

    The τ proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, τ has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire τ molecule in the isolated nuclei of neuroblastoma cells. Nuclear τ proteins, like the τ proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that τ may function in processes not directly associated with microtubules and that highly insoluble complexes of τ may also play a role in normal cellular physiology

  11. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

    International Nuclear Information System (INIS)

    Miyazaki, Takamichi; Futaki, Sugiko; Hasegawa, Kouichi; Kawasaki, Miwa; Sanzen, Noriko; Hayashi, Maria; Kawase, Eihachiro; Sekiguchi, Kiyotoshi; Nakatsuji, Norio; Suemori, Hirofumi

    2008-01-01

    Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin α6β1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin α6β1 expressed on hESCs

  12. Dual roles for coactivator activator and its counterbalancing isoform coactivator modulator in human kidney cell tumorigenesis.

    Science.gov (United States)

    Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y; Tsai, Ming-Jer; O'Malley, Bert W

    2008-10-01

    Coactivator activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein, we show that CoAA is a dual-function coregulator that inhibits G(1)-S transition in human kidney cells and suppresses anchorage-independent growth and xenograft tumor formation. Suppression occurs in part by down-regulating c-myc and its downstream effectors ccnd1 and skp2 and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, coactivator modulator (CoAM), antagonizes CoAA-induced G(1)-S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma compared with normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice isoform. This is, thus far, the only example of a nuclear receptor coregulator involved in suppression of kidney cancer and suggests potentially significant new roles for coregulators in renal cancer biology.

  13. Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease.

    Science.gov (United States)

    Salter, D M; Godolphin, J L; Gourlay, M S; Lawson, M F; Hughes, D E; Dunne, E

    1996-08-01

    Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

  14. 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.

  15. Role of cyclooxygenase isoforms in the altered excitatory motor pathways of human colon with diverticular disease.

    Science.gov (United States)

    Fornai, M; Colucci, R; Antonioli, L; Ippolito, C; Segnani, C; Buccianti, P; Marioni, A; Chiarugi, M; Villanacci, V; Bassotti, G; Blandizzi, C; Bernardini, N

    2014-08-01

    The COX isoforms (COX-1, COX-2) regulate human gut motility, although their role under pathological conditions remains unclear. This study examines the effects of COX inhibitors on excitatory motility in colonic tissue from patients with diverticular disease (DD). Longitudinal muscle preparations, from patients with DD or uncomplicated cancer (controls), were set up in organ baths and connected to isotonic transducers. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor) or DFU (COX-2 inhibitor) were assayed on electrically evoked, neurogenic, cholinergic and tachykininergic contractions, or carbachol- and substance P (SP)-induced myogenic contractions. Distribution and expression of COX isoforms in the neuromuscular compartment were assessed by RT-PCR, Western blot and immunohistochemical analysis. In control preparations, neurogenic cholinergic contractions were enhanced by COX inhibitors, whereas tachykininergic responses were blunted. Carbachol-evoked contractions were increased by indomethacin or SC-560, but not DFU, whereas all inhibitors reduced SP-induced motor responses. In preparations from DD patients, COX inhibitors did not affect electrically evoked cholinergic contractions. Both indomethacin and DFU, but not SC-560, decreased tachykininergic responses. COX inhibitors did not modify carbachol-evoked motor responses, whereas they counteracted SP-induced contractions. COX-1 expression was decreased in myenteric neurons, whereas COX-2 was enhanced in glial cells and smooth muscle. In control colon, COX-1 and COX-2 down-regulate cholinergic motility, whereas both isoforms enhance tachykininergic motor activity. In the presence of DD, there is a loss of modulation by both COX isoforms on the cholinergic system, whereas COX-2 displays an enhanced facilitatory control on tachykininergic contractile activity. © 2014 The British Pharmacological Society.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  1. Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography.

    Science.gov (United States)

    Sharp, Christopher A; Linder, Cecilia; Magnusson, Per

    2007-04-01

    Several isoforms of alkaline phosphatase (ALP) can be identified in human tissues and serum after separation by anion-exchange HPLC and isoelectric focusing (IEF). We purified four soluble bone ALP (BALP) isoforms (B/I, B1x, B1 and B2) from human SaOS-2 cells, determined their specific pI values by broad range IEF (pH 3.5-9.5), compared these with commercial preparations of bone, intestinal and liver ALPs and established the effects of neuraminidase and wheat germ lectin (WGA) on enzyme activity. Whilst the isoforms B1x (pI=4.48), B1 (pI=4.32) and B2 (pI=4.12) resolved as well-defined bands, B/I resolved as a complex (pI=4.85-6.84). Neuraminidase altered the migration of all BALP isoforms to pI=6.84 and abolished their binding to the anion-exchange matrix, but increased their enzymatic activities by 11-20%. WGA precipitated the BALP isoforms in IEF gels and the HPLC column and attenuated their enzymatic activities by 54-73%. IEF resolved the commercial BALP into 2 major bands (pI=4.41 and 4.55). Migration of BALP isoforms is similar in IEF and anion-exchange HPLC and dependent on sialic acid content. HPLC is preferable in smaller scale research applications where samples containing mixtures of BALP isoforms are analysed. Circulating liver ALP (pI=3.85) can be resolved from BALP by either method. IEF represents a simpler approach for routine purposes even though some overlapping of the isoforms may occur.

  2. KCC isoforms in a human lens epithelial cell line (B3) and lens tissue extracts.

    Science.gov (United States)

    Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C; Warwar, Ronald; Brown, Thomas L; Lauf, Peter K

    2006-11-01

    We recently reported potassium-chloride cotransporter activity in human lens epithelial B3 (HLE-B3) cells. The purpose of the present study was to demonstrate in these cells as well as in human lens tissue the potassium-chloride cotransport (KCC) isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence microscopy. Of the four KCC genes known to encode the respective proteins and their spliced variants, RT-PCR with both rat and human primers revealed the predicted cDNA fragments of KCC1, KCC3a, KCC3b, and KCC4 but not KCC2 in both HLE-B3 cells and in human lens tissue extracts from cataractous patients. Polyclonal rabbit (rb) anti-rat (rt) and anti-human (hm) antibodies against rtKCC1 and hmKCC3, respectively, and a commercially available rb-anti-mouse (ms) KCC4 antibody were used. Rb anti-rtKCC1-ECL3 [against epitopes within the large extracellular loop 3 (ECL3)] revealed a 150kDa band in HLE-B3 cells consistent with the known molecular weight of KCC1. Rb anti-hmKCC3-ECL3 yielded three bands of 150, 122 and 105kDa, evidence for the presence of KCC3a, KCC3b and possibly KCC3c isoforms. The 122 and 112kDa bands were also demonstrated by rb anti-hmKCC3-CTD [the C-terminal domain (CTD)]. Rb anti-msKCC4 antibody only showed a 100kDa band in HLE-B3 cells. In the human lens tissues, a 115kDa protein was detected with rb anti-rtKCC1-ECL3 and a 100kDa band with rb anti-msKCC4, however, no bands with rb anti-hmKCC3-ECL3 or rb anti-hmKCC3-CTD. Fluorescence microscopy revealed immunocytochemical cytoplasmic and membrane labeling of HLE-B3 cells with anti-KCC1, -KCC3 (laser confocal microscopy) and -KCC4 antibodies and a Cy3-tagged secondary antibody. Hence HLE-B3 cells expressed proteins of the KCC1, KCC3a, b, and KCC4 isoforms, whereas surgically removed cataractous lens tissue expressed only those of KCC1 and KCC4.

  3. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450

    International Nuclear Information System (INIS)

    Burkina, Viktoriia; Rasmussen, Martin Krøyer; Pilipenko, Nadezhda; Zamaratskaia, Galia

    2017-01-01

    Highlights: • The percent identity of porcine, murine and piscine CYPs was compared with human CYPs. • Main similarities and differences were reviewed. • Understanding of molecular mechanisms of CYP system will provide further insights into the CYP regulatory processes, and responses to different factors. - Abstract: Cytochrome P450 proteins (CYP450s) are present in most domains of life and play a critical role in the metabolism of endogenous compounds and xenobiotics. The effects of exposure to xenobiotics depend heavily on the expression and activity of drug-metabolizing CYP450s, which is determined by species, genetic background, age, gender, diet, and exposure to environmental pollutants. Numerous reports have investigated the role of different vertebrate CYP450s in xenobiotic metabolism. Model organisms provide powerful experimental tools to investigate Phase I metabolism. The aim of the present review is to compare the existing data on human CYP450 proteins (1–3 families) with those found in pigs, mice, and fish. We will highlight differences and similarities and identify research gaps which need to be addressed in order to use these species as models that mimic human traits. Moreover, we will discuss the roles of nuclear receptors in the cellular regulation of CYP450 expression in select organisms.

  4. Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

    Directory of Open Access Journals (Sweden)

    Piero Leone

    2018-01-01

    Full Text Available FAD synthase (FADS, EC 2.7.7.2 is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf. Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3′-phosphoadenosine 5′-phosphosulfate (PAPS reductase domain (named FADS6. This isoform has been previously detected in Riboflavin-Responsive (RR-MADD and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L−1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min−1, as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

  5. 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...

  6. Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

    NARCIS (Netherlands)

    Kusters, B.; Waal, R.M.W. de; Wesseling, P.; Verrijp, K.; Maass, C.N.; Heerschap, A.; Barentsz, J.O.; Sweep, C.G.J.; Ruiter, D.J.; Leenders, W.P.J.

    2003-01-01

    We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood

  7. Foetal and adult human CYP3A isoforms in the bioactivation of organophosphorothionate insecticides.

    Science.gov (United States)

    Buratti, Franca M; Leoni, Claudia; Testai, Emanuela

    2006-12-15

    In humans organophosphorothionate pesticides (OPT) prenatal exposure has been demonstrated. Since OPT-induced neurodevelopmental effects may be due to in situ bioactivation by foetal enzymes, the catalytic activity of the foetal CYP3A7 toward chlorpyrifos (CPF), parathion (PAR), malathion (MAL) and fenthion (FEN) has been assessed by using recombinant enzymes. A comparison with the adult isoforms CYP3A4 and CYP3A5 has been also carried out. CYP3A7 was able to produce significant levels of oxon or sulfoxide from the four OPTs in the range of tested concentrations (0.05-200 microM). When the efficiencies of CYP3A isoforms were compared, the ranking, expressed as CLi values, were: CPF=3A4>3A5>3A7; PAR=3A4>3A7>3A5; MAL=3A4>3A7>3A5; FEN (sulfoxide formation)=3A4>3A5>3A7. The CYP3A5 efficiency appeared to be more dependent on the single insecticide than its related isozyme CYP3A4. Our results indicate that the levels of toxic metabolite formed in situ by CYP3A7 from CPF, MAL and PAR but not from FEN have the chance to inhibit acetylcholinesterase, following prenatal exposure to OPTs. However, due to the smaller weight of foetal liver, the contribution to total OPT biotransformation is relatively low. On the other hand, our results clearly indicate that at low CPF concentrations, the formation of the non-toxic metabolites is highly favoured in the foetus.

  8. Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

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    Helena Hernández

    2009-09-01

    Full Text Available Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

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

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    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

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

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    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.

  11. Isoforms of thyroxine-binding globulin as a model for molecular epidemiology of human cancer risk

    International Nuclear Information System (INIS)

    Golovaty, A.S.; Lapko, A.G.

    2000-01-01

    -binding globulin (TBG) by 50-60%, but no elevation in free T4, as compared with similar control groups of from Minsk. One of the reasons for the euthyroid syndrome in teenagers from the contaminated region was shown to be the blood isoforms in TBG. These isoforms differed in the structure of the carbohydrate components and as a rule are characterized by a prolonged circulation time. Using specific affinity chromatography, with subsequent immunological assay we shown that a certain amount of serum TBG-molecules in persons from Khojniki contains a high level of TBG (about 30% higher than the control group) containing fucosyalated biantennary sugar chains with more prolonged survival time. Previous findings showed that increased levels of fucosylation are observed for some serum glycoproteins in the blood of tumour patients. We provide evidence for variations in the TBGfuc which are contained in the serum TBG pool of patients suffering from cancer with various localisation. Thus, analysis of TBGfuc may be regarded as a prognostic marker in the determination of risk groups. The molecular mechanism of increased activity of fucosyl transferases will be discussed. We propose to measure the level of TBG fucosylated to assess the human cancer risk and habitants of contaminated region of Belarus. (authors)

  12. Effects of Andrographis paniculata and Orthosiphon stamineus extracts on the glucuronidation of 4-methylumbelliferone in human UGT isoforms.

    Science.gov (United States)

    Ismail, Sabariah; Hanapi, Nur Aziah; Ab Halim, Mohd Rohaimi; Uchaipichat, Verawan; Mackenzie, Peter I

    2010-05-14

    The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU) by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA) as the cofactor, MgCl(2), cell lysate of respective isoform, and 4MU at the approximate apparent K(m) or S(50) value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 microg/mL and 0.01, 0.10, 1.0, 10 and 50 microg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC(50) 1.70 microg/mL for UGT1A3, 2.57 microg/mL for UGT1A8, 2.82 microg/mL for UGT2B7, 5.00 micorg/mL for UGT1A1, 5.66 microg/mL for UGT1A6, 9.88 microg/mL for UGT1A7 and 15.66 microg/mL for UGT1A10). Both extracts showed less than 70% inhibition of UGT2B15, so the IC(50) values were >50 microg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

  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. FGF2 is an alternative translation product expressed as different isoforms termed LMW and HMW. FGF2 is involved in melanoma development and progression. HMW FGF2 isoforms enhance in vitro motility of melanoma cells. LMW FGF2 confers stem-like features and increases in vivo metastasization. LMW FGF2 promotes angiogenesis while HMW FGF2 induces vasculogenic mimicry.

  14. 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.

    2018-01-01

    Objective 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. Method 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. Results 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. Conclusions

  15. Dual roles for CoAA and its counterbalancing isoform CoAM in human kidney cell tumorigenesis

    Science.gov (United States)

    Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y.; Tsai, Ming-Jer; W. O’Malley, Bert

    2008-01-01

    Co-Activator Activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein we show that CoAA is a dual-function coregulator that inhibits G1/S transition in human kidney cells and suppresses anchorage independent growth and xenograft tumor formation. Suppression occurs in part by downregulating c-myc and its downstream effectors ccnd1 and skp2, and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene, c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, Coactivator Modulator (CoAM), antagonizes CoAA-induced G1/S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma as compared to normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice-isoform. This is so far the only example of a nuclear receptor coregulator involved in suppression of kidney cancer, and suggests potentially significant new roles for coregulators in renal cancer biology. PMID:18829545

  16. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    Science.gov (United States)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  17. 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

    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...

  18. A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

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    Ioannis Gavvovidis

    Full Text Available Biallelic mutations in MCPH1 cause primary microcephaly (MCPH with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL and a second transcript lacking the six 3' exons (MCPH1Δe9-14. Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.

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

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    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

  20. Effects of Andrographis paniculata and Orthosiphon stamineus Extracts on the Glucuronidation of 4-Methylumbelliferone in Human UGT Isoforms

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    Sabariah Ismail

    2010-05-01

    Full Text Available The effects of Andrographis paniculata and Orthosiphon stamineus extracts on the in vitro glucuronidation of 4-methylumbelliferone (4MU by recombinant human UGTs, UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7 and UGT2B15 were determined. The potential inhibitory effects of both of the extracts on the activity of each of the UGT isoforms were investigated using 4MU as the substrate. Incubations contained UDP-glucuronic acid (UDPGA as the cofactor, MgCl2, cell lysate of respective isoform, and 4MU at the approximate apparent Km or S50 value of each isoform. Final concentrations of Andrographis paniculata and Orthosiphon stamineus extracts used were 0.025, 0.25, 2.5, 25 and 50 μg/mL and 0.01, 0.10, 1.0, 10 and 50 μg/mL respectively. Both extracts variably inhibited the activity of most of the isoforms in a concentration dependent manner. Andrographis paniculata extract was the better inhibitor of all the isoforms studied (IC50 1.70 μg/mL for UGT1A3, 2.57 μg/mL for UGT1A8, 2.82 μg/mL for UGT2B7, 5.00 μg/mL for UGT1A1, 5.66 μg/mL for UGT1A6, 9.88 μg/mL for UGT1A7 and 15.66 μg/mL for UGT1A10. Both extracts showed less than 70% inhibition of UGT2B15, so the IC50 values were >50μg/mL. The inhibition of human UGTs by Andrographis paniculata and Orthosiphon stamineus extracts in vitro suggests a potential for drug-herbal extract interactions in the therapeutic setting.

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

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    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.

  2. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

    DEFF Research Database (Denmark)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien

    2012-01-01

    site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial...... diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites...... and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain...

  3. Smoking specifically induces metallothionein-2 isoform in human placenta at term

    International Nuclear Information System (INIS)

    Ronco, Ana Maria; Garrido, Fernando; Llanos, Miguel N.

    2006-01-01

    Recently, we reported the presence of higher levels of metallothionein (MT) in placentas of smokers compared to non-smokers. In the present study, we designed experiments to separate and evaluate two isoforms of MT (MT-1 and MT-2) in placentas of smokers and non-smokers. Metallothionein was extracted and separated by ion-exchange high performance liquid chromatography (HPLC), previous saturation with cadmium chloride. Two peaks eluting at 6 and 12.5 min, corresponding to MT-1 and MT-2, respectively, were obtained. Metallothionein present in both peaks was identified by Western blot analysis using a monoclonal antibody directed against MT-1 and MT-2. Each isoform concentration was calculated after measuring its cadmium content by atomic absorption spectrometry with inductively coupled-plasma. In placentas of smokers, MT-2 levels increased by seven-fold compared to non-smokers, whereas MT-1 was not changed. Total placental cadmium and zinc concentrations, determined by atomic absorption spectrometry and neutron activation analysis, respectively, were higher in smokers. Metallothioneins levels were clearly in excess to bind all cadmium ions present in placentas. However, most of placental zinc remains unbound to MTs, although as much as twice zinc ions could be bound to MT in smokers. In conclusion, MT-2 is the main isoform induced by smoking, suggesting that this isoform could be involved in placental cadmium and zinc retention. This fact, which could contribute to reduce the transference of zinc to the fetus, may be associated to detrimental effects on fetal growth and development

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

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    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.

  5. Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms

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    Jasleen K. Sodhi

    2017-07-01

    Full Text Available In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM fortified with β-nicotinamide adenine dinucleotide phosphate (NADPH, resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A. Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM. Three trapping agents (dimedone, N-acetyl-lysine and arsenite did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine and trapping agent (NEM were chosen for further studies. For the same amount of ticlopidine (1 μM, increasing concentrations of the trapping agent NEM (0.007–40 mM resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM, due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

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

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    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.

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

    DEFF Research Database (Denmark)

    Nissen, Jakob D; Lykke, Kasper; Bryk, Jaroslaw

    2017-01-01

    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...

  8. Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat

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    Jin Hong

    2009-12-01

    Full Text Available Abstract Background The dystrophin glycoprotein complex is disrupted in Duchenne muscular dystrophy and many other neuromuscular diseases. The principal heterodimeric partner of dystrophin at the heart of the dystrophin glycoprotein complex in the main clinically affected tissues (skeletal muscle, heart and brain is its distant relative, α-dystrobrevin. The α-dystrobrevin gene is subject to complex transcriptional and post-transcriptional regulation, generating a substantial range of isoforms by alternative promoter use, alternative polyadenylation and alternative splicing. The choice of isoform is understood, amongst other things, to determine the stoichiometry of syntrophins (and their ligands in the dystrophin glycoprotein complex. Results We show here that, contrary to the literature, most α-dystrobrevin genes, including that of humans, encode three distinct syntrophin-binding sites, rather than two, resulting in a greatly enhanced isoform repertoire. We compare in detail the quantitative tissue-specific expression pattern of human and mouse α-dystrobrevin isoforms, and show that two major gene features (the novel syntrophin-binding site-encoding exon and the internal promoter and first exon of brain-specific isoforms α-dystrobrevin-4 and -5 are present in most mammals but specifically ablated in mouse and rat. Conclusion Lineage-specific mutations in the murids mean that the mouse brain has fewer than half of the α-dystrobrevin isoforms found in the human brain. Our finding that there are likely to be fundamental functional differences between the α-dystrobrevins (and therefore the dystrophin glycoprotein complexes of mice and humans raises questions about the current use of the mouse as the principal model animal for studying Duchenne muscular dystrophy and other related disorders, especially the neurological aspects thereof.

  9. Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

    Science.gov (United States)

    Zoraghi, Roya; Campbell, Sara; Kim, Catrina; Dullaghan, Edie M; Blair, Lachlan M; Gillard, Rachel M; Reiner, Neil E; Sperry, Jonathan

    2014-11-01

    Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. 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

    Full Text Available The involvement of cholinergic system and the reactive oxygen species (ROS in the pathogenesis of some degenerative diseases has been widely reported; however, the specific impact of hydrogen peroxide (H2O2 on the acetylcholinesterase (AChE activity as well as AChE isoform levels has not been clearly established. Hence, the purpose of present study is to clarify whether H2O2 alters these parameters.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. Keywords: Acetylcholinesterase, Hydrogen peroxide, Alternative splicing, Cell culture, Cell death

  12. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

    Science.gov (United States)

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

    2012-05-01

    Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

  13. Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.

    Science.gov (United States)

    Jiang, Wenting; Liu, Liang; Chen, Yun

    2018-03-06

    Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.

  14. Biochemical characterization of individual human glycosylated pro-insulin-like growth factor (IGF)-II and big-IGF-II isoforms associated with cancer.

    Science.gov (United States)

    Greenall, Sameer A; Bentley, John D; Pearce, Lesley A; Scoble, Judith A; Sparrow, Lindsay G; Bartone, Nicola A; Xiao, Xiaowen; Baxter, Robert C; Cosgrove, Leah J; Adams, Timothy E

    2013-01-04

    Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.

  15. Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

    Science.gov (United States)

    Greenall, Sameer A.; Bentley, John D.; Pearce, Lesley A.; Scoble, Judith A.; Sparrow, Lindsay G.; Bartone, Nicola A.; Xiao, Xiaowen; Baxter, Robert C.; Cosgrove, Leah J.; Adams, Timothy E.

    2013-01-01

    Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling. PMID:23166326

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

    Science.gov (United States)

    Liang, Ping; Nair, Jayakumar R; Song, Lei; McGuire, John J; Dolnick, Bruce J

    2005-01-01

    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. PMID:16162288

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. Enantioselective N-demethylation and hydroxylation of sibutramine in human liver microsomes and recombinant cytochrome p-450 isoforms.

    Science.gov (United States)

    Shinde, Dhananjay D; Kim, Min-Jung; Jeong, Eun-Sook; Kim, Yang-Weon; Lee, Ji-Woo; Shin, Jae-Gook; Kim, Dong-Hyun

    2014-01-01

    The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Clint) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Clint ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Clint ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.

  20. To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms.

    Science.gov (United States)

    Duque, Harry Morales; Mourão, Caroline Barbosa Farias; Tibery, Diogo Vieira; Barbosa, Eder Alves; Campos, Leandro Ambrósio; Schwartz, Elisabeth Ferroni

    2017-09-01

    Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNa v 1.1-hNa v 1.7) at low concentrations, and has more pronounced activity on hNa v 1.1, hNa v 1.2 and hNa v 1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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    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.

  2. Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Whitehead, Lewis; Dobler, Markus R.; Radetich, Branko; Zhu, Yanyi; Atadja, Peter W.; Claiborne, Tavina; Grob, Jonathan E.; McRiner, Andrew; Pancost, Margaret R.; Patnaik, Anup; Shao, Wenlin; Shultz, Michael; Tichkule, Ritesh; Tommasi, Ruben A.; Vash, Brian; Wang, Ping; Stams, Travis (Novartis)

    2013-11-20

    Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhibitors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through novel structural features revealed at the atomic level.

  3. Genes influenced by the non-muscle isoform of Myosin light chain kinase impact human cancer prognosis.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome-wide expression in wild type and nmMLCK knockout (KO mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature (GeneCards definition and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001, colon cancer (P<0.001, glioma (P<0.001, and lung cancer (P<0.001. In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002, colon cancer (P = 0.035, glioma (P = 0.023, and lung cancer (P = 0.023. The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.

  4. Effect of resistance exercise intensity on the expression of PGC-1α isoforms and the anabolic and catabolic signaling mediators, IGF-1 and myostatin, in human skeletal muscle.

    Science.gov (United States)

    Schwarz, Neil A; McKinley-Barnard, Sarah K; Spillane, Mike B; Andre, Thomas L; Gann, Joshua J; Willoughby, Darryn S

    2016-08-01

    The purpose of this study was to investigate the acute messenger (mRNA) expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) isoforms, insulin-like growth factor-1Ea (IGF-1Ea), and myostatin in response to 2 resistance exercise intensities. In a uniform-balanced, crossover design, 10 participants performed 2 separate testing sessions involving a lower body resistance exercise component consisting of a lower intensity (50% of 1-repetition maximum; 1RM) protocol and a higher intensity (80% of 1RM) protocol of equal volumes. Muscle samples were obtained at before exercise, 45 min, 3 h, 24 h, and 48 h postexercise. Resistance exercise did not alter total PGC-1α mRNA expression; however, distinct responses of each PGC-1α isoform were observed. The response of each isoform was consistent between sessions, suggesting no effect of resistance exercise intensity on the complex transcriptional expression of the PGC-1α gene. IGF-1Ea mRNA expression significantly increased following the higher intensity session compared with pre-exercise and the lower intensity session. Myostatin mRNA expression was significantly reduced compared with pre-exercise values at all time points with no difference between exercise intensity. Further research is needed to determine the effects of the various isoforms of PGC-1α in human skeletal muscle on the translational level as well as their relation to the expression of IGF-1 and myostatin.

  5. The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

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    Deepshi Thakral

    Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

  6. 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.

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

    International Nuclear Information System (INIS)

    Louis, Simon N.S.; Chow, Laurie T.C.; Varghayee, Naghmeh; Rezmann, Linda A.; Frauman, Albert G.; Louis, William J.

    2011-01-01

    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 2 -) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT 2 -receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT 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 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

  8. Opposite Effects of Two Human ATG10 Isoforms on Replication of a HCV Sub-genomic Replicon Are Mediated via Regulating Autophagy Flux in Zebrafish

    Directory of Open Access Journals (Sweden)

    Yu-Chen Li

    2018-04-01

    Full Text Available Autophagy is a host mechanism for cellular homeostatic control. Intracellular stresses are symptoms of, and responses to, dysregulation of the physiological environment of the cell. Alternative gene transcription splicing is a mechanism potentially used by a host to respond to physiological or pathological challenges. Here, we aimed to confirm opposite effects of two isoforms of the human autophagy-related protein ATG10 on an HCV subgenomic replicon in zebrafish. A liver-specific HCV subreplicon model was established and exhibited several changes in gene expression typically induced by HCV infection, including overexpression of several HCV-dependent genes (argsyn, leugpcr, rasgbd, and scaf-2, as well as overexpression of several ER stress related genes (atf4, chop, atf6, and bip. Autophagy flux was blocked in the HCV model. Our results indicated that the replication of the HCV subreplicon was suppressed via a decrease in autophagosome formation caused by the autophagy inhibitor 3MA, but enhanced via dysfunction in the lysosomal degradation caused by another autophagy inhibitor CQ. Human ATG10, a canonical isoform in autophagy, facilitated the amplification of the HCV-subgenomic replicon via promoting autophagosome formation. ATG10S, a non-canonical short isoform of the ATG10 protein, promoted autophagy flux, leading to lysosomal degradation of the HCV-subgenomic replicon. Human ATG10S may therefore inhibit HCV replication, and may be an appropriate target for future antiviral drug screening.

  9. Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms.

    Science.gov (United States)

    Brill, Shirley S; Furimsky, Anna M; Ho, Mark N; Furniss, Michael J; Li, Yi; Green, Adam G; Bradford, Wallace W; Green, Carol E; Kapetanovic, Izet M; Iyer, Lalitha V

    2006-04-01

    Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.

  10. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms

    Directory of Open Access Journals (Sweden)

    Bogdan Iorga

    2018-01-01

    Full Text Available Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of β-myosin heavy chain (βMyHC isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs differentiated in vitro resemble those of human ventricular myofibrils (hvMFs isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s−1, reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa than for hvMFs (94 kPa. At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04 than for hvMFs (pCa50 = 5.80. At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s−1 than for hvMFs (0.28 s−1. During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s−1 than for hvMFs (0.21 s−1, while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins

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

    Science.gov (United States)

    He, Xian-hui; Xu, Li-hui; Liu, Yi

    2005-04-01

    To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). 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. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? 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 intracellular 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. 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.

  13. Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

    Science.gov (United States)

    Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

    2015-01-01

    The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  14. A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform.

    Science.gov (United States)

    Hirz, Melanie; Richter, Gerald; Leitner, Erich; Wriessnegger, Tamara; Pichler, Harald

    2013-11-01

    The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.

  15. 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.

  16. 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.

  17. 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

    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...... of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant...

  18. The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

    Science.gov (United States)

    van Keimpema, Martine; Grüneberg, Leonie J; Schilder-Tol, Esther J M; Oud, Monique E C M; Beuling, Esther A; Hensbergen, Paul J; de Jong, Johann; Pals, Steven T; Spaargaren, Marcel

    2017-03-01

    The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients. Copyright© Ferrata Storti Foundation.

  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 CO 2 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 CO 2 , 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. © 2016 Wiley Periodicals, Inc.

  20. 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.

  1. A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

    Science.gov (United States)

    Balbi, Pietro; Massobrio, Paolo; Hellgren Kotaleski, Jeanette

    2017-09-01

    Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The

  2. The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

    International Nuclear Information System (INIS)

    Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

    2008-01-01

    Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

  3. The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450.

    Science.gov (United States)

    Ho, Shirley H Y; Singh, Mohini; Holloway, Alison C; Crankshaw, Denis J

    2011-07-01

    The study set out to determine the potential for commercially available preparations of black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus) and false unicorn (Chamaelirium luteum) to inhibit the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 as well as CYP1A1 which activates some carcinogens. In vitro microplate-based assays using cDNA-expressed CYP450 isoforms and fluorogenic substrates were used. Components of the commercial herbal preparations interfered with the assays and limited the concentration ranges that could be tested. Nevertheless, the fluorogenic assays were robust, reproducible and easy to perform and thus are still useful for initial screening for potential herb-drug interactions. None of the preparations affected CYPs 1A1 or 2C9 at the concentrations tested but all preparations inhibited some of the enzymes with potencies around 1 μg/mL. The three most potent interactions were: chaste tree berry and CYP2C19 (IC₅₀) 0.22 μg/mL); chaste tree berry and CYP3A4 (IC₅₀) 0.3 μg/mL); black cohosh and CYP2C19 (IC₅₀) 0.37 μg/mL,). Thus, the study successfully identified the potential for the commercial herbal preparations to inhibit human drug metabolizing enzymes. Whether this potential translates into clinically significant herb-drug interactions can only be confirmed by appropriate in vivo studies. Copyright © 2011 John Wiley & Sons, Ltd.

  4. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

    Science.gov (United States)

    Izuogu, Osagie G; Alhasan, Abd A; Mellough, Carla; Collin, Joseph; Gallon, Richard; Hyslop, Jonathon; Mastrorosa, Francesco K; Ehrmann, Ingrid; Lako, Majlinda; Elliott, David J; Santibanez-Koref, Mauro; Jackson, Michael S

    2018-04-20

    Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

  5. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, Sep 18 (2015), s. 223-232 ISSN 0223-5234 Institutional support: RVO:61388963 Keywords : human carbonic anhydrase * isoquinoline * quinoline * X-ray * molecular docking Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2015

  6. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, SEP 18 (2015), s. 223-232 ISSN 0223-5234 R&D Projects: GA ČR GA15-05677S Grant - others:Fondo di Ateneo per la Ricerca (PRA)(IT) ORME09SPNC Institutional support: RVO:68378050 Keywords : Human carbonic anhydrase * Isoquinoline * Quinoline * X-ray * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.902, year: 2015

  7. 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.

  8. 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

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

    DEFF Research Database (Denmark)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte

    2006-01-01

    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...... 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...

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

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    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

  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

    Objective 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. Methods 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. Results 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±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the

  13. 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.

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    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.

  14. Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

    Science.gov (United States)

    Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

    1998-01-01

    Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

  15. Human ApoE Isoforms Differentially Modulate Glucose and Amyloid Metabolic Pathways in Female Brain: Evidence of the Mechanism of Neuroprotection by ApoE2 and Implications for Alzheimer's Disease Prevention and Early Intervention.

    Science.gov (United States)

    Keeney, Jeriel Thomas-Richard; Ibrahimi, Shaher; Zhao, Liqin

    2015-01-01

    Three major genetic isoforms of apolipoprotein E (ApoE), ApoE2, ApoE3, and ApoE4, exist in humans and lead to differences in susceptibility to Alzheimer's disease (AD). This study investigated the impact of human ApoE isoforms on brain metabolic pathways involved in glucose utilization and amyloid-β (Aβ) degradation, two major areas that are significantly perturbed in preclinical AD. Hippocampal RNA samples from middle-aged female mice with targeted human ApoE2, ApoE3, and ApoE4 gene replacement were comparatively analyzed with a qRT-PCR custom array for the expression of 85 genes involved in insulin/insulin-like growth factor (Igf) signaling. Consistent with its protective role against AD, ApoE2 brain exhibited the most metabolically robust profile among the three ApoE genotypes. When compared to ApoE2 brain, both ApoE3 and ApoE4 brains exhibited markedly reduced levels of Igf1, insulin receptor substrates (Irs), and facilitated glucose transporter 4 (Glut4), indicating reduced glucose uptake. Additionally, ApoE4 brain exhibited significantly decreased Pparg and insulin-degrading enzyme (Ide), indicating further compromised glucose metabolism and Aβ dysregulation associated with ApoE4. Protein analysis showed significantly decreased Igf1, Irs, and Glut4 in ApoE3 brain, and Igf1, Irs, Glut4, Pparg, and Ide in ApoE4 brain compared to ApoE2 brain. These data provide the first documented evidence that human ApoE isoforms differentially affect brain insulin/Igf signaling and downstream glucose and amyloid metabolic pathways, illustrating a potential mechanism for their differential risk in AD. A therapeutic strategy that enhances brain insulin/Igf1 signaling activity to a more robust ApoE2-like phenotype favoring both energy production and amyloid homeostasis holds promise for AD prevention and early intervention.

  16. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

    Science.gov (United States)

    Wisniewska, Magdalena; Karlberg, Tobias; Lehtiö, Lari; Johansson, Ida; Kotenyova, Tetyana; Moche, Martin; Schüler, Herwig

    2010-01-11

    The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  17. Crystal structures of the ATPase domains of four human Hsp70 isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78.

    Directory of Open Access Journals (Sweden)

    Magdalena Wisniewska

    2010-01-01

    Full Text Available The 70-kDa heat shock proteins (Hsp70 are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD with ATPase activity, and a C-terminal substrate binding domain (SBD. We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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 of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3β, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of β-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 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 G 0 /G 1 phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells

  19. Characterisation of Cdkl5 transcript isoforms in rat.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Ritakari, Tuula E; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2017-03-01

    CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically and experimentally, the rat Cdkl5 gene structure and its associated transcript isoforms. New exonic regions, splice sites and UTRs are described, confirming the presence of four distinct transcript isoforms. The predominant isoform in the brain, which we name rCdkl5_1, is orthologous to the human hCDKL5_1 and mouse mCdkl5_1 isoforms and is the most highly expressed isoform across all brain regions tested. This updated gene model of Cdkl5 in rat provides a framework for studies into its protein products and provides a reference for the development of molecular therapies for testing in rat models of CDKL5 disorder. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Human cytochrome P450 and personalized medicine.

    Science.gov (United States)

    Chen, Qi; Wei, Dongqing

    2015-01-01

    Personalized medicine has become a hot topic ascribed to the development of Human Genome Project. And currently, bioinformatics methodology plays an essential role in personal drug design. Here in this review we mainly focused on the basic introduction of the SNPs of human drug metabolic enzymes and their relationships with personalized medicine. Some common bioinformatics analysis methods and latest progresses and applications in personal drug design have also been discussed. Thus bioinformatics studies on SNPs of human CYP450 genes will contribute to indicate the most possible genes that are associated with human diseases and relevant therapeutic targets, identify and predict the drug efficacy and adverse drug response, investigate individual gene specific properties and then provide personalized and optimal clinic therapies.

  1. Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

    OpenAIRE

    Huang, Zhiquan; Tan, Ning; Guo, Weijie; Wang, Lili; Li, Haigang; Zhang, Tianyu; Liu, Xiaojia; Xu, Qin; Li, Jinsong; Guo, Zhongmin

    2014-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and n...

  2. Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

    Science.gov (United States)

    Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J; Zhou, Lixin; Nguyen, Nhan L T; Lokareddy, Ravi K; Cingolani, Gino; Panté, Nelly

    2017-09-12

    The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.

  3. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    International Nuclear Information System (INIS)

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-01-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility

  4. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  5. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    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.

  6. In silico prediction of potential chemical reactions mediated by human enzymes.

    Science.gov (United States)

    Yu, Myeong-Sang; Lee, Hyang-Mi; Park, Aaron; Park, Chungoo; Ceong, Hyithaek; Rhee, Ki-Hyeong; Na, Dokyun

    2018-06-13

    Administered drugs are often converted into an ineffective or activated form by enzymes in our body. Conventional in silico prediction approaches focused on therapeutically important enzymes such as CYP450. However, there are more than thousands of different cellular enzymes that potentially convert administered drug into other forms. We developed an in silico model to predict which of human enzymes including metabolic enzymes as well as CYP450 family can catalyze a given chemical compound. The prediction is based on the chemical and physical similarity between known enzyme substrates and a query chemical compound. Our in silico model was developed using multiple linear regression and the model showed high performance (AUC = 0.896) despite of the large number of enzymes. When evaluated on a test dataset, it also showed significantly high performance (AUC = 0.746). Interestingly, evaluation with literature data showed that our model can be used to predict not only enzymatic reactions but also drug conversion and enzyme inhibition. Our model was able to predict enzymatic reactions of a query molecule with a high accuracy. This may foster to discover new metabolic routes and to accelerate the computational development of drug candidates by enabling the prediction of the potential conversion of administered drugs into active or inactive forms.

  7. Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences

    Energy Technology Data Exchange (ETDEWEB)

    Iwanari, M.; Nakajima, M.; Yokoi, T. [Div. of Drug Metabolism, Kanazawa Univ., Kanazawa (Japan); Kizu, R.; Hayakawa, K. [Lab. of Hygienic Chemistry, Kanazawa Univ., Kanazawa (Japan)

    2002-06-01

    Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene were weaker than those of their parents benzo[a]pyrene and benz[a]anthracene, respectively. This

  8. Antimicrobial actions of the human epididymis 2 (HE2 protein isoforms, HE2alpha, HE2beta1 and HE2beta2

    Directory of Open Access Journals (Sweden)

    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

  9. 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.

  10. Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology

    Directory of Open Access Journals (Sweden)

    Michael G. Harrington

    2006-01-01

    Full Text Available Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

  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. Modulation of neuronal differentiation by CD40 isoforms

    International Nuclear Information System (INIS)

    Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

    2008-01-01

    Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40 -/- deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40 -/- mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may represent

  13. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-01-01

    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 m...... 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...

  15. 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. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.

    Science.gov (United States)

    Halling Linder, Cecilia; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

    2009-11-01

    Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP

  17. 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...

  18. Plectin isoforms as organizers of intermediate filament cytoarchitecture.

    Science.gov (United States)

    Wiche, Gerhard; Winter, Lilli

    2011-01-01

    Intermediate filaments (IFs) form cytoplamic and nuclear networks that provide cells with mechanical strength. Perturbation of this structural support causes cell and tissue fragility and accounts for a number of human genetic diseases. In recent years, important additional roles, nonmechanical in nature, were ascribed to IFs, including regulation of signaling pathways that control survival and growth of the cells, and vectorial processes such as protein targeting in polarized cellular settings. The cytolinker protein plectin anchors IF networks to junctional complexes, the nuclear envelope and cytoplasmic organelles and it mediates their cross talk with the actin and tubulin cytoskeleton. These functions empower plectin to wield significant influence over IF network cytoarchitecture. Moreover, the unusual diversity of plectin isoforms with different N termini and a common IF-binding (C-terminal) domain enables these isoforms to specifically associate with and thereby bridge IF networks to distinct cellular structures. Here we review the evidence for IF cytoarchitecture being controlled by specific plectin isoforms in different cell systems, including fibroblasts, endothelial cells, lens fibers, lymphocytes, myocytes, keratinocytes, neurons and astrocytes, and discuss what impact the absence of these isoforms has on IF cytoarchitecture-dependent cellular functions.

  19. Regulation of cardiac remodeling by cardiac Na/K-ATPase isoforms

    Directory of Open Access Journals (Sweden)

    Lijun Catherine Liu

    2016-09-01

    Full Text Available Cardiac remodeling occurs after cardiac pressure/volume overload or myocardial injury during the development of heart failure and is a determinant of heart failure. Preventing or reversing remodeling is a goal of heart failure therapy. Human cardiomyocyte Na+/K+-ATPase has multiple α isoforms (1-3. The expression of the α subunit of the Na+/K+-ATPase is often altered in hypertrophic and failing hearts. The mechanisms are unclear. There are limited data from human cardiomyocytes. Abundant evidences from rodents show that Na+/K+-ATPase regulates cardiac contractility, cell signaling, hypertrophy and fibrosis. The α1 isoform of the Na+/K+-ATPase is the ubiquitous isoform and possesses both pumping and signaling functions. The α2 isoform of the Na+/K+-ATPase regulates intracellular Ca2+ signaling, contractility and pathological hypertrophy. The α3 isoform of the Na+/K+-ATPase may also be a target for cardiac hypertrophy. Restoration of cardiac Na+/K+-ATPase expression may be an effective approach for prevention of cardiac remodeling. In this article, we will overview: (1 the distribution and function of isoform specific Na+/K+-ATPase in the cardiomyocytes. (2 the role of cardiac Na+/K+-ATPase in the regulation of cell signaling, contractility, cardiac hypertrophy and fibrosis in vitro and in vivo. Selective targeting of cardiac Na+/K+-ATPase isoform may offer a new target for the prevention of cardiac remodeling.

  20. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

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    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.

  1. Complex p63 mRNA isoform expression patterns in squamous cell carcinoma of the head and neck

    DEFF Research Database (Denmark)

    Thurfjell, N.; Coates, P.J.; Uusitalo, T.

    2004-01-01

    on the role of p63 expression in human tumours, we used quantitative real-time RT-PCR to study individual p63 isoforms in squamous cell carcinomas of the head and neck (SCCHN). In keeping with previous reports, expression of the deltaN- and p63alpha-isoforms predominated and deltaNp63 mRNA was expressed...

  2. The Related Transcriptional Enhancer Factor-1 Isoform, TEAD4216, Can Repress Vascular Endothelial Growth Factor Expression in Mammalian Cells

    Science.gov (United States)

    Appukuttan, Binoy; McFarland, Trevor J.; Stempel, Andrew; Kassem, Jean B.; Hartzell, Matthew; Zhang, Yi; Bond, Derek; West, Kelsey; Wilson, Reid; Stout, Andrew; Pan, Yuzhen; Ilias, Hoda; Robertson, Kathryn; Klein, Michael L.; Wilson, David; Smith, Justine R.; Stout, J. Timothy

    2012-01-01

    Increased cellular production of vascular endothelial growth factor (VEGF) is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4) protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4216, which represses VEGF promoter activity. The TEAD4216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE), which is the sequence critical to hypoxia inducible factor (HIF)-mediated effects. The TEAD4216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4216 isoform can competitively repress the stimulatory activity of the TEAD4434 and TEAD4148 enhancers. Synthesis of the native VEGF165 protein and cellular proliferation is suppressed by the TEAD4216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases. PMID:22761647

  3. The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Feng Li

    2018-05-01

    Full Text Available Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM using metabolomic approaches. In human liver microsomes (HLM, we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

  4. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

    DEFF Research Database (Denmark)

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T

    2014-01-01

    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......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...

  5. Proliferation marker pKi-67 occurs in different isoforms with various cellular effects.

    Science.gov (United States)

    Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Finniss, Susan; Bögler, Oliver; Duchrow, Michael

    2004-04-15

    The Ki-67 antigen, pKi-67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi-67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi-67 N-terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi-67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi-67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi-67 N-terminus is differentially spliced resulting in at least five different isoforms with different functions. Copyright 2004 Wiley-Liss, Inc.

  6. Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

    Science.gov (United States)

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

  7. CYP450 genotype and aggressive behavior on selective serotonin reuptake inhibitors

    NARCIS (Netherlands)

    Ekhart, Corine; Matic, Maja; Kant, Agnes; Schaik, Ron van; van Puijenbroek, Eugène

    2017-01-01

    AIM: Genetic variants for selective serotonin reuptake inhibitor (SSRI) metabolizing enzymes have been hypothesized to be a risk factor for aggression as adverse drug effect of SSRIs. Our aim was to assess the possible involvement of these polymorphisms on aggression when using SSRIs. MATERIALS &

  8. Effect of Fruit/Vegetable-Drug Interactions on CYP450, OATP and p ...

    African Journals Online (AJOL)

    Purpose: To review the concomitant use of certain drugs with fruit/vegetable juices that may lead to drug-juice interactions resulting in medication-related problems. Method: In this systematic review, online databases (PubMed, Google Scholar and Science Direct) were searched for information on juices derived from fruits ...

  9. Mammary Gland Tumor Development in Transgenic Mice Overexpressing Different Isoforms of the CDP/Cux Transcription Factor

    National Research Council Canada - National Science Library

    Cadieux, Chantal

    2008-01-01

    Short CUX1 isoforms were found to be overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas, suggesting that these proteins play a key role in tumor development and progression...

  10. Mammary Gland Tumor Development in Transgenic Mice Overexpressing Different Isoforms of the CDP/Cux Transcription Factor

    National Research Council Canada - National Science Library

    Cadieux, Chantal

    2007-01-01

    Short CDP/Cux isoforms were found to be overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas, suggesting that these proteins play a key role in tumor development and progression...

  11. 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.

  12. A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

    DEFF Research Database (Denmark)

    Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

    2008-01-01

    Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined...

  13. Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

    International Nuclear Information System (INIS)

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois

    2005-01-01

    Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins

  14. Characterisation of Cdkl5transcript isoforms in rat

    OpenAIRE

    Hector, Ralph D.; Dando, Owen; Ritakari, Tuula E.; Kind, Peter C.; Bailey, Mark E.S.; Cobb, Stuart R.

    2017-01-01

    CDKL5 deficiency is a severe neurological disorder caused by mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5). The predominant human CDKL5 brain isoform is a 9.7kb transcript comprised of 18 exons with a large 6.6kb 3'-untranslated region (UTR). Mammalian models of CDKL5 disorder are currently limited to mouse, and little is known about Cdkl5 in other organisms used to model neurodevelopmental disorders, such as rat. In this study we characterise, both bioinformatically a...

  15. 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...... unique expression profiles and specialized functional features. We use a Two Electrode Voltage Clamp setup to determine pre-steady-state and steady-state characteristics of each isoform and design chimeras to pin-point the structural elements responsible for observed differences. With this strategy we...

  16. Discovery of novel isoforms of huntingtin reveals a new hominid-specific exon.

    Directory of Open Access Journals (Sweden)

    Albert Ruzo

    Full Text Available Huntington's disease (HD is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT. HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.

  17. Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

    Science.gov (United States)

    Popowski, Melissa; Haremaki, Tomomi; Croft, Gist F.; Deglincerti, Alessia; Brivanlou, Ali H.

    2015-01-01

    Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel HTT mRNA splice isoforms that are expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease. PMID:26010866

  18. P1 promoter-driven HNF4α isoforms are specifically repressed by β-catenin signaling in colorectal cancer cells.

    Science.gov (United States)

    Babeu, Jean-Philippe; Jones, Christine; Geha, Sameh; Carrier, Julie C; Boudreau, François

    2018-06-13

    HNF4α is a key nuclear receptor for regulating gene expression in the gut. While both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms may regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism while P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by β-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms are rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome thereby promoting colorectal cancer progression. © 2018. Published by The Company of Biologists Ltd.

  19. Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man

    NARCIS (Netherlands)

    Bosma, P. J.; Seppen, J.; Goldhoorn, B.; Bakker, C.; Oude Elferink, R. P.; Chowdhury, J. R.; Chowdhury, N. R.; Jansen, P. L.

    1994-01-01

    Crigler-Najjar syndrome type I (CN-I) is caused by an inherited absence of UDP-glucuronosyltransferase activity toward bilirubin (B-UGT), resulting in severe non-hemolytic unconjugated hyperbilirubinemia. Based on the expression of cDNAs in COS cells, two UGT isoforms in human liver, B-UGT1 and

  20. Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

    2010-07-23

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

  1. Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

    2010-01-01

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

  2. WT1 isoform expression pattern in acute myeloid leukemia.

    Science.gov (United States)

    Luna, Irene; Such, Esperanza; Cervera, Jose; Barragán, Eva; Ibañez, Mariam; Gómez-Seguí, Inés; López-Pavía, María; Llop, Marta; Fuster, Oscar; Dolz, Sandra; Oltra, Silvestre; Alonso, Carmen; Vera, Belén; Lorenzo, Ignacio; Martínez-Cuadrón, David; Montesinos, Pau; Senent, M Leonor; Moscardó, Federico; Bolufer, Pascual; Sanz, Miguel A

    2013-12-01

    WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. FoxP2 isoforms delineate spatiotemporal transcriptional networks for vocal learning in the zebra finch

    Science.gov (United States)

    Day, Nancy F; Kimball, Todd Haswell; Aamodt, Caitlin M; Heston, Jonathan B; Hilliard, Austin T; Xiao, Xinshu; White, Stephanie A

    2018-01-01

    Human speech is one of the few examples of vocal learning among mammals yet ~half of avian species exhibit this ability. Its neurogenetic basis is largely unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. We manipulated FoxP2 isoforms in Area X, a song-specific region of the avian striatopallidum analogous to human anterior striatum, during a critical period for song development. We delineate, for the first time, unique contributions of each isoform to vocal learning. Weighted gene coexpression network analysis of RNA-seq data revealed gene modules correlated to singing, learning, or vocal variability. Coexpression related to singing was found in juvenile and adult Area X whereas coexpression correlated to learning was unique to juveniles. The confluence of learning and singing coexpression in juvenile Area X may underscore molecular processes that drive vocal learning in young zebra finches and, by analogy, humans. PMID:29360038

  4. 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.

  5. Redundancy of IL-1 Isoform Signaling and Its Implications for Arterial Remodeling.

    Directory of Open Access Journals (Sweden)

    Marina Beltrami-Moreira

    Full Text Available Mice deficient in IL-1 receptor 1 (hence unresponsive to both IL-1 isoforms α and β have impaired expansive arterial remodeling due to diminished expression of matrix-degrading enzymes, especially MMP-3. Emergence of IL-1 as a target in cardiovascular disease prompted the investigation of the redundancy of IL-1α and IL-1β in the induction of MMP-3 and other matrix-remodeling enzymes in human cells.Human primary vascular smooth muscle cells (VSMCs and carotid endarterectomy specimens were stimulated with equimolar concentrations of IL-1α or IL-1β and analyzed protease expression by immunoblot and ELISA. Either IL-1α or IL-1β increased the expression of pro-MMP-3 in VSMCs, facilitated VSMC migration through Matrigel, and induced MMP-3 production in specimens from atheromatous plaques. VSMCs also secreted MMP-1 and Cathepsin S (CatS upon stimulation with IL-1α or IL-1β. IL-1 isoforms similarly increased MMP-1 and MMP-9 expression in carotid endarterectomy specimens. We examined the expression of MMP-3 and IL-1 isoforms by immunostaining of carotid atheromata, calculated the % positive areas, and tested associations by linear regression. MMP-3 colocalized with IL-1 isoforms in atheromata. MMP-3+ area in plaques positively associated with IL-1α+ (R2 = 0.61, P<0.001 and with IL-1β + areas (R2 = 0.68, P<0.001. MMP-3+ area within atheroma also associated with CD68+ area, but not with α-smooth muscle actin area.Either IL-1α or IL-1β can induce the expression of enzymes implicated in remodeling of the arterial extracellular matrix, and facilitate human VSMC migration in vitro. Human atheromata contain both IL-1 isoforms in association with immunoreactive MMP-3. This redundancy of IL-1 isoforms suggests that selective blocking of one IL-1 isoform should not impair expansive arterial remodeling, a finding with important clinical implications for therapeutic targeting of IL-1 in atherosclerosis.

  6. Frataxin mRNA Isoforms in FRDA Patients and Normal Subjects: Effect of Tocotrienol Supplementation

    Directory of Open Access Journals (Sweden)

    Provvidenza Maria Abruzzo

    2013-01-01

    Full Text Available Friedreich’s ataxia (FRDA is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3 in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor-γ (PPARG, PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role.

  7. Specific Profile of Tau Isoforms in Argyrophylic Grain Disease

    Directory of Open Access Journals (Sweden)

    Alberto Rábano

    2013-01-01

    Full Text Available Argyrophylic grain disease (AGD is a neurodegenerative condition that has been classified among the sporadic tauopathies. Entities in this group present intracellular aggregates of hyperphosphorylated tau, giving rise to characteristic neuronal and glial inclusions. In different tauopathies, the proportion of several tau isoforms present in the aggregates shows specific patterns. AGD has been tentatively classified in the 4R group (predominance of 4R tau isoforms together with progressive supranuclear palsy and corticobasal degeneration. Pick's disease is included in the 3R group (predominance of 3R isoforms, whereas tau pathology of Alzheimer's disease represents and intermediate group (3 or 4 repeats [3R plus 4R, respectively] isoforms. In this work, we have analyzed tau present in aggregates isolated from brain samples of patients with argyrophylic grain disease. Our results indicate that the main tau isoform present in aggregates obtained from patients with AGD is a hyperphosphorylated isoform containing exons 2 and 10 but lacking exon 3.

  8. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Sergey V., E-mail: Sergey.Ivanov@med.nyu.edu [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States); Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I. [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States)

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  9. 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.

  10. Oxygenation properties and isoform diversity of snake hemoglobins

    DEFF Research Database (Denmark)

    Storz, Jay F.; Natarajan, Chandrasekhar; Moriyama, Hideaki

    2015-01-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer- dimer dissociation. However, standardized comparative data are lacking fo...... isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform....

  11. A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

    DEFF Research Database (Denmark)

    Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

    2008-01-01

    Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics...

  12. Carcinogen biomonitoring in human exposures and laboratory research: validation and application to human occupational exposures.

    Science.gov (United States)

    Talaska, Glenn; Maier, Andrew; Henn, Scott; Booth-Jones, Angela; Tsuneoka, Yutaka; Vermeulen, Roel; Schumann, Brenda L

    2002-08-05

    A multiple biomarker approach is required to integrate for metabolism, temporal response and exposure-response kinetics, biological relevance, and positive predictive value. Carcinogen DNA adduct analysis can be used in animal and in vitro studies to detect absorption permutations caused by mixture interactions, and to control metabolic variation when specific CYP450 genes (1A1 or 1A2) are knocked out. These enzymes are not critical to the metabolic activation of model Polycyclic Aromatic Compounds (PAC) and aromatic amines, respectively, as suggested by in vitro analysis. Several human studies have been carried out where multiple biomarkers have been measured. In a study of benzidine workers, the similarities in elimination kinetics between urinary metabolites and mutagenicity is likely responsible for a better correlation between these markers than to BZ-DNA adducts in exfoliated cells. In a study of rubber workers, the relationship between specific departments, urinary 1 HP and DNA adducts in exfoliated cells coincided with the historical urinary bladder cancer risk in these departments; the same relationship did not hold for urinary mutagenicity. In a study of automotive mechanics, biomarkers were used to monitor the effectiveness of exposure interventions. These data reinforce the notion that carcinogen biomarkers are useful to monitor exposure, but that a complementary approaches involving effect and perhaps susceptibility biomarkers is necessary to obtain the necessary information.

  13. Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

    Science.gov (United States)

    Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

    2014-07-01

    p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. 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

  15. APPRIS 2017: principal isoforms for multiple gene sets

    Science.gov (United States)

    Rodriguez-Rivas, Juan; Di Domenico, Tomás; Vázquez, Jesús; Valencia, Alfonso

    2018-01-01

    Abstract The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding genes. APPRIS selects a single protein isoform, the ‘principal’ isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants. PMID:29069475

  16. Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1.

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    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

  17. In Vitro Inhibition of Human UDP-Glucuronosyl-Transferase (UGT Isoforms by Astaxanthin, β-Cryptoxanthin, Canthaxanthin, Lutein, and Zeaxanthin: Prediction of in Vivo Dietary Supplement-Drug Interactions

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    Yu Fen Zheng

    2016-08-01

    Full Text Available Despite the widespread use of the five major xanthophylls astaxanthin, β-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs. Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15 or recombinant supersomes (UGT2B15. We also predicted potential dietary supplement-drug interactions for β-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. β-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40 and 34.9 ± 5.98 μM, respectively. Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 μM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 μM, respectively. Among the tested xanthophyll-UGT pairs, β-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 (Ki, 12.2 ± 0.985 μM. In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of β-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls.

  18. In Vitro Inhibition of Human UDP-Glucuronosyl-Transferase (UGT) Isoforms by Astaxanthin, β-Cryptoxanthin, Canthaxanthin, Lutein, and Zeaxanthin: Prediction of in Vivo Dietary Supplement-Drug Interactions.

    Science.gov (United States)

    Zheng, Yu Fen; Min, Jee Sun; Kim, Doyun; Park, Jung Bae; Choi, Sung-Wook; Lee, Eun Seong; Na, Kun; Bae, Soo Kyung

    2016-08-12

    Despite the widespread use of the five major xanthophylls astaxanthin, β-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs). Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15) or recombinant supersomes (UGT2B15). We also predicted potential dietary supplement-drug interactions for β-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. β-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40 and 34.9 ± 5.98 μM, respectively. Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 μM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 μM, respectively. Among the tested xanthophyll-UGT pairs, β-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 (Ki, 12.2 ± 0.985 μM). In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of β-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls.

  19. 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.

  20. Neuronal glucose transporter isoform 3 deficient mice demonstrate features of autism spectrum disorders.

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    Zhao, Y; Fung, C; Shin, D; Shin, B-C; Thamotharan, S; Sankar, R; Ehninger, D; Silva, A; Devaskar, S U

    2010-03-01

    Neuronal glucose transporter (GLUT) isoform 3 deficiency in null heterozygous mice led to abnormal spatial learning and working memory but normal acquisition and retrieval during contextual conditioning, abnormal cognitive flexibility with intact gross motor ability, electroencephalographic seizures, perturbed social behavior with reduced vocalization and stereotypies at low frequency. This phenotypic expression is unique as it combines the neurobehavioral with the epileptiform characteristics of autism spectrum disorders. This clinical presentation occurred despite metabolic adaptations consisting of an increase in microvascular/glial GLUT1, neuronal GLUT8 and monocarboxylate transporter isoform 2 concentrations, with minimal to no change in brain glucose uptake but an increase in lactate uptake. Neuron-specific glucose deficiency has a negative impact on neurodevelopment interfering with functional competence. This is the first description of GLUT3 deficiency that forms a possible novel genetic mechanism for pervasive developmental disorders, such as the neuropsychiatric autism spectrum disorders, requiring further investigation in humans.

  1. The related transcriptional enhancer factor-1 isoform, TEAD4(216, can repress vascular endothelial growth factor expression in mammalian cells.

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    Binoy Appukuttan

    Full Text Available Increased cellular production of vascular endothelial growth factor (VEGF is responsible for the development and progression of multiple cancers and other neovascular conditions, and therapies targeting post-translational VEGF products are used in the treatment of these diseases. Development of methods to control and modify the transcription of the VEGF gene is an alternative approach that may have therapeutic potential. We have previously shown that isoforms of the transcriptional enhancer factor 1-related (TEAD4 protein can enhance the production of VEGF. In this study we describe a new TEAD4 isoform, TEAD4(216, which represses VEGF promoter activity. The TEAD4(216 isoform inhibits human VEGF promoter activity and does not require the presence of the hypoxia responsive element (HRE, which is the sequence critical to hypoxia inducible factor (HIF-mediated effects. The TEAD4(216 protein is localized to the cytoplasm, whereas the enhancer isoforms are found within the nucleus. The TEAD4(216 isoform can competitively repress the stimulatory activity of the TEAD4(434 and TEAD4(148 enhancers. Synthesis of the native VEGF(165 protein and cellular proliferation is suppressed by the TEAD4(216 isoform. Mutational analysis indicates that nuclear or cytoplasmic localization of any isoform determines whether it acts as an enhancer or repressor, respectively. The TEAD4(216 isoform appears to inhibit VEGF production independently of the HRE required activity by HIF, suggesting that this alternatively spliced isoform of TEAD4 may provide a novel approach to treat VEGF-dependent diseases.

  2. 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

  3. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

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    Francesca Rappa

    Full Text Available 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.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.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.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.

  4. 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

  5. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    International Nuclear Information System (INIS)

    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. Finally, I

  6. The Role of Akt Isoforms in Colorectal Cancer

    Science.gov (United States)

    2015-09-01

    AD_________________ Award Number: W81XWH-13-1-0198 TITLE: The Role of Akt Isoforms in Colorectal Cancer PRINCIPAL INVESTIGATOR: Jatin Roper...CONTRACT NUMBER The Role of Akt Isoforms in Colorectal Cancer 5b. GRANT NUMBER W81XWH-13-1-0198 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER...substantially reduces colorectal tumorigenesis in our genetically engineered mouse model. We also successfully ablated novel downstream targets of Akt in our

  7. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes

    DEFF Research Database (Denmark)

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B

    2015-01-01

    understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each...

  8. 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

  9. The Correlation between CYP450 Ile462Val Polymorphism and Prostate Cancer in a Group of Iranian Men

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    Maryam Seydali Route

    2016-04-01

    Full Text Available Background: Cytochrome P450 plays an important role in pharmaceutical metabolism, steroidal hormones and procarcinogens. CYP1A1 is an enzyme, which is very active in the formation of reactive mediators or injurious agents for DNA. The aim of this study is to evaluate the prevalence rate of genetic polymorphism RS 1048943 gene CYP1A1 in men diagnosed with prostate cancer compared to a control group of healthy men. Methods: This case-control study analysed blood samples from 79 patients with prostate cancer as well as 79 healthy men. Genomic DNA was extracted by the salting out method. After selecting the suitable primers from the papers, the samples were amplified for the considered segment and genotypes of the participants were determined by PCR-RFLP. Results: Individuals with prostate cancer had the following genotypes: AA (31.64%, GG (59.49% and AG/GA (8.86% compared to the control group that had genotypes AA (55.69%, GG (29.11% and AG/GA (15.18%. According to the Hardy–Weinberg equilibrium, the frequency of allele A was 36.7% in the cancer group and 63.29% in the control group. The frequency of allele G was 63.92% in the cancer group and 36.70% in the control group. There were meaningful differences in the frequencies of homozygotes GG (P<0.001 and AA (P=0.002 between patients and controls. Conclusion: Polymorphism RS 1048943 in gene CYP1A1 is related to the risk of developing prostate cancer and it is likely one of the major factors in its occurrence.

  10. CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine

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    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.

  11. CYP-450 isoenzymes catalyze the generation of hazardous aromatic amines after reaction with the azo dye Sudan III.

    Science.gov (United States)

    Zanoni, Thalita Boldrin; Lizier, Thiago M; Assis, Marilda das Dores; Zanoni, Maria Valnice B; de Oliveira, Danielle Palma

    2013-07-01

    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(-4)molL(-1) and generation of 7.6 × 10(-7)molL(-1) to 0.31 × 10(-4)molL(-1) of aniline, o-anisidine, 2-methoxi-5-methylaniline, 4-aminobiphenyl, 4,4'-oxydianiline; 4,4'-diaminodiphenylmethane and 2,6-dimethylaniline. The results were confirmed by LC-MS-MS experiments. We also correlate the mutagenic effects of Sudan III using S. typhimurium with the strain TA1535 in the presence of exogenous metabolic activation (S9) with the metabolization products of this compound. Our findings clearly indicate that aromatic amines are formed due to oxidative reactions that can be promoted by hepatic cells, after the ingestion of Sudan III. Considering that, the use of azo compounds as food dyestuffs should be carefully controlled. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. 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

  13. Identification of a cytochrome P450 gene in the earthworm Eisenia fetida and its mRNA expression under enrofloxacin stress.

    Science.gov (United States)

    Li, Yinsheng; Zhao, Chun; Lu, Xiaoxu; Ai, Xiaojie; Qiu, Jiangping

    2018-04-15

    Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477bp. The 3'-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500mgkg -1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

    Directory of Open Access Journals (Sweden)

    James E J Bedard

    Full Text Available Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

  15. In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

    Science.gov (United States)

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

  16. Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

    Science.gov (United States)

    Oudin, Madeleine J; Hughes, Shannon K; Rohani, Nazanin; Moufarrej, Mira N; Jones, Joan G; Condeelis, John S; Lauffenburger, Douglas A; Gertler, Frank B

    2016-03-01

    Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

  17. Differential Signature of the Centrosomal MARK4 Isoforms in Glioma

    Directory of Open Access Journals (Sweden)

    Ivana Magnani

    2011-01-01

    Full Text Available Background: MAP/microtubule affinity-regulating kinase 4 (MARK4 is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. Methods: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs and mouse neural stem cells (NSCs by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. Results: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. Conclusions: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.

  18. Expression of various sarcomeric tropomyosin isoforms in equine striated muscles

    Directory of Open Access Journals (Sweden)

    Syamalima Dube

    2017-06-01

    Full Text Available In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM, a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4 generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

  19. Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

    Science.gov (United States)

    Zempel, Hans; Dennissen, Frank J A; Kumar, Yatender; Luedtke, Julia; Biernat, Jacek; Mandelkow, Eva-Maria; Mandelkow, Eckhard

    2017-07-21

    Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    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...

  1. 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 isof...

  2. The prognostic value of Her4 receptor isoform expression in triple-negative and Her2 positive breast cancer patients

    International Nuclear Information System (INIS)

    Machleidt, Anna; Buchholz, Stefan; Diermeier-Daucher, Simone; Zeman, Florian; Ortmann, Olaf; Brockhoff, Gero

    2013-01-01

    Not only four but rather seven different human epidermal growth factor receptor related (Her) receptor tyrosine kinases (RTKs) have been described to be expressed in a variety of normal and neoplastic tissues: Her1, Her2, Her3, and additionally four Her4 isoforms have been identified. A differential expression of Her4 isoforms does not, however, play any role in either the molecular diagnostics or treatment decision for breast cancer patients. The prognostic and predictive impact of Her4 expression in breast cancer is basically unclear. We quantified the Her4 variants JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, and JM-b/CYT2 by isoform-specific polymerase chain reaction (qPCR) in (i) triple-negative, (ii) Her2 positive breast cancer tissues and (iii) in benign breast tissues. In all three tissue collectives we never found the JM-b/CYT1 or the JM-b/CYT2 isoform expressed. In contrast, the two JM-a/CYT1 and JM-a/CYT2 isoforms were always simultaneously expressed but at different ratios. We identified a positive prognostic impact on overall survival (OS) in triple-negative and event-free survival (EFS) in Her2 positive patients. This finding is independent of the absolute JM-a/CYT1 to JM-a/CYT2 expression ratio. In Her2 positive patients, Her4 expression only has a favorable effect in estrogen-receptor (ER)-positive but not in ER-negative individuals. In summary, JM-a/CYT1 and JM-a/CYT2 but not JM-b isoforms of the Her4 receptor are simultaneously expressed in both triple-negative and Her2 positive breast cancer tissues. Although different expression ratios of the two JM-a isoforms did not reveal any additional information, Her4 expression basically indicates a prolonged EFS and OFS. An extended expression analysis that takes all Her receptor homologs, including the Her4 isoforms, into account might render more precisely the molecular diagnostics required for the development of optimized targeted therapies

  3. Increased dysbindin-1B isoform expression in schizophrenia and its propensity in aggresome formation

    Science.gov (United States)

    Xu, Yiliang; Sun, Yuhui; Ye, Haihong; Zhu, Li; Liu, Jianghong; Wu, Xiaofeng; Wang, Le; He, Tingting; Shen, Yan; Wu, Jane Y; Xu, Qi

    2015-01-01

    Genetic variations in the human dysbindin-1 gene (DTNBP1) have been associated with schizophrenia. As a result of alternative splicing, the human DTNBP1 gene generates at least three distinct protein isoforms, dysbindin-1A, -1B and -1C. Significant effort has focused on dysbindin-1A, an important player in multiple steps of neurodevelopment. However, the other isoforms, dysbindin-1B and dysbindin-1C have not been well characterized. Nor have been associated with human diseases. Here we report an increase in expression of DTNBP1b mRNA in patients with paranoid schizophrenia as compared with healthy controls. A single-nucleotide polymorphism located in intron 9, rs117610176, has been identified and associated with paranoid schizophrenia, and its C allele leads to an increase of DTNBP1b mRNA splicing. Our data show that different dysbindin splicing isoforms exhibit distinct subcellular distribution, suggesting their distinct functional activities. Dysbindin-1B forms aggresomes at the perinuclear region, whereas dysbindin-1A and -1C proteins exhibit diffused patterns in the cytoplasm. Dysbindin-1A interacts with dysbindin-1B, getting recruited to the aggresome structure when co-expressed with dysbindin-1B. Moreover, cortical neurons over-expressing dysbindin-1B show reduction in neurite outgrowth, suggesting that dysbindin-1B may interfere with dysbindin-1A function in a dominant-negative manner. Taken together, our study uncovers a previously unknown association of DTNBP1b expression with schizophrenia in addition to its distinct biochemical and functional properties. PMID:27462430

  4. Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein

    OpenAIRE

    Minervini, Giovanni; Mazzotta, Gabriella M.; Masiero, Alessandro; Sartori, Elena; Corr?, 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 t...

  5. Oxygenation properties and isoform diversity of snake hemoglobins.

    Science.gov (United States)

    Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E

    2015-11-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. Copyright © 2015 the American Physiological Society.

  6. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  7. Functions of PDE3 Isoforms in Cardiac Muscle

    Science.gov (United States)

    Movsesian, Matthew; Ahmad, Faiyaz

    2018-01-01

    Isoforms in the PDE3 family of cyclic nucleotide phosphodiesterases have important roles in cyclic nucleotide-mediated signalling in cardiac myocytes. These enzymes are targeted by inhibitors used to increase contractility in patients with heart failure, with a combination of beneficial and adverse effects on clinical outcomes. This review covers relevant aspects of the molecular biology of the isoforms that have been identified in cardiac myocytes; the roles of these enzymes in modulating cAMP-mediated signalling and the processes mediated thereby; and the potential for targeting these enzymes to improve the profile of clinical responses. PMID:29415428

  8. Modified expression of cytoplasmic isocitrate dehydrogenase electrophoretic isoforms in seminal plasma of men with sertoli-cell-only syndrome and seminoma.

    Science.gov (United States)

    Starita-Geribaldi, Mireille; Samson, Michel; Guigonis, Jean-Marie; Pointis, Georges; Fenichel, Patrick

    2008-06-01

    Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated. (c) 2007 Wiley-Liss, Inc.

  9. Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

    Science.gov (United States)

    Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

    2017-09-01

    Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Roles of the troponin isoforms during indirect flight muscle ...

    Indian Academy of Sciences (India)

    IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed ...

  11. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  12. Molecular characters and expression analysis of a new isoform of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-10-20

    Oct 20, 2008 ... isoform of the myocyte enhancer factor 2 gene from the silkworm, Bombyx mori. Qing-zhi Ling1, 2, ... BMEF2B mRNA content in the brain was measured using the combined method of quantitative RT-PCR and Southern ... specific cofactors to control gene expression in pheno- typically different muscles.

  13. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the

  14. N Termini of apPDE4 Isoforms Are Responsible for Targeting the Isoforms to Different Cellular Membranes

    Science.gov (United States)

    Jang, Deok-Jin; Park, Soo-Won; Lee, Jin-A; Lee, Changhoon; Chae, Yeon-Su; Park, Hyungju; Kim, Min-Jeong; Choi, Sun-Lim; Lee, Nuribalhae; Kim, Hyoung; Kaang, Bong-Kiun

    2010-01-01

    Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the…

  15. Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study

    Directory of Open Access Journals (Sweden)

    Laura L. Perissinotti

    2018-04-01

    Full Text Available IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS (Lees-Miller et al., 1997 in humans are linked to functional modifications in the Kv11.1 (hERG ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero- channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells were analyzed simultaneously and fitted to Markov-chain models (M

  16. Characterization of ß-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production

    NARCIS (Netherlands)

    Warmerdam, A.; Paudel, E.; Wanqing, J.; Boom, R.M.; Janssen, A.E.M.

    2013-01-01

    A ß-galactosidase preparation from Bacillus circulans consists of four isoforms called ß-gal-A, ß-gal-B, ß-gal-C, and ß-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms

  17. Overexpression of EMMPRIN isoform 2 is associated with head and neck cancer metastasis.

    Directory of Open Access Journals (Sweden)

    Zhiquan Huang

    Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN, a plasma membrane protein of the immunoglobulin (Ig superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2 was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2, urokinase-type plasminogen activator(uPA and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel

  18. Overexpression of EMMPRIN isoform 2 is associated with head and neck cancer metastasis.

    Science.gov (United States)

    Huang, Zhiquan; Tan, Ning; Guo, Weijie; Wang, Lili; Li, Haigang; Zhang, Tianyu; Liu, Xiaojia; Xu, Qin; Li, Jinsong; Guo, Zhongmin

    2014-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2), urokinase-type plasminogen activator(uPA) and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel mechanism

  19. Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms.

    Science.gov (United States)

    Zhi, Li; Mans, Janet; Paskow, Michael J; Brown, Patrick H; Schuck, Peter; Jonjić, Stipan; Natarajan, Kannan; Margulies, David H

    2010-03-23

    Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 to prevent NK cell control in vivo. So far, it is unclear if there is a direct interaction between m152 and RAE-1 and, if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or -resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152 and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (K(d) RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1beta, although contributing to its affinity for m152, does not influence the affinity of RAE-1gamma or RAE-1delta, suggesting that other differences contribute to the RAE-1-m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.

  20. Isoform-specific inhibition of cyclophilins.

    Science.gov (United States)

    Daum, Sebastian; Schumann, Michael; Mathea, Sebastian; Aumüller, Tobias; Balsley, Molly A; Constant, Stephanie L; de Lacroix, Boris Féaux; Kruska, Fabian; Braun, Manfred; Schiene-Fischer, Cordelia

    2009-07-07

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

  1. Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence.

    Science.gov (United States)

    Dentovskaya, Svetlana V; Platonov, Mikhail E; Svetoch, Tat'yana E; Kopylov, Pavel Kh; Kombarova, Tat'yana I; Ivanov, Sergey A; Shaikhutdinova, Rima Z; Kolombet, Lyubov' V; Chauhan, Sadhana; Ablamunits, Vitaly G; Motin, Vladimir L; Uversky, Vladimir N; Anisimov, Andrey P

    2016-01-01

    It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla-strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification

  2. Synaptojanin 1: localization on coated endocytic intermediates in nerve terminals and interaction of its 170 kDa isoform with Eps15

    DEFF Research Database (Denmark)

    Haffner, C; Takei, K; Chen, H

    1997-01-01

    that synaptojanin-170, an alternatively spliced isoform of synaptojanin 1, binds Eps15, a clathrin coat-associated protein. Binding is mediated by the COOH-terminal region of synaptojanin-170 which we show here to be poorly conserved from rat to humans, but to contain in both species three asparagine...

  3. Total and isoform-specific quantitative assessment of circulating Fibulin-1 using selected reaction monitoring mass spectrometry and time-resolved immunofluorometry

    DEFF Research Database (Denmark)

    Overgaard, Martin; Cangemi, Claudia; Jensen, Martin L

    2015-01-01

    biomarker fibulin-1 and its circulating isoforms in human plasma. EXPERIMENTAL DESIGN:: We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time......PURPOSE:: Targeted proteomics using SRM-MS combined with stable isotope dilution has emerged as a promising quantitative technique for the study of circulating protein biomarkers. The purpose of this study was to develop and characterize robust quantitative assays for the emerging cardiovascular......-resolved immunofluorometric assay (TRIFMA). Both methods were validated and compared to a commercial ELISA (CircuLex). Molecular size determination was performed under native conditions by SEC analysis coupled to SRM-MS and TRIFMA. RESULTS:: Absolute quantitation of total fibulin-1, isoforms -1C and -1D was performed by SRM...

  4. Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Vakhrushev, Sergey Y; Kong, Yun

    2012-01-01

    to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific O-glycosylation inhibits proprotein...... activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme...

  5. Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis.

    Science.gov (United States)

    Kyriazis, George A; Wei, Zelan; Vandermey, Miriam; Jo, Dong-Gyu; Xin, Ouyang; Mattson, Mark P; Chan, Sic L

    2008-09-12

    Central to the pathogenesis of Alzheimer disease is the aberrant processing of the amyloid precursor protein (APP) to generate amyloid beta-peptide (Abeta), the principle component of amyloid plaques. The cell fate determinant Numb is a phosphotyrosine binding domain (PTB)-containing endocytic adapter protein that interacts with the carboxyl-terminal domain of APP. The physiological relevance of this interaction is unknown. Mammals produce four alternatively spliced variants of Numb that differ in the length of their PTB and proline-rich region. In the current study, we determined the influence of the four human Numb isoforms on the intracellular trafficking and processing of APP. Stable expression of Numb isoforms that differ in the PTB but not in the proline-rich region results in marked differences in the sorting of APP to the recycling and degradative pathways. Neural cells expressing Numb isoforms that lack the insert in the PTB (short PTB (SPTB)) exhibited marked accumulation of APP in Rab5A-labeled early endosomal and recycling compartments, whereas those expressing isoforms with the insertion in the PTB (long PTB (LPTB)) exhibited reduced amounts of cellular APP and its proteolytic derivatives relative to parental control cells. Neither the activities of the beta- and gamma-secretases nor the expression of APP mRNA were significantly different in the stably transfected cells, suggesting that the differential effects of the Numb proteins on APP metabolism is likely to be secondary to altered APP trafficking. In addition, the expression of SPTB-Numb increases at the expense of LPTB-Numb in neuronal cultures subjected to stress, suggesting a role for Numb in stress-induced Abeta production. Taken together, these results suggest distinct roles for the human Numb isoforms in APP metabolism and may provide a novel potential link between altered Numb isoform expression and increased Abeta generation.

  6. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    M. R. Aquino-Silva

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  7. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    Aquino-Silva M. R.

    2003-01-01

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  8. Isoform-Selective Disruption of AKAP-Localized PKA Using Hydrocarbon Stapled Peptides

    Science.gov (United States)

    2015-01-01

    A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. PMID:24422448

  9. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma.

    Science.gov (United States)

    Pritchard-Jones, R O; Dunn, D B A; Qiu, Y; Varey, A H R; Orlando, A; Rigby, H; Harper, S J; Bates, D O

    2007-07-16

    Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Pxxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

  10. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    Science.gov (United States)

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

    2016-04-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

  11. Effect of inhibition of the ROCK isoform on RT2 malignant glioma cells.

    Science.gov (United States)

    Inaba, Nobuharu; Ishizawa, Sho; Kimura, Masaki; Fujioka, Kouki; Watanabe, Michiko; Shibasaki, Toshiaki; Manome, Yoshinobu

    2010-09-01

    Malignant glioma is one of the most intractable diseases in the human body. Rho-kinase (ROCK) is overexpressed and has been proposed as the main cause for the refractoriness of the disease. Since efficacious treatment is required, this study investigated the effect of inhibition of ROCK isoforms. The short hairpin RNA transcription vector was transfected into the RT2 rat glioma cell line and the characteristics of the cells were investigated. The effect of nimustine hydrochloride (ACNU) anti-neoplastic agent on cells was also measured. Inhibition of ROCK isoforms did not alter cell growth. Cell cycle analysis revealed that ROCK1 down-regulation reduced the G(0) phase population and ROCK2 down-regulation reduced the G(2)/M phase population. When ROCK1-down-regulated cells were exposed to ACNU, they demonstrated susceptibility to the agent. The roles of ROCK1 and ROCK2 may be different in glioma cells. Furthermore, the combination of ROCK1 down-regulation and an anti-neoplastic agent may be useful for the therapy of malignant glioma.

  12. Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms.

    Science.gov (United States)

    Kern, J S; Herz, C; Haan, E; Moore, D; Nottelmann, S; von Lilien, T; Greiner, P; Schmitt-Graeff, A; Opitz, O G; Bruckner-Tuderman, L; Has, C

    2007-12-01

    Kindlin-1 is an epithelium-specific phosphoprotein and focal adhesion adaptor component. Mutations in the corresponding gene (KIND1) cause Kindler syndrome (KS), which is manifested by skin blistering, poikiloderma, photosensitivity and carcinogenesis. Some patients also exhibit gastrointestinal symptoms, but it has remained unclear whether these represent a feature of Kindler syndrome or a coincidence. We examined kindlin-1 in human gastrointestinal epithelia and showed that it is involved in the aetiopathology of Kindler syndrome-associated colitis. Kindlin-1 expression was assessed by indirect immunofluorescence, western blot and RT-PCR. Kindlin-1 is expressed in oral mucosa, colon and rectum. Both the full-length 74 kDa kindlin-1 protein and a 43 kDa isoform were detected in CaCo2 cells, the latter resulting from alternative splicing. In the first months of life, patients (homozygous for null mutations) had severe intestinal involvement with haemorrhagic diarrhoea and showed morphological features of severe ulcerative colitis. Later in childhood, histopathology demonstrated focal detachment of the epithelium in all segments of the colon, chronic inflammation and mucosal atrophy. These findings define an intestinal phenotype for Kindler syndrome as a consequence of a primary epithelial barrier defect. The different clinical intestinal manifestations in Kindler syndrome patients may be explained by partial functional compensation of kindlin-1 deficiency by the intestinal isoform or by the presence of truncated mutant kindlin-1. (c) 2007 Pathological Society of Great Britain and Ireland

  13. Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

    Directory of Open Access Journals (Sweden)

    Yanqi Hao

    2015-07-01

    Full Text Available Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse, which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing.

  14. Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis.

    Directory of Open Access Journals (Sweden)

    Bo G Lindberg

    2018-03-01

    Full Text Available Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB, JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic

  15. Nitric oxide synthase isoforms in spontaneous and salt hypertension

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Kuneš, Jaroslav; Zicha, Josef

    2007-01-01

    Roč. 25, Suppl. 2 (2007), S 338-S 338 ISSN 0263-6352. [European Meeting on Hypertension /17./. 15.06.2007-19.06.2007, Milan] R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : nitric oxide synthase isoforms * spontaneous and salt hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  16. Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a ...

    African Journals Online (AJOL)

    Plasma lipoprotein (a) Concentrations and apo(a) isoforms were determined in 101 healthy Nigerian subjects (M=63), F=38; age range 17-68 years), and coronary heart disease (CHD) patients (M=19, F=17, age range 30-79 years). Median Lp(a) level was 24.4 mg/di in the CHD patients and 22.1 mg/di in the controls.

  17. RON kinase isoforms demonstrate variable cell motility in normal cells.

    Science.gov (United States)

    Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

    2016-09-01

    Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

  18. [Characterization of a malic enzyme isoform V from Mucor circinelloides].

    Science.gov (United States)

    Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

    2016-02-04

    We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

  19. Differential expression of syndecan isoforms during mouse incisor amelogenesis.

    Science.gov (United States)

    Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

    2007-08-01

    Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

  20. Gestational exposure to BDE-99 produces toxicity through upregulation of CYP isoforms and ROS production in the fetal rat liver.

    Science.gov (United States)

    Blanco, Jordi; Mulero, Miquel; Domingo, José L; Sánchez, Domènec J

    2012-05-01

    On gestation day (GD) 6 to GD 19, pregnant Sprague Dawley rats were orally exposed to 0, 0.5, 1, and 2 mg/kg/day to one of the most prevalent polybrominated diphenyl ethers congeners found in humans, 2,2',4,4',5-pentaBDE (BDE-99). All dams were euthanized on GD 20, and live fetuses were evaluated for sex, body weight, and external, internal, and skeletal malformations and developmental variations. The liver from one fetus of each litter was excised for the evaluation of oxidative stress markers and the messenger RNA expression of multiple cytochrome P450 (CYP) isoforms. Exposure to BDE-99 during the gestational period produced delayed ossification, slight hypertrophy of the heart, and enlargement of the liver in fetuses. A transplacental effect of BDE-99, evidenced by the activation of nuclear hormones receptors that induce the upregulation of CYP1A1, CYP1A2, CYP2B1, and CYP3A2 isoforms, was also found in fetal liver. These isoforms are correlated with the activity level of the enzyme catalase and the levels of thiobarbituric acid reactive substances. However, teratogenic effects from BDE-99 exposure were not observed. Clear signs of embryo/fetal toxicity, due to a possible hormonal disruption, were evidenced by a large increase in the CYP system and the production of reactive oxygen species in fetal liver.

  1. Novel VEGF decoy receptor fusion protein conbercept targeting multiple VEGF isoforms provide remarkable anti-angiogenesis effect in vivo.

    Directory of Open Access Journals (Sweden)

    Qin Wang

    Full Text Available VEGF family factors are known to be the principal stimulators of abnormal angiogenesis, which play a fundamental role in tumor and various ocular diseases. Inhibition of VEGF is widely applied in antiangiogenic therapy. Conbercept is a novel decoy receptor protein constructed by fusing VEGF receptor 1 and VEGF receptor 2 extracellular domains with the Fc region of human immunoglobulin. In this study, we systematically evaluated the binding affinity of conbercept with VEGF isoforms and PlGF by using anti-VEGF antibody (Avastin as reference. BIACORE and ELISA assay results indicated that conbercept could bind different VEGF-A isoforms with higher affinity than reference. Furthermore, conbercept could also bind VEGF-B and PlGF, whereas Avastin showed no binding. Oxygen-induced retinopathy model showed that conbercept could inhibit the formation of neovasularizations. In tumor-bearing nude mice, conbercept could also suppress tumor growth very effectively in vivo. Overall, our study have demonstrated that conbercept could bind with high affinity to multiple VEGF isoforms and consequently provide remarkable anti-angiogenic effect, suggesting the possibility to treat angiogenesis-related diseases such as cancer and wet AMD etc.

  2. Ablation of whirlin long isoform disrupts the USH2 protein complex and causes vision and hearing loss.

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2010-05-01

    Full Text Available Mutations in whirlin cause either Usher syndrome type II (USH2, a deafness-blindness disorder, or nonsyndromic deafness. The molecular basis for the variable disease expression is unknown. We show here that only the whirlin long isoform, distinct from a short isoform by virtue of having two N-terminal PDZ domains, is expressed in the retina. Both long and short isoforms are expressed in the inner ear. The N-terminal PDZ domains of the long whirlin isoform mediates the formation of a multi-protein complex that includes usherin and VLGR1, both of which are also implicated in USH2. We localized this USH2 protein complex to the periciliary membrane complex (PMC in mouse photoreceptors that appears analogous to the frog periciliary ridge complex. The latter is proposed to play a role in photoreceptor protein trafficking through the connecting cilium. Mice carrying a targeted disruption near the N-terminus of whirlin manifest retinal and inner ear defects, reproducing the clinical features of human USH2 disease. This is in contrast to mice with mutations affecting the C-terminal portion of whirlin in which the phenotype is restricted to the inner ear. In mice lacking any one of the USH2 proteins, the normal localization of all USH2 proteins is disrupted, and there is evidence of protein destabilization. Taken together, our findings provide new insights into the pathogenic mechanism of Usher syndrome. First, the three USH2 proteins exist as an obligatory functional complex in vivo, and loss of one USH2 protein is functionally close to loss of all three. Second, defects in the three USH2 proteins share a common pathogenic process, i.e., disruption of the PMC. Third, whirlin mutations that ablate the N-terminal PDZ domains lead to Usher syndrome, but non-syndromic hearing loss will result if they are spared.

  3. The short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction.

    Science.gov (United States)

    Chen, Lanfen; Chen, Zhangguo; Baker, Kristi; Halvorsen, Elizabeth M; da Cunha, Andre Pires; Flak, Magdalena B; Gerber, Georg; Huang, Yu-Hwa; Hosomi, Shuhei; Arthur, Janelle C; Dery, Ken J; Nagaishi, Takashi; Beauchemin, Nicole; Holmes, Kathryn V; Ho, Joshua W K; Shively, John E; Jobin, Christian; Onderdonk, Andrew B; Bry, Lynn; Weiner, Howard L; Higgins, Darren E; Blumberg, Richard S

    2012-11-16

    Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer

    International Nuclear Information System (INIS)

    Young, Gavin M.; Radhakrishnan, Vijayababu M.; Centuori, Sara M.; Gomes, Cecil J.; Martinez, Jesse D.

    2015-01-01

    The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers. DNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression. Here we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells. To

  5. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    International Nuclear Information System (INIS)

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn

    2013-01-01

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  6. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  7. NHS-A isoform of the NHS gene is a novel interactor of ZO-1.

    Science.gov (United States)

    Sharma, Shiwani; Koh, Katrina S Y; Collin, Caitlin; Dave, Alpana; McMellon, Amy; Sugiyama, Yuki; McAvoy, John W; Voss, Anne K; Gécz, Jozef; Craig, Jamie E

    2009-08-15

    Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.

  8. Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

    Science.gov (United States)

    Dave, Mihika B; Dherai, Alpa J; Udani, Vrajesh P; Hegde, Anaita U; Desai, Neelu A; Ashavaid, Tester F

    2018-01-01

    Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects. © 2017 Wiley Periodicals, Inc.

  9. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    Directory of Open Access Journals (Sweden)

    Senthil kumar M

    2011-05-01

    Full Text Available Abstract The Metallothionein (MT is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems. Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios. The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

  10. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  11. 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 Ahmed; Poulsen, Steen Seier

    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...... and half of those (18/36) expressed both isoforms. JM-a/CYT2 expression correlated to improved survival (p = 0.004), but not when ER-α was co-expressed (p = 0.897). Immunohistochemistry revealed protein expression of HER4 and ER-α in tumor cells. Growth of RT4 bladder cancer cells, expressing both JM...

  12. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

    International Nuclear Information System (INIS)

    Acikgoez, Ali; Karim, Najibulla; Giri, Shibashish; Schmidt-Heck, Wolfgang; Bader, Augustinus

    2009-01-01

    Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c i,j (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p k (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the

  13. Metabolism of ginger component [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human.

    Science.gov (United States)

    Chen, Huadong; Soroka, Dominique; Zhu, Yingdong; Sang, Shengmin

    2013-05-01

    There are limited data on the metabolism of [6]-shogaol (6S), a major bioactive component of ginger. This study demonstrates metabolism of 6S in liver microsomes from mouse, rat, dog, monkey, and human. The in vitro metabolism of 6S was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with 6S, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E,4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites, M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than 6S. We conclude that 6S is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning preclinical trials toward 6S chemoprevention. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Science.gov (United States)

    Béziau, Delphine M; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  15. Identification and characterization of novel smoothelin isoforms in vascular smooth muscle.

    Science.gov (United States)

    Krämer, J; Quensel, C; Meding, J; Cardoso, M C; Leonhardt, H

    2001-01-01

    Smoothelin is a cytoskeletal protein specifically expressed in differentiated smooth muscle cells and has been shown to colocalize with smooth muscle alpha actin. In addition to the small smoothelin isoform of 59 kD, we recently identified a large smoothelin isoform of 117 kD. The aim of this study was to identify and characterize novel smoothelin isoforms. The genomic structure and sequence of the smoothelin gene were determined by genomic PCR, RT-PCR and DNA sequencing. Comparison of the cDNA and genomic sequences shows that the small smoothelin isoform is generated by transcription initiation 10 kb downstream of the start site of the large isoform. In addition to the known smoothelin cDNA (c1 isoform) we identified two novel cDNA variants (c2 and c3 isoform) that are generated by alternative splicing within a region, which shows similarity to the spectrin family of F-actin cross-linking proteins. Visceral organs express the c1 form, while the c2 form prevails in well-vascularized tissue as analyzed by RT-PCR. We then generated specific antibodies against the major smoothelin isoforms and could show by Western blotting and immunohistochemistry that the large isoform is specifically expressed in vascular smooth muscle cells, while the small isoform is abundant in visceral smooth muscle. These results strongly suggest that the smoothelin gene contains a vascular and a visceral smooth muscle promoter. The cell-type-specific expression of smoothelin isoforms that are associated with actin filaments may play a role in the modulation of the contractile properties of different smooth muscle cell types. Copyright 2001 S. Karger AG, Basel

  16. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  17. Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

    Directory of Open Access Journals (Sweden)

    Olga Romanets-Korbut

    Full Text Available CD150 (IPO3/SLAM belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS. Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150, containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.

  18. Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure.

    Science.gov (United States)

    Katz, Adriana; Tal, Daniel M; Heller, Dan; Habeck, Michael; Ben Zeev, Efrat; Rabah, Bilal; Bar Kana, Yaniv; Marcovich, Arie L; Karlish, Steven J D

    2015-11-03

    The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1β1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2β3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2β3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2β1 over α1β1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the β1 subunit, Gln84, suggesting a role for β in digoxin binding. Gln84 in β1 is replaced by Val88 in β3. Assuming that alkyl substituents might interact with β3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2β3 over α1β1 (22-33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2β3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.

  19. Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2.

    Science.gov (United States)

    Peach, Chloe J; Mignone, Viviane W; Arruda, Maria Augusta; Alcobia, Diana C; Hill, Stephen J; Kilpatrick, Laura E; Woolard, Jeanette

    2018-04-23

    Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGF xxx a or VEGF xxx b isoforms. Alternative splicing events at exons 5⁻7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF 165 a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology.

  20. Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Susi Zatti

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.

  1. RAGE receptor and its soluble isoforms in diabetes mellitus complications

    Directory of Open Access Journals (Sweden)

    Mauren Isfer Anghebem Oliveira

    2013-04-01

    Full Text Available Chronic hyperglycemia, which is present in all types of diabetes, increases the formation of advanced glycation end-products (AGEs. The interaction of AGEs with receptor of advanced glycation end-products (RAGE initiates a cascade of pro-inflammatory and pro-coagulant processes that result in oxidative stress, stimulating the formation and accumulation of more AGE molecules. This cyclic process, denominated metabolic memory, may explain the persistency of diabetic vascular complications in patients with satisfactory glycemic control. The RAGE found in several cell membranes is also present in soluble isoforms (esRAGE and cRAGE, which are generated by alternative deoxyribonucleic acid splicing or by proteolytic cleavage. This review focuses on new research into these mediators as potential biomarkers for vascular complications in diabetes.

  2. Metallothionein Isoform Expression in Benign and Malignant Thyroid Lesions.

    Science.gov (United States)

    Wojtczak, Beata; Pula, Bartosz; Gomulkiewicz, Agnieszka; Olbromski, Mateusz; Podhorska-Okolow, Marzena; Domoslawski, Paweł; Bolanowski, Marek; Daroszewski, Jacek; Dziegiel, Piotr

    2017-09-01

    Metallothioneins (MTs) are involved in numerous cell processes such as binding and transport of zinc and copper ions, differentiation, proliferation and apoptosis, therefore contributing to carcinogenesis. Scarce data exist on their expression in benign and malignant lesions of the thyroid. mRNA expression of functional isoforms of MT genes (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT4) was studied in 17 nodular goiters (NG), 12 follicular adenomas (FA) and 26 papillary thyroid carcinomas (PTC). One-way ANOVA revealed significant differences in mRNA expression levels of MT1A (pbenign and malignant lesions. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  3. Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

    Directory of Open Access Journals (Sweden)

    Llewellyn Lynda

    2007-06-01

    Full Text Available Abstract Background Flatfish metamorphosis is a thyroid hormone (TH driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT, a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. Results In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Conclusion Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

  4. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    OpenAIRE

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine r...

  5. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

    Science.gov (United States)

    Banerjee, Subhrajit; Kane, Patricia M

    2017-09-15

    Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  7. VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

    Science.gov (United States)

    Rennel, E S; Varey, A H R; Churchill, A J; Wheatley, E R; Stewart, L; Mather, S; Bates, D O; Harper, S J

    2009-10-06

    The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

  8. The Schistosoma mansoni Cytochrome P450 (CYP3050A1 Is Essential for Worm Survival and Egg Development.

    Directory of Open Access Journals (Sweden)

    Peter D Ziniel

    2015-12-01

    Full Text Available Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450 gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA silencing of S. mansoni (SmCYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl-2-(1H-imidazol-1-ylethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.

  9. Inulin isoforms differ by repeated additions of one crystal unit cell

    Science.gov (United States)

    Cooper, Peter D.; Barclay, Thomas G.; Ginic-Markovic, Milena; Gerson, Andrea R.; Petrovsky, Nikolai

    2014-01-01

    Inulin isoforms, especially delta inulin, are important biologically as immune activators and clinically as vaccine adjuvants. In exploring action mechanisms, we previously found regular increments in thermal properties of the seven-member inulin isoform series that suggested regular additions of some energetic structural unit. Because the previous isolates carried additional longer chains that masked defining ranges, these were contrasted with new isoform isolates comprising only inulin chain lengths defining that isoform. The new series began with 19 fructose units per chain (alpha-1 inulin), increasing regularly by 6 fructose units per isoform. Thus the ‘energetic unit’ equates to 6 fructose residues per chain. All isoforms showed indistinguishable X-ray diffraction patterns that were also identical with known inulin crystals. We conclude that an ‘energetic unit’ equates to one helix turn of 6 fructose units per chain as found in one unit cell of the inulin crystal. Each isoform chain comprised progressively more helix turns plus one additional fructose and glucose residues per chain. PMID:24528745

  10. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2016-05-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145 promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

  11. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    Science.gov (United States)

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-05-15

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. © 2016. Published by The Company of Biologists Ltd.

  12. High Molecular Weight Isoforms of Growth Hormone In Cells of the Immune System

    Science.gov (United States)

    Weigent, Douglas A.

    2013-01-01

    A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100 kDa, 65 kDa, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 kDa and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm whereas the lower molecular weight 65 kDa and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost three-fold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress. PMID:21741628

  13. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes.

    Science.gov (United States)

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse K

    2015-01-01

    Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen. © 2014 Wiley Periodicals, Inc.

  14. Myosin isoform fiber type and fiber size in the tail of the Virginia opossum (Didelphis virginiana).

    Science.gov (United States)

    Hazimihalis, P J; Gorvet, M A; Butcher, M T

    2013-01-01

    Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. Copyright © 2012 Wiley Periodicals, Inc.

  15. The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions

    Science.gov (United States)

    Cooper, Peter D; Barclay, Thomas G; Ginic-Markovic, Milena; Petrovsky, Nikolai

    2013-01-01

    In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous → alpha-1 (AI-1) → alpha-2 (AI-2) → gamma (GI) → delta (DI) → zeta (ZI) → epsilon (EI) → omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) “melting” or “freezing” points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators. PMID:23853206

  16. Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

    Directory of Open Access Journals (Sweden)

    Hisayo Sadamoto

    2010-05-01

    Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

  17. Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

    DEFF Research Database (Denmark)

    Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils

    2014-01-01

    RNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n¿=¿22) or vehicle (n¿=¿5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor......-receptor positive and isoform-A positive tumours in aglepristone-treated dogs.ConclusionsThese findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A....

  18. Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Qingbo; Man, Cong; Li, Danning; Tan, Huijuan; Xie, Ye; Huang, Jirong

    2016-12-05

    Anthocyanins, a group of L-phenylalanine (Phe)-derived flavonoids, have been demonstrated to play important roles in plant stress resistance and interactions between plants and insects. Although the anthocyanin biosynthetic pathway and its regulatory mechanisms have been extensively studied, it remains unclear whether the level of Phe supply affects anthocyanin biosynthesis. Here, we investigated the roles of arogenate dehydratases (ADTs), the key enzymes that catalyze the conversion of arogenate into Phe, in sucrose-induced anthocyanin biosynthesis in Arabidopsis. Genetic analysis showed that all six ADT isoforms function redundantly in anthocyanin biosynthesis but have differential contributions. ADT2 contributes the most to anthocyanin accumulation, followed by ADT1 and ADT3, and ADT4-ADT6. We found that anthocyanin content is positively correlated with the levels of Phe and sucrose-induced ADT transcripts in seedlings. Consistently, addition of Phe to the medium could dramatically increase anthocyanin content in the wild-type plants and rescue the phenotype of the adt1 adt3 double mutant regarding the anthocyanin accumulation. Moreover, transgenic plants overexpressing ADT4, which appears to be less sensitive to Phe than overexpression of ADT2, hyperaccumulate Phe and produce elevated level of anthocyanins. Taken together, our results suggest that the level of Phe is an important regulatory factor for sustaining anthocyanin biosynthesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  19. Differential susceptibility of RAE-1 isoforms to mouse cytomegalovirus.

    Science.gov (United States)

    Arapovic, Jurica; Lenac, Tihana; Antulov, Ronald; Polic, Bojan; Ruzsics, Zsolt; Carayannopoulos, Leonidas N; Koszinowski, Ulrich H; Krmpotic, Astrid; Jonjic, Stipan

    2009-08-01

    The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1delta, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1delta and RAE-1gamma in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1delta form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1delta compared to RAE-1gamma but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1delta. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.

  20. Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

    International Nuclear Information System (INIS)

    Hu, Xiaolan; Yu, Yingnian; Li, Qian; Wu, Danxiao; Tan, Zhengning; Wang, Cheng; Wang, Jvping; Wu, Meiping

    2011-01-01

    Highlights: → MNNG-induced appearance of DUT-N in the extracellular fluid has cellular specificity. → MNNG alters the subcellular distribution of DUT-N in human cells in different ways. → DUT-N may be a potential biomarker to assess the risk of alkylating agents exposure. -- Abstract: Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

  1. A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

    Science.gov (United States)

    Vicario, Mattia; Zanni, Ginevra; Vallese, Francesca; Santorelli, Filippo; Grinzato, Alessandro; Cieri, Domenico; Berto, Paola; Frizzarin, Martina; Lopreiato, Raffaele; Zonta, Francesco; Ferro, Stefania; Sandre, Michele; Marin, Oriano; Ruzzene, Maria; Bertini, Enrico; Zanotti, Giuseppe; Brini, Marisa; Calì, Tito; Carafoli, Ernesto

    2018-04-12

    The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca 2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca 2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca 2+ signaling in neurons demands the continuous activity of Ca 2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca 2+ ATPases (PMCA pumps) play a key role in the regulation of Ca 2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca 2+ ejection. Copyright © 2018. Published by Elsevier Inc.

  2. The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

    International Nuclear Information System (INIS)

    Abreu, Maria M.; Sealy, Linda

    2010-01-01

    Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

  3. The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Abreu, Maria M. [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Sealy, Linda, E-mail: Linda.sealy@vanderbilt.edu [Department of Cancer Biology, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States); Department of Molecular Physiology and Biophysics, 752 Preston Research Building, Vanderbilt University, Nashville, TN 37232 (United States)

    2010-11-15

    Autophagy is a process involving the bulk degradation of cellular components in the cytoplasm via the lysosomal degradation pathway. Autophagy manifests a protective role in stressful conditions such as nutrient or growth factor depletion; however, extensive degradation of regulatory molecules or organelles essential for survival can lead to the demise of the cell, or autophagy-mediated cell death. The role of autophagy in cancer is complex with roles in both tumor suppression and tumor promotion proposed. Here we report that an isoform of the C/EBPbeta transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Overexpression of LIP is incompatible with cell growth and when cell cycle analysis was performed, a DNA profile of cells undergoing apoptosis was not observed. Instead, LIP expressing cells appeared to have large autophagic vesicles when examined via electron microscopy. Autophagy was further assessed in LIP expressing cells by monitoring the development of acidic vesicular organelles and conversion of LC3 from the cytoplasmic form to the membrane-bound form. Our work shows that C/EBPbeta isoform, LIP, is another member of the group of transcription factors, including E2F1 and p53, which are capable of playing a role in autophagy.

  4. Influences of Realgar-Indigo naturalis, A Traditional Chinese Medicine Formula, on the Main CYP450 Activities in Rats Using a Cocktail Method

    Directory of Open Access Journals (Sweden)

    Huan-Hua Xu

    2017-01-01

    Full Text Available The purpose of this work was to study the influences of Realgar-Indigo naturalis (RIF and its principal element realgar on 4 main cytochrome P450 enzymes activities in rats. A simple and efficient cocktail method was developed to detect the four probe drugs simultaneously. In this study, Wistar rats were administered intragastric RIF and realgar for 14 days; mixed probe drugs were injected into rats by caudal vein. Through analyzing the pharmacokinetic parameter of mixed probe drugs in rats, we can calculate the CYPs activities. The results showed that RIF could inhibit CYP1A2 enzyme activity and induce CYP2C11 enzyme activity significantly. Interestingly, in realgar high dosage group, CYP3A1/2 enzyme activity was inhibited significantly, and different dosage of realgar manifested a good dose-dependent manner. The RIF results indicated that drug coadministrated with RIF may need to be paid attention in relation to drug-drug interactions (DDIs. Realgar, a toxic traditional Chinese medicine (TCM, does have curative effect on acute promyelocytic leukemia (APL. Its toxicity studies should be focused on. We found that, in realgar high dosage group, CYP3A1/2 enzymes activity was inhibited. This phenomenon may explain its potential toxicity mechanism.

  5. E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting P-glycoprotein in small intestine and CYP450 3A in liver and kidney of broilers.

    Science.gov (United States)

    Guo, Mengjie; Sun, Yong; Zhang, Yu; Bughio, Shamsuddin; Dai, Xiaohua; Ren, Weilong; Wang, Liping

    2014-01-01

    P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (Penrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL(-1), P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL(-1) h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h(-1), P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers.

  6. [Age 89 years, depression, fall with pelvic fracture, severe confusion - serotonin syndrome : Differential diagnosis, importance of CYP450 and economic considerations].

    Science.gov (United States)

    Weinrebe, W; Moutaouakil, A; Risz, K; Martin, M; Jeckelmann, K; Goetz, S

    2017-04-18

    A case study of an 89-year-old patient is reported, who was admitted to hospital because of immobility due to pain. After the cause of the pain could initially be clarified and treated, the further clinical course in this very old woman showed an alarming symptom complex of agitation, confusion and cognitive deterioration, which took on grave forms. The work-up of this case showed a typical constellation of pain and depression in old age; however, the pharmaceutical treatment in this case is not atypical and could lead to a severe serotonin syndrome. The interaction, diagnostics, differential diagnosis, pharmacological, functional, codification and economic aspects of the course of the disease are discussed.

  7. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

    Directory of Open Access Journals (Sweden)

    Michael V. Clausen

    2017-06-01

    Full Text Available The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  8. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

    Science.gov (United States)

    Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

    2017-01-01

    The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  9. An abnormally glycosylated isoform of erythropoietin in hemangioblastoma is associated with polycythemia.

    Science.gov (United States)

    Delanghe, Sigurd E; Dierick, Jan; Maenhout, Thomas M; Zabeau, Lennart; Tavernier, Jan; Claes, Kathleen; Bleyen, Joris; Delanghe, Joris R

    2015-01-01

    Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Altered β-Amyloid Precursor Protein Isoforms in Mexican Alzheimer’s Disease Patients

    Directory of Open Access Journals (Sweden)

    V. J. Sánchez-González

    2006-01-01

    Full Text Available Objective: To determine the β-amyloid precursor protein (βAPP isoforms ratio as a risk factor for Alzheimer’s Disease and to assess its relationship with demographic and genetic variables of the disease.

  11. Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms.

    Directory of Open Access Journals (Sweden)

    Pavel Kh Kopylov

    Full Text Available Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117, type Caf1NT2 (Ser48 Phe117 found in Transcaucasian-highland and Pre-Araks natural plague foci #4-7, and a novel Caf1NT3 type (Ala48 Val117 endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL, which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection.

  12. Characterization of heparan sulfate N-deacetylase/N-sulfotransferase isoform 4 using synthetic oligosaccharide substrates.

    Science.gov (United States)

    Li, Yi-Jun; Yin, Feng-Xin; Zhang, Xin-Ke; Yu, Jie; Zheng, Shuang; Song, Xin-Lei; Wang, Feng-Shan; Sheng, Ju-Zheng

    2018-03-01

    The final structure of heparan sulfate chains is strictly regulated in vivo, though the biosynthesis is not guided by a template process. N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme in the HS biosynthetic pathway. The N-sulfo groups introduced by NDST are reportedly involved in determination of the susceptibility to subsequent processes catalyzed by C 5 -epimerse and 3-O-sulfotransferases. Understanding the substrate specificities of the four human NDST isoforms has become central to uncovering the regulatory mechanism of HS biosynthesis. Highly-purified recombinant NDST-4 (rNDST-4) and a selective library of structurally-defined oligosaccharides were employed to determine the substrate specificity of rNDST-4. Full-length rNDST-4 lacks obvious N-deacetylase activity, and displays only N-sulfotransferase activity. Unlike NDST-1, NDST-4 did not show directional N-sulfotransferase activity while the N-deacetylase domain was inactive. Individual NDST-4 could not effectively assume the key role in the distribution of N-S domains and N-Ac domains in HS biosynthesis in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Each individual isoform of the dopamine D2 receptor protects from lactotroph hyperplasia.

    Science.gov (United States)

    Radl, Daniela; De Mei, Claudia; Chen, Eric; Lee, Hyuna; Borrelli, Emiliana

    2013-06-01

    Dopamine acting through D2 receptors (D2Rs) controls lactotroph proliferation and prolactin (PRL) levels. Ablation of this receptor in mice results in lactotroph hyperplasia and prolactinomas in aged females. Alternative splicing of the Drd2 gene generates 2 independent isoforms, a long (D2L) and a short (D2S) isoform, which are present in all D2R-expressing cells. Here, we addressed the role of D2L and D2S on lactotroph physiology through the generation and analysis of D2S-null mice and their comparison with D2L-null animals. These mice represent a valuable tool with which to investigate dopamine-dependent isoform-specific signaling in the pituitary gland. We sought to assess the existence of a more prominent role of D2L or D2S in controlling PRL expression and lactotroph hyperplasia. Importantly, we found that D2L and D2S are specifically linked to independent transduction pathways in the pituitary. D2L-mediated signaling inhibits the AKT/protein kinase B kinase activity whereas D2S, in contrast, is required for the activation of the ERK 1/2 pathway. Under normal conditions, presence of only 1 of the 2 D2R isoforms in vivo prevents hyperprolactinemia, formation of lactotroph's hyperplasia, and tumorigenesis that is observed when both isoforms are deleted as in D2R-/- mice. However, the protective function of the single D2R isoforms is overridden when single isoform-knockout mice are challenged by chronic estrogen treatments as they show increased PRL production and lactotroph hyperplasia. Our study indicates that signaling from each of the D2R isoforms is sufficient to maintain lactotroph homeostasis in physiologic conditions; however, signaling from both is necessary in conditions simulating pathologic states.

  14. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2015-12-01

    Full Text Available High-throughput mRNA sequencing (RNA-Seq is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA, the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.

  15. Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

    Czech Academy of Sciences Publication Activity Database

    Arnoštová, P.; Jedelsky, P. L.; Soukup, Tomáš; Žurmanová, J.

    2011-01-01

    Roč. 2011, - (2011), e634253 ISSN 1110-7243 R&D Projects: GA AV ČR IAAX01110901; GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : cardiac MyHC isoforms * MyHC isoform mobility * effect of thyroid hormones * mass spectrometry * SDS-PAGE and western blot Subject RIV: ED - Physiology Impact factor: 2.436, year: 2011

  16. Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk

    DEFF Research Database (Denmark)

    Jacobsen, Stine; Niewold, T.A.; Kornalijnslijper, E.

    2005-01-01

    The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis.......The aim of the present study was to characterise the serum amyloid A (SAA) response to intramammary inoculation of Escherichia coli and to examine the distribution of hepatically and extrahepatically pruduced SAA isoforms in plasma and milk fra cows with mastitis....

  17. Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.

    OpenAIRE

    Seasholtz, A F; Gamm, D M; Ballestero, R P; Scarpetta, M A; Uhler, M D

    1995-01-01

    Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many ...

  18. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    Science.gov (United States)

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. Copyright © 2014 the American Physiological Society.

  19. Robust stratification of breast cancer subtypes using differential patterns of transcript isoform expression.

    Directory of Open Access Journals (Sweden)

    Thomas P Stricker

    2017-03-01

    Full Text Available Breast cancer, the second leading cause of cancer death of women worldwide, is a heterogenous disease with multiple different subtypes. These subtypes carry important implications for prognosis and therapy. Interestingly, it is known that these different subtypes not only have different biological behaviors, but also have distinct gene expression profiles. However, it has not been rigorously explored whether particular transcriptional isoforms are also differentially expressed among breast cancer subtypes, or whether transcript isoforms from the same sets of genes can be used to differentiate subtypes. To address these questions, we analyzed the patterns of transcript isoform expression using a small set of RNA-sequencing data for eleven Estrogen Receptor positive (ER+ subtype and fourteen triple negative (TN subtype tumors. We identified specific sets of isoforms that distinguish these tumor subtypes with higher fidelity than standard mRNA expression profiles. We found that alternate promoter usage, alternative splicing, and alternate 3'UTR usage are differentially regulated in breast cancer subtypes. Profiling of isoform expression in a second, independent cohort of 68 tumors confirmed that expression of splice isoforms differentiates breast cancer subtypes. Furthermore, analysis of RNAseq data from 594 cases from the TCGA cohort confirmed the ability of isoform usage to distinguish breast cancer subtypes. Also using our expression data, we identified several RNA processing factors that were differentially expressed between tumor subtypes and/or regulated by estrogen receptor, including YBX1, YBX2, MAGOH, MAGOHB, and PCBP2. RNAi knock-down of these RNA processing factors in MCF7 cells altered isoform expression. These results indicate that global dysregulation of splicing in breast cancer occurs in a subtype-specific and reproducible manner and is driven by specific differentially expressed RNA processing factors.

  20. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

    Directory of Open Access Journals (Sweden)

    Valeria Hansberg-Pastor

    2015-01-01

    Full Text Available The CCAAT/enhancer-binding protein beta (C/EBPβ is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

  1. Developmental changes in circulating IL-8/CXCL8 isoforms in neonates.

    Science.gov (United States)

    Maheshwari, Akhil; Voitenok, Nikolai N; Akalovich, Svetlana; Shaik, Sadiq S; Randolph, David A; Sims, Brian; Patel, Rakesh P; Killingsworth, Cheryl R; Fallon, Michael B; Ohls, Robin K

    2009-04-01

    Interleukin-8 (IL-8/CXCL8) is widely expressed in fetal tissues although inflammatory changes are not seen. Circulating IL-8 is comprised of an endothelial-derived [ala-IL-8](77) isoform and another, more potent [ser-IL-8](72) secreted by most other cells; [ala-IL-8](77) can be converted into [ser-IL-8](72) by proteolytic removal of an N-terminal pentapeptide from [ala-IL-8](77). In this study, we show [ala-IL-8](77) is the predominant circulating isoform of IL-8 in premature neonates but not in term neonates/adults, who have [ser-IL-8](72) as the major isoform. This isoform switch from the less potent [ala-IL-8](77) to [ser-IL-8](72) correlates with a maturational increase in the neutrophil chemotactic potency of plasma IL-8. The emergence of [ser-IL-8](72) as the major isoform is likely due to increased plasma [ala-IL-8](77)-convertase activity and/or changes in the cellular sources of IL-8. Developmental changes in IL-8 isoforms may serve to minimize its inflammatory effects in the fetus and also provide a mechanism to restore its full activity after birth.

  2. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    Science.gov (United States)

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-03

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  3. Molecular modeling study on tunnel behavior in different histone deacetylase isoforms.

    Directory of Open Access Journals (Sweden)

    Sundarapandian Thangapandian

    Full Text Available Histone deacetylases (HDACs have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E in HDAC10 and leucine (L in HDAC 11 based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.

  4. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    Science.gov (United States)

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  5. Relation of Transcriptional Factors to the Expression and Activity of Cytochrome P450 and UDP-Glucuronosyltransferases 1A in Human Liver: Co-Expression Network Analysis.

    Science.gov (United States)

    Zhong, Shilong; Han, Weichao; Hou, Chuqi; Liu, Junjin; Wu, Lili; Liu, Menghua; Liang, Zhi; Lin, Haoming; Zhou, Lili; Liu, Shuwen; Tang, Lan

    2017-01-01

    Cytochrome P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism of exogenous and endogenous compounds. The gene transcription of CYPs and UGTs can be enhanced or reduced by transcription factors (TFs). This study aims to explore novel TFs involved in the regulatory network of human hepatic UGTs/CYPs. Correlations between the transcription levels of 683 key TFs and CYPs/UGTs in three different human liver expression profiles (n = 640) were calculated first. Supervised weighted correlation network analysis (sWGCNA) was employed to define hub genes among the selected TFs. The relationship among 17 defined TFs, CYPs/UGTs expression, and activity were evaluated in 30 liver samples from Chinese patients. The positive controls (e.g., PPARA, NR1I2, NR1I3) and hub TFs (NFIA, NR3C2, and AR) in the Grey sWGCNA Module were significantly and positively associated with CYPs/UGTs expression. And the cancer- or inflammation-related TFs (TEAD4, NFKB2, and NFKB1) were negatively associated with mRNA expression of CYP2C9/CYP2E1/UGT1A9. Furthermore, the effect of NR1I2, NR1I3, AR, TEAD4, and NFKB2 on CYP450/UGT1A gene transcription translated into moderate influences on enzyme activities. To our knowledge, this is the first study to integrate Gene Expression Omnibus (GEO) datasets and supervised weighted correlation network analysis (sWGCNA) for defining TFs potentially related to CYPs/UGTs. We detected several novel TFs involved in the regulatory network of hepatic CYPs and UGTs in humans. Further validation and investigation may reveal their exact mechanism of CYPs/UGTs regulation.

  6. P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

    International Nuclear Information System (INIS)

    Liu Yang; Dong Qianze; Zhao Yue; Dong Xinjun; Miao Yuan; Dai Shundong; Yang Zhiqiang; Zhang Di; Wang Yan; Li Qingchang; Zhao Chen; Wang Enhua

    2009-01-01

    Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and β-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms

  7. A novel KCNQ1 nonsense variant in the isoform-specific first exon causes both jervell and Lange-Nielsen syndrome 1 and long QT syndrome 1: a case report.

    Science.gov (United States)

    Nishimura, Motoi; Ueda, Marehiko; Ebata, Ryota; Utsuno, Emi; Ishii, Takuma; Matsushita, Kazuyuki; Ohara, Osamu; Shimojo, Naoki; Kobayashi, Yoshio; Nomura, Fumio

    2017-06-08

    According to previous KCNQ1 (potassium channel, voltage gated, KQT-like subfamily, member 1) gene screening studies, missense variants, but not nonsense or frame-shift variants, cause the majority of long QT syndrome (LQTS; Romano-Ward syndrome [RWS]) 1 cases. Several missense variants are reported to cause RWS by a dominant-negative mechanism, and some KCNQ1 variants can cause both Jervell and Lange-Nielsen Syndrome (JLNS; in an autosomal recessive manner) and LQTS1 (in an autosomal dominant manner), while other KCNQ1 variants cause only JLNS. The human KCNQ1 gene is known to have two transcript isoforms (kidney isoform and pancreas isoform), and both isoforms can form a functional cardiac potassium channel. Here, we report a novel nonsense KCNQ1 variant causing not only JLNS, but also significant QTc prolongation identical to RWS in an autosomal dominant manner. Our case study supports that haploinsufficiency in the KCNQ1 gene is causative of significant QTc prolongation identical to RWS. Interestingly, the nonsense variant (NM_000218.2:c.115G > T [p.Glu39X]) locates in exon 1a of KCNQ1, which is a kidney-isoform specific exon. The variant is located closer to the N-terminus than previously identified nonsense or frame-shift variants. To the best of our knowledge, this is the first report showing that a nonsense variant in exon 1a of KCNQ1, which is the kidney-isoform specific exon, causes JLNS. Our findings may be informative to the genetic pathogenesis of RWS and JLNS caused by KCNQ1 variants.

  8. Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

    Directory of Open Access Journals (Sweden)

    De Santanu

    2012-01-01

    Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

  9. Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation.

    Science.gov (United States)

    Achariyar, Thiyagaragan M; Li, Baoman; Peng, Weiguo; Verghese, Philip B; Shi, Yang; McConnell, Evan; Benraiss, Abdellatif; Kasper, Tristan; Song, Wei; Takano, Takahiro; Holtzman, David M; Nedergaard, Maiken; Deane, Rashid

    2016-12-08

    Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it's expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation. We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student's t- test. We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state. Thus, choroid plexus

  10. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

  11. Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

    Science.gov (United States)

    Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

    2015-01-01

    The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

  12. Gene duplication and the evolution of hemoglobin isoform differentiation in birds.

    Science.gov (United States)

    Grispo, Michael T; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Storz, Jay F

    2012-11-02

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.

  13. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    Science.gov (United States)

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

  14. Complexities in human herpesvirus-6A and -6B binding to host cells

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Höllsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...

  15. Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

  16. Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.

    Science.gov (United States)

    Bhalla, Akhil; Chicka, Michael C; Chapman, Edwin R

    2008-08-01

    Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.

  17. Elevated serum tartrate-resistant acid phosphatase isoform 5a levels in metabolic syndrome.

    Science.gov (United States)

    Huang, Yi-Jhih; Huang, Tsai-Wang; Chao, Tsu-Yi; Sun, Yu-Shan; Chen, Shyi-Jou; Chu, Der-Ming; Chen, Wei-Liang; Wu, Li-Wei

    2017-09-29

    Tartrate-resistant phosphatase isoform 5a is expressed in tumor-associated macrophages and is a biomarker of chronic inflammation. Herein, we correlated serum tartrate-resistant phosphatase isoform 5a levels with metabolic syndrome status and made comparisons with traditional markers of inflammation, including c-reactive protein and interleukin-6. One hundred healthy volunteers were randomly selected, and cut-off points for metabolic syndrome related inflammatory biomarkers were determined using receiver operating characteristic curves. Linear and logistic regression models were subsequently used to correlate inflammatory markers with the risk of metabolic syndrome. Twenty-two participants met the criteria for metabolic syndrome, and serum tartrate-resistant phosphatase isoform 5a levels of >5.8 μg/L were associated with metabolic syndrome (c-statistics, 0.730; p = 0.001; 95% confidence interval, 0.618-0.842). In addition, 1 μg/L increases in tartrate-resistant phosphatase isoform 5a levels were indicative of a 1.860 fold increase in the risk of metabolic syndrome (p = 0.012). Elevated serum tartrate-resistant phosphatase isoform 5a levels are associated with the risk of metabolic syndrome, with a cut-off level of 5.8 μg/L.

  18. Novel frataxin isoforms may contribute to the pathological mechanism of Friedreich ataxia.

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    Haiyan Xia

    Full Text Available Friedreich ataxia (FRDA is an inherited neurodegenerative disease caused by frataxin (FXN deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III, which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.

  19. Proteomic Analysis of Parkin Isoforms Expression in Different Rat Brain Areas.

    Science.gov (United States)

    D'Amico, Agata Grazia; Maugeri, Grazia; Reitano, Rita; Cavallaro, Sebastiano; D'Agata, Velia

    2016-10-01

    PARK2 gene's mutations are related to the familial form of juvenile Parkinsonism, also known as the autosomic recessive juvenile Parkinsonism. This gene encodes for parkin, a 465-amino acid protein. To date, a large number of parkin isoforms, generated by an alternative splicing mechanism, have been described. Currently, Gene Bank lists 27 rat PARK2 transcripts, which matches to 20 exclusive parkin alternative splice variants. Despite the existence of these isoforms, most of the studies carried out so far, have been focused only on the originally cloned parkin. In this work we have analyzed the expression profile of parkin isoforms in some rat brain areas including prefrontal cortex, hippocampus, substantia nigra and cerebellum. To discriminate among these isoforms, we detected their localization through the use of two antibodies that are able to identify different domains of the parkin canonical sequence. Our analysis has revealed that at least fourteen parkin isoforms are expressed in rat brain with a various distribution in the regions analyzed. Our study might help to elucidate the pathophysiological role of these proteins in the central nervous system.

  20. Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

    2017-03-01

    Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

  1. Multiple isoforms for the catalytic subunit of PKA in the basal fungal lineage Mucor circinelloides.

    Science.gov (United States)

    Fernández Núñez, Lucas; Ocampo, Josefina; Gottlieb, Alexandra M; Rossi, Silvia; Moreno, Silvia

    2016-12-01

    Protein kinase A (PKA) activity is involved in dimorphism of the basal fungal lineage Mucor. From the recently sequenced genome of Mucor circinelloides we could predict ten catalytic subunits of PKA. From sequence alignment and structural prediction we conclude that the catalytic core of the isoforms is conserved, and the difference between them resides in their amino termini. This high number of isoforms is maintained in the subdivision Mucoromycotina. Each paralogue, when compared to the ones form other fungi is more homologous to one of its orthologs than to its paralogs. All of these fungal isoforms cannot be included in the class I or II in which fungal protein kinases have been classified. mRNA levels for each isoform were measured during aerobic and anaerobic growth. The expression of each isoform is differential and associated to a particular growth stage. We reanalyzed the sequence of PKAC (GI 20218944), the only cloned sequence available until now for a catalytic subunit of M. circinelloides. PKAC cannot be classified as a PKA because of its difference in the conserved C-tail; it shares with PKB a conserved C2 domain in the N-terminus. No catalytic activity could be measured for this protein nor predicted bioinformatically. It can thus be classified as a pseudokinase. Its importance can not be underestimated since it is expressed at the mRNA level in different stages of growth, and its deletion is lethal. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  2. MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

    Science.gov (United States)

    Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

    2011-07-01

    Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

  3. Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

    Science.gov (United States)

    Isakov, Noah

    2018-02-01

    The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its

  4. Differential expression of a new isoform of DLG2 in renal oncocytoma

    International Nuclear Information System (INIS)

    Zubakov, Dmitry; Stupar, Zorica; Kovacs, Gyula

    2006-01-01

    Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma

  5. The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

    Science.gov (United States)

    Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

    2017-06-26

    Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

  6. Branchial Expression Patterns of Claudin Isoforms in Atlantic Salmon During Seawater Acclimation and Smoltification

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Kiilerich, Pia; Nilsen, Tom O

    2008-01-01

    in epithelia. We identified Atlantic salmon genes belonging to the claudin family by screening expressed sequence tag libraries available at NCBI and classification was performed with aid of maximum likelihood and neighbour-joining analysis. In gill libraries, five isoforms (10e, 27a, 28a, 28b and 30) were...... present and QPCR analysis confirmed tissue-specific expression in gill when compared to kidney, intestine, heart, muscle, brain and liver. Expression patterns during acclimation of freshwater salmon to seawater (SW) and during the smoltification process were examined. Acclimation to SW reduced...... induced no significant changes in expression of the other isoforms. This study demonstrates the expression of an array of salmon claudin isoforms and shows that SW acclimation involves inverse regulation, in the gill, of claudin 10e versus claudin 27a and 30. It is possible, that claudin 10e...

  7. The Р60-S6K1 isoform of ribosomal protein S6 kinase 1 is a product of alternative mRNA translation

    Directory of Open Access Journals (Sweden)

    I. V. Zaiets

    2018-07-01

    Full Text Available Ribosomal protein S6 kinase 1 (S6K1 is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1 participating primarily in the regulation of cell growth and metabolism. Deregulation of mTOR/S6K1 signaling can promote numerous human pathologies, including cancer, neurodegeneration, cardiovascular disease, and metabolic disorders. As existing data suggest, the S6K1 gene encodes several protein isoforms, including p85-S6K1, p70-S6K1, and p60-S6K1. The two of these isoforms, p85-S6K1 and p70-S6K1, were extensively studied to date. The origin and functional significance of the p60-S6K1 isoform remains a mystery, however, it was suggested that the isoform could be a product of alternative S6K1 mRNA translation. Herein we report the generation of HEK-293 cells exclusively expressing p60-S6K1 as a result of CRISPR/Cas9-mediated inactivation of p85/p70-S6K1 translation. Moreover, the generated modified cells displayed the elevated level of p60-S6K1 expression compared to that in wild-type HEK-293 cells. Our data confirm an assumption that p60-S6K1 is alternatively translated, most probably, from the common for both p70- and p85-S6K1 mRNA transcript and reveal a link between p60-S6K1 expression and such cellular processes as cell proliferation and motility. In addition, our findings indicate that the p60-S6K1 isoform of S6K1 may undergo a mode of regulation distinct from p70- and p85-S6K1 due to the absence of mTOR-regulated p60-S6K1 phosphorylation at T389 that is important for S6K1 activation.

  8. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

    Science.gov (United States)

    Seeger, Tanja; Hart, Melanie; Patarroyo, Manuel; Rolauffs, Bernd; Aicher, Wilhelm K.; Klein, Gerd

    2015-01-01

    Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells. PMID:26406476

  9. Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation.

    Directory of Open Access Journals (Sweden)

    Tanja Seeger

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are well known for their tri-lineage potential and ability to differentiate in vitro into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated in vitro into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521 showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.

  10. GSK3β isoform-selective regulation of depression, memory and hippocampal cell proliferation.

    Science.gov (United States)

    Pardo, M; Abrial, E; Jope, R S; Beurel, E

    2016-03-01

    Abnormally active glycogen synthase kinase-3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood-relevant behaviors, impairing performance in several cognitive tasks and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine-to-alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression-like behavior. Three cognitive measures impaired in GSK3α/β knockin mice showed differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not in GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and co-ordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of overexpression or disease pathology, is sufficient to impair mood regulation, novel object recognition and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results show that hyperactivity of the two GSK3 isoforms execute non-redundant effects on these processes. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  11. BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer

    Science.gov (United States)

    Link, Petra A.; Zhang, Wa; Odunsi, Kunle; Karpf, Adam R.

    2013-01-01

    Cancer germline (CG) genes are normally expressed in germ cells and aberrantly expressed in a variety of cancers; their immunogenicity has led to the widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL is an autosomal CG antigen and promising cancer vaccine target. BORIS is the only known paralog of CTCF, a gene intimately involved in genomic imprinting, chromatin insulation, and nuclear regulation. We have previously shown that BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression coincides with promoter and global DNA hypomethylation. Recently, 23 different BORIS mRNA variants have been described, and have been functionally grouped into six BORIS isoform families (sf1–sf6). In the present study, we have characterized the expression of BORIS isoform families in normal ovary (NO) and EOC, the latter of which were selected to include two groups with widely varying global DNA methylation status. We find selective expression of BORIS isoform families in NO, which becomes altered in EOC, primarily by the activation of BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we find that BORIS sf1 and sf2 isoform families are selectively activated in globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform families was induced to varying extents by epigenetic modulatory drugs in EOC cell lines, particularly when DNMT and HDAC inhibitors were used in combination. PMID:23390377

  12. Expression of Metallothionein and Vascular Endothelial Growth Factor Isoforms in Breast Cancer Cells.

    Science.gov (United States)

    Wierzowiecka, Barbara; Gomulkiewicz, Agnieszka; Cwynar-Zajac, Lucja; Olbromski, Mateusz; Grzegrzolka, Jedrzej; Kobierzycki, Christopher; Podhorska-Okolow, Marzenna; Dziegiel, Piotr

    2016-01-01

    Metallothioneins (MTs) are low-molecular-weight and cysteine-rich proteins that bind heavy metal ions and oxygen-free radicals. MTs are commonly expressed in various tissues of mammals and are involved in regulation of cell proliferation and differentiation, and may be engaged in angiogenesis. Expression of MTs has been studied in many cancer types, especially breast cancer. The research results indicate that MTs may play important, although not yet fully known, roles in cancer angiogenesis. The aim of this study was to analyze the level of gene expression of selected MT isoforms induced with zinc ions in correlation with vascular endothelial growth factor (VEGF) isoforms in in vitro models of breast cancer. The studies were carried out in three breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231). An epithelial cell line derived from normal breast tissue (Me16c) was used as a control. The levels of expression of selected MT isoforms and selected genes involved in angiogenesis were studied with real-time PCR. Expression of different MT isoforms was induced by zinc ions to differing degrees in individual breast cancer cell lines. An increase in the expression of some MT isoforms was associated with a slight increase in the level of expression of VEGFA. The research results may indicate certain correlation between an increased expression of selected MT isoforms and a pro-angiogenic factor VEGF in specific types of breast cancer cells. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-β Isoforms for Early and Differential Dementia Diagnosis.

    Science.gov (United States)

    Struyfs, Hanne; Van Broeck, Bianca; Timmers, Maarten; Fransen, Erik; Sleegers, Kristel; Van Broeckhoven, Christine; De Deyn, Peter P; Streffer, Johannes R; Mercken, Marc; Engelborghs, Sebastiaan

    2015-01-01

    Overlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimer's disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-β (Aβ) isoforms might improve the AD versus non-AD differential diagnosis. To determine the added diagnostic value of Aβ isoforms, Aβ(1-37), Aβ(1-38), and Aβ(1-40), as compared to the AD CSF biomarkers Aβ(1-42), T-tau, and P-tau(181P). CSF from patients with dementia due to AD (n = 50), non-AD dementias (n = 50), mild cognitive impairment due to AD (n = 50) and non-demented controls (n = 50) was analyzed with a prototype multiplex assay using MSD detection technology. The non-AD group consisted of frontotemporal dementia (FTD; n = 17), dementia with Lewy bodies (DLB; n = 17), and vascular dementia (n = 16). Aβ(1-37) and Aβ(1-38) increased accuracy to differentiate AD from FTD or DLB. Aβ(1-37), Aβ(1-38), and Aβ(1-40) levels correlated with Mini-Mental State Examination scores and disease duration in dementia due to AD. The Aβ(1-42)/Aβ(1-40) ratio improved diagnostic performance of Aβ(1-42) in most differential diagnostic situations. Aβ(1-42) levels were lower in APOE ε4 carriers compared to non-carriers. Aβ isoforms help to differentiate AD from FTD and DLB. Aβ isoforms increase diagnostic performance of Aβ(1-42). In contrast to Aβ1-42, Aβ isoforms seem to be correlated with disease severity in AD. Adding the Aβ isoforms to the current biomarker panel could enhance diagnostic accuracy.

  14. Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

    Science.gov (United States)

    Bhattacharyya, Dipto; Hazra, Saptarshi; Banerjee, Anindyajit; Datta, Riddhi; Kumar, Deepak; Chakrabarti, Saikat; Chattopadhyay, Sharmila

    2016-09-01

    Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

  15. The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

    Directory of Open Access Journals (Sweden)

    Lili Li

    Full Text Available Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB(αΔ mice. Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ mice. CREB(αΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ. CREB(αΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

  16. Three-dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte-like cells: a comparative study.

    Science.gov (United States)

    Chitrangi, Swati; Nair, Prabha; Khanna, Aparna

    2017-08-01

    Stem cell-based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro-environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three-dimensional (3D) micro-environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran-gelatin (DG1), chitosan-hyaluronic acid (CH1) and gelatin-vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was induced by culturing hUC-MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro-environment and architecture to facilitate 3D cell-cell and cell-matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC-MSCs. The differentiated hUC-MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel-coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well-characterized scaffold (GEVAC) for enhanced differentiation of hUC-MSCs to hepatocyte-like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    Science.gov (United States)

    2011-01-01

    Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H

  18. Effect of renal replacement therapy on retinol-binding protein 4 isoforms

    DEFF Research Database (Denmark)

    Frey, Simone K; Henze, Andrea; Nagl, Britta

    2009-01-01

    Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney dis...... diseases but not in serum of patients with various liver diseases. The aim of this study was to investigate the effect of renal replacement therapy on RBP4 isoforms....

  19. Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

    Science.gov (United States)

    Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

    2014-01-01

    The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. © 2013 Anatomical Society.

  20. VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A

    Directory of Open Access Journals (Sweden)

    Pio Ruben

    2010-12-01

    Full Text Available Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189 have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p xxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033 between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken

  1. The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion

    Science.gov (United States)

    Fink, Annette; Renzaho, Angeliqué; Reddehase, Matthias J.; Lemmermann, Niels A. W.

    2013-01-01

    The MHC-class I (MHC-I)-like viral (MHC-Iv) m152 gene product of murine cytomegalovirus (mCMV) was the first immune evasion molecule described for a member of the β-subfamily of herpesviruses as a paradigm for analogous functions of human cytomegalovirus proteins. Notably, by interacting with classical MHC-I molecules and with MHC-I-like RAE1 family ligands of the activatory natural killer (NK) cell receptor NKG2D, it inhibits presentation of antigenic peptides to CD8 T cells and the NKG2D-dependent activation of NK cells, respectively, thus simultaneously interfering with adaptive and innate immune recognition of infected cells. Although the m152 gene product exists in differentially glycosylated isoforms whose individual contributions to immune evasion are unknown, it has entered the scientific literature as m152/gp40, based on the quantitatively most prominent isoform but with no functional justification. By construction of a recombinant mCMV in which all three N-glycosylation sites are mutated (N61Q, N208Q, and N241Q), we show here that N-linked glycosylation is not essential for functional interaction of the m152 immune evasion protein with either MHC-I or RAE1. These data add an important functional detail to recent structural analysis of the m152/RAE1γ complex that has revealed N-glycosylations at positions Asn61 and Asn208 of m152 distant from the m152/RAE1γ interface. PMID:24351798

  2. The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion

    Directory of Open Access Journals (Sweden)

    Annette Fink

    2013-12-01

    Full Text Available The MHC-class I (MHC-I-like viral (MHC-Iv m152 gene product of murine cytomegalovirus (mCMV was the first immune evasion molecule described for a member of the β-subfamily of herpesviruses as a paradigm for analogous functions of human cytomegalovirus proteins. Notably, by interacting with classical MHC-I molecules and with MHC-I-like RAE1 family ligands of the activatory natural killer (NK cell receptor NKG2D, it inhibits presentation of antigenic peptides to CD8 T cells and the NKG2D-dependent activation of NK cells, respectively, thus simultaneously interfering with adaptive and innate immune recognition of infected cells. Although the m152 gene product exists in differentially glycosylated isoforms whose individual contributions to immune evasion are unknown, it has entered the scientific literature as m152/gp40, based on the quantitatively most prominent isoform but with no functional justification. By construction of a recombinant mCMV in which all three N-glycosylation sites are mutated (N61Q, N208Q, and N241Q, we show here that N-linked glycosylation is not essential for functional interaction of the m152 immune evasion protein with either MHC-I or RAE1. These data add an important functional detail to recent structural analysis of the m152/RAE1g complex that has revealed N-glycosylations at positions Asn61 and Asn208 of m152 distant from the m152/RAE1g interface.

  3. Skeletal muscle myosin heavy chain isoform content in relation to gonadal hormones and anabolic-catabolic balance in trained and untrained men.

    Science.gov (United States)

    Grandys, Marcin; Majerczak, Joanna; Karasinski, Janusz; Kulpa, Jan; Zoladz, Jerzy A

    2012-12-01

    Gonadal hormones and anabolic-catabolic hormone balance have potent influence on skeletal muscle tissue, but little is known about their action with regard to myosin heavy chain (MHC) transformation in humans. We investigated the relationship between skeletal muscle MHC isoform content in the vastus lateralis muscle and basal testosterone (T) concentration in 3 groups of subjects: endurance trained (E), sprint/strength trained (S), and untrained (U) young men. We have also determined basal sex hormone-binding globulin and cortisol (C) concentrations in untrained subjects to examine the relationship between MHC composition and the anabolic-catabolic hormone balance. Moreover, basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations were calculated for this subgroup. Despite significant differences in MHC isoform content (69.4 ± 2.39%, 61.4 ± 8.04%, and 37.5 ± 13.80% of MHC-2 for groups S, U, and E, respectively, Kruskal-Wallis: H = 18.58, p 0.5). We have also found that in the U group, type 2 MHC in the vastus lateralis muscle is positively correlated with basal fT:C ratio (r = 0.63, p = 0.01). It is concluded that the differences in the training history and training specificity can be distinguished with regard to the MHC composition but not with regard to the basal T concentration. Simultaneously, it has been shown that MHC isoform content in human vastus lateralis muscle may be related to basal anabolic-catabolic hormone balance, and this hypothesis needs further investigation.

  4. Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

    Science.gov (United States)

    Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

    2014-10-10

    Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

  5. Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

    Science.gov (United States)

    The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) [Lp(a)], Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examinati...

  6. Distinct transthyretin oxidation isoform profile in spinal fluid from patients with Alzheimer’s disease and mild cognitive impairment

    DEFF Research Database (Denmark)

    Poulsen, Keld; Bahl, Justyna Mc; Simonsen, Anja H

    2014-01-01

    BACKGROUND: Transthyretin (TTR), an abundant protein in cerebrospinal fluid (CSF), contains a free, oxidation-prone cysteine residue that gives rise to TTR isoforms. These isoforms may reflect conditions in vivo. Since increased oxidative stress has been linked to neurodegenerative disorders such...

  7. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    Science.gov (United States)

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  8. D-serine : The right or wrong isoform?

    NARCIS (Netherlands)

    Fuchs, Sabine A; Berger, Ruud; de Koning, Tom J

    2011-01-01

    Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to

  9. SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box

    International Nuclear Information System (INIS)

    Short, Stephen; Malartre, Marianne; Sharpe, Colin

    2005-01-01

    SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT

  10. The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

    Science.gov (United States)

    Cao, Yanyun; Zhao, Luping; Ying, Yusang; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-06-15

    Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Sodium valproate increases the brain isoform of glycogen phosphorylase: looking for a compensation mechanism in McArdle disease using a mouse primary skeletal-muscle culture in vitro

    Directory of Open Access Journals (Sweden)

    Noemí de Luna

    2015-05-01

    Full Text Available McArdle disease, also termed ‘glycogen storage disease type V’, is a disorder of skeletal muscle carbohydrate metabolism caused by inherited deficiency of the muscle-specific isoform of glycogen phosphorylase (GP-MM. It is an autosomic recessive disorder that is caused by mutations in the PYGM gene and typically presents with exercise intolerance, i.e. episodes of early exertional fatigue frequently accompanied by rhabdomyolysis and myoglobinuria. Muscle biopsies from affected individuals contain subsarcolemmal deposits of glycogen. Besides GP-MM, two other GP isoforms have been described: the liver (GP-LL and brain (GP-BB isoforms, which are encoded by the PYGL and PYGB genes, respectively; GP-BB is the main GP isoform found in human and rat foetal tissues, including the muscle, although its postnatal expression is dramatically reduced in the vast majority of differentiated tissues with the exception of brain and heart, where it remains as the major isoform. We developed a cell culture model from knock-in McArdle mice that mimics the glycogen accumulation and GP-MM deficiency observed in skeletal muscle from individuals with McArdle disease. We treated mouse primary skeletal muscle cultures in vitro with sodium valproate (VPA, a histone deacetylase inhibitor. After VPA treatment, myotubes expressed GP-BB and a dose-dependent decrease in glycogen accumulation was also observed. Thus, this in vitro model could be useful for high-throughput screening of new drugs to treat this disease. The immortalization of these primary skeletal muscle cultures could provide a never-ending source of cells for this experimental model. Furthermore, VPA could be considered as a gene-expression modulator, allowing compensatory expression of GP-BB and decreased glycogen accumulation in skeletal muscle of individuals with McArdle disease.

  12. Comparison of inhibition capability of scutellarein and scutellarin towards important liver UDP-glucuronosyltransferase (UGT) isoforms.

    Science.gov (United States)

    Ma, Guang-You; Cao, Yun-Feng; Hu, Cui-Min; Fang, Zhong-Ze; Sun, Xiao-Yu; Hong, Mo; Zhu, Zhi-Tu

    2014-03-01

    Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand-Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP-glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (Ki ) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the Ki values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro-in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Isoform 1 of TPD52 (PC-1) promotes neuroendocrine transdifferentiation in prostate cancer cells

    KAUST Repository

    Moritz, Tom; Venz, Simone; Junker, Heike; Kreuz, Sarah; Walther, Reinhard; Zimmermann, Uwe

    2016-01-01

    The tumour protein D52 isoform 1 (PC-1), a member of the tumour protein D52 (TPD52) protein family, is androgen-regulated and prostate-specific expressed. Previous studies confirmed that PC-1 contributes to malignant progression in prostate cancer

  14. Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

    NARCIS (Netherlands)

    West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi

    2015-01-01

    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,

  15. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    NARCIS (Netherlands)

    Eilers, W.; Gevers, W.; van Overbeek, D.; de Haan, A.; Jaspers, R.T.; Hilbers, P.A.; van Riel, A.C.R.; Flueck, M.

    2014-01-01

    We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its

  16. Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

    DEFF Research Database (Denmark)

    Naik, M U; Benedikz, Eirikur; Hernandez, I

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region...

  17. Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

    DEFF Research Database (Denmark)

    Ross, Fiona A; Jensen, Thomas Elbenhardt; Hardie, D Grahame

    2016-01-01

    The g subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different g isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged ve...

  18. Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis

    Directory of Open Access Journals (Sweden)

    Xianghe Yuan

    2016-02-01

    Full Text Available The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50–60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0–8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat were determined by using a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.

  19. Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms

    DEFF Research Database (Denmark)

    Krintel, Christian; Klint, Cecilia; Lindvall, Håkan

    2010-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependen...

  20. Role of the AMPKgamma3 isoform in hypoxia-stimulated glucose transport in glycolytic skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Glund, Stephan; Tom, Robby Z

    2009-01-01

    , phosphorylation of CaMKII, AMPK, ACC, and TBC1D1/D4 as well as isoform-specific AMPK activity was determined. Basal and hypoxia-mediated phosphorylation of CaMKII, AMPK, and ACC as well as alpha1- and alpha2-associated AMPK activity was comparable between AMPKgamma3-KO and wild-type mice. KN-93 reduced hypoxia...

  1. TGF-β's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

    International Nuclear Information System (INIS)

    Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

    2009-01-01

    Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-β (TGF-β) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-β on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-β1, -β2 and -β3 on wound repair in other tissues. In the current study we compared the effect of TGF-β1, -β2 and -β3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-β increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-β1, -β2 and -β3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-β promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner

  2. Thick filament length and isoform composition determine self-organized contractile units in actomyosin bundles.

    Science.gov (United States)

    Thoresen, Todd; Lenz, Martin; Gardel, Margaret L

    2013-02-05

    Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Neuronal Glucose Transporter Isoform 3 Deficient Mice Demonstrate Features of Autism Spectrum Disorders

    OpenAIRE

    Zhao, Yuanzi; Fung, Camille; Shin, Don; Shin, Bo-Chul; Thamotharan, Shanthie; Sankar, Raman; Ehninger, Dan; Silva, Alcino; Devaskar, Sherin U.

    2009-01-01

    Neuronal glucose transporter (GLUT) isoform 3 deficiency in null heterozygous mice led to abnormal spatial learning and working memory but normal acquisition and retrieval during contextual conditioning, abnormal cognitive flexibility with intact gross motor ability, electroencephalographic seizures, perturbed social behavior with reduced vocalization and stereotypies at low frequency. This phenotypic expression is unique as it combines the neurobehavioral with the epileptiform characteristic...

  4. Alternative NF-κB Isoforms in the Drosophila Neuromuscular Junction and Brain.

    Directory of Open Access Journals (Sweden)

    Bo Zhou

    Full Text Available The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, we demonstrate that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. We find that Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. We further find that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-κB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, we demonstrate that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.

  5. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    International Nuclear Information System (INIS)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

    2006-01-01

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  6. The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

    Science.gov (United States)

    Obis, Teresa; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Priego, Mercedes; Simon, Anna; Garcia, Neus; Santafe, Manel M; Lanuza, Maria A; Tomàs, Josep

    2015-12-01

    Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction. We use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca(2+), stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion. Together, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

  7. The Invasion and Metastasis Promotion Role of CD97 Small Isoform in Gastric Carcinoma

    DEFF Research Database (Denmark)

    Liu, Daren; Trojanowicz, Bogusz; Ye, Longyun

    2012-01-01

    CD97 is over-expressed in the majority of gastric adenocarcinomas and is associated with its dedifferentiation and aggressiveness. Our previous results demonstrated that out of three CD97 isoforms tested, only the small one was able to promote increased invasiveness in vitro. Based on these data ...

  8. Roles of SGK Isoform Signaling in Breast Cancer Migration and Invasion

    Science.gov (United States)

    2011-04-01

    significant number of overlapping substrates, and deregulation in breast carcinoma (5,6). To date, no studies have investigated any role for SGK in cell...isoforms in breast carcinoma cell lines (months 2-3) To insure specificity of SGK knockdown in breast cancer cell lines I made two different specific

  9. The activity and isoforms of NADP-malic enzyme in Nicotiana benthamiana plants under biotic stress

    Czech Academy of Sciences Publication Activity Database

    Doubnerová, V.; Jirásková, A.; Janošková, M.; Müller, Karel; Baťková, Petra; Synková, Helena; Čeřovská, Noemi; Ryšlavá, H.

    2007-01-01

    Roč. 26, č. 4 (2007), s. 281-289 ISSN 0231-5882 Institutional research plan: CEZ:AV0Z50380511 Keywords : NADP * malic enzyme isoforms * Nicotiana benthamiana Subject RIV: EF - Botanics Impact factor: 1.286, year: 2007 http://www.gpb.sav.sk/2007-4.htm

  10. Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2.

    Directory of Open Access Journals (Sweden)

    Sebastien A Gauthier

    Full Text Available The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR and Ultraspiracle (USP. In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4 is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC, the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH, an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.

  11. Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

    Directory of Open Access Journals (Sweden)

    Ma Xin-Ming

    2011-02-01

    Full Text Available Abstract Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of

  12. Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

    Science.gov (United States)

    Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

    2018-06-03

    Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

  13. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail: kawamots@mail.nih.gov

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  14. Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

    Science.gov (United States)

    Orfanos, Zacharias; Sparrow, John C

    2013-01-01

    During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

  15. Statistical modeling of isoform splicing dynamics from RNA-seq time series data.

    Science.gov (United States)

    Huang, Yuanhua; Sanguinetti, Guido

    2016-10-01

    Isoform quantification is an important goal of RNA-seq experiments, yet it remains problematic for genes with low expression or several isoforms. These difficulties may in principle be ameliorated by exploiting correlated experimental designs, such as time series or dosage response experiments. Time series RNA-seq experiments, in particular, are becoming increasingly popular, yet there are no methods that explicitly leverage the experimental design to improve isoform quantification. Here, we present DICEseq, the first isoform quantification method tailored to correlated RNA-seq experiments. DICEseq explicitly models the correlations between different RNA-seq experiments to aid the quantification of isoforms across experiments. Numerical experiments on simulated datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. On real datasets, our results show that DICEseq provides substantially more reproducible and robust quantifications, increasing the correlation of estimates from replicate datasets by up to 10% on genes with low or moderate expression levels (bottom third of all genes). Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Our results have strong implications for the design of RNA-seq experiments, and offer a novel tool for improved analysis of such datasets. Python code is freely available at http://diceseq.sf.net G.Sanguinetti@ed.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

    Science.gov (United States)

    Masroori, Nasser; Merindol, Natacha; Berthoux, Lionel

    2016-03-22

    The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has

  17. DMPD: The role of C/EBP isoforms in the control of inflammatory and native immunityfunctions. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9792624 The role of C/EBP isoforms in the control of inflammatory and native immunityfunction...f C/EBP isoforms in the control of inflammatory and native immunityfunctions. PubmedID 9792624 Title The rol...e of C/EBP isoforms in the control of inflammatory and native immunityfunctions.

  18. Crystallization and Identification of the Glycosylated Moieties of Two Isoforms of the Main Allergen Hev b 2 and Preliminary X-ray Analysis of Two Polymorphs of Isoform ll

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva,D.; Mendoza-Hernandez, G.; Stojanoff, V.; Palomares, L.; Zenteno, E.; Torres-Larios, A.; Rodriguez-Romero, A.

    2007-01-01

    Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a {beta}-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapor-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 {angstrom} were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 {angstrom}, {beta}= 113.6{sup o}. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

  19. Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva, D. [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Mendoza-Hernández, G. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Stojanoff, V. [Brookhaven National Laboratory, National Synchrotron Light Source, Upton, NY (United States); Palomares, L. A. [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Zenteno, E. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Torres-Larios, A. [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Rodríguez-Romero, A., E-mail: adela@servidor.unam.mx [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico)

    2007-09-01

    Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

  20. Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

    International Nuclear Information System (INIS)

    Fuentes-Silva, D.; Mendoza-Hernández, G.; Stojanoff, V.; Palomares, L. A.; Zenteno, E.; Torres-Larios, A.; Rodríguez-Romero, A.

    2007-01-01

    Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4 1 with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units

  1. Novel P2 promoter-derived HNF4α isoforms with different N-terminus generated by alternate exon insertion

    International Nuclear Information System (INIS)

    Huang, Jianmin; Levitsky, Lynne L.; Rhoads, David B.

    2009-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is a critical transcription factor for pancreas and liver development and functions in islet β cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4α variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4α variants designated HNF4α10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4α10-α12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding α7-α9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4α10-α12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4α than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4α mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  2. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jianmin, E-mail: jmhuang@partners.org [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Levitsky, Lynne L. [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Rhoads, David B., E-mail: rhoads@helix.mgh.harvard.edu [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States)

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  3. A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

    Directory of Open Access Journals (Sweden)

    Andrew L Mammen

    Full Text Available Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish, UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

  4. Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Christine Chaponnier

    2016-06-01

    Full Text Available Higher vertebrates (mammals and birds express six different highly conserved actin isoforms that can be classified in three subgroups: 1 sarcomeric actins, α-skeletal (α-SKA and α-cardiac (α-CAA, 2 smooth muscle actins (SMAs, α-SMA and γ-SMA, and 3 cytoplasmic actins (CYAs, β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb against an actin isoform (α-SMA was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986 . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS. In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

  5. Functional properties of human neuronal Kv11 channels

    DEFF Research Database (Denmark)

    Einarsen, Karoline; Calloe, Kirstine; Grunnet, Morten

    2009-01-01

    Kv11 potassium channels are important for regulation of the membrane potential. Kv11.2 and Kv11.3 are primarily found in the nervous system, where they most likely are involved in the regulation of neuronal excitability. Two isoforms of human Kv11.2 have been published so far. Here, we present...... current characteristics of the isoforms presented in this work may contribute to the regulation of neuronal excitability....

  6. 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

    -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...

  7. Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

    Directory of Open Access Journals (Sweden)

    Chryso Kanthou

    Full Text Available Vascular endothelial growth factor-A (VEGF is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120 on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188 or wild type controls (fswt were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine

  8. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

    Directory of Open Access Journals (Sweden)

    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  9. SSP: an interval integer linear programming for de novo transcriptome assembly and isoform discovery of RNA-seq reads.

    Science.gov (United States)

    Safikhani, Zhaleh; Sadeghi, Mehdi; Pezeshk, Hamid; Eslahchi, Changiz

    2013-01-01

    Recent advances in the sequencing technologies have provided a handful of RNA-seq datasets for transcriptome analysis. However, reconstruction of full-length isoforms and estimation of the expression level of transcripts with a low cost are challenging tasks. We propose a novel de novo method named SSP that incorporates interval integer linear programming to resolve alternatively spliced isoforms and reconstruct the whole transcriptome from short reads. Experimental results show that SSP is fast and precise in determining different alternatively spliced isoforms along with the estimation of reconstructed transcript abundances. The SSP software package is available at http://www.bioinf.cs.ipm.ir/software/ssp. © 2013.

  10. Hypomethylation and Over-Expression of the Beta Isoform of BLIMP1 is Induced by Epstein-Barr Virus Infection of B Cells; Potential Implications for the Pathogenesis of EBV-Associated Lymphomas

    Directory of Open Access Journals (Sweden)

    Katerina Vrzalikova

    2012-10-01

    Full Text Available B-lymphocyte-induced maturation protein 1 (BLIMP1 exists as two major isoforms, α and β, which arise from alternate promoters. Inactivation of the full length BLIMP1α isoform is thought to contribute to B cell lymphomagenesis by blocking post-germinal centre (GC B cell differentiation. In contrast, the shorter β isoform is functionally impaired and over-expressed in several haematological malignancies, including diffuse large B cell lymphomas (DLBCL. We have studied the influence on BLIMP1β expression of the Epstein-Barr virus (EBV, a human herpesvirus that is implicated in the pathogenesis of several GC-derived lymphomas, including a subset of DLBCL and Hodgkin’s lymphoma (HL. We show that BLIMP1β expression is increased following the EBV infection of normal human tonsillar GC B cells. We also show that this change in expression is accompanied by hypomethylation of the BLIMP1β-specific promoter. Furthermore, we confirmed previous reports that the BLIMP1β promoter is hypomethylated in DLBCL cell lines and show for the first time that BLIMP1β is hypomethylated in the Hodgkin/Reed-Sternberg (HRS cells of HL. Our results provide evidence in support of a role for BLIMP1β in the pathogenesis of EBV-associated B cell lymphomas.

  11. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    Science.gov (United States)

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  12. Examination of Gelatinase Isoforms in Rodent Models of Acute Neurodegenerative Diseases Using Two-Dimensional Zymography.

    Science.gov (United States)

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Qu, Zhe; Cui, Jiankun; Gu, Zezong

    2017-01-01

    Pathological activation of gelatinases (matrix metalloproteinase-2 and -9; MMP-2/-9) has been shown to cause a number of detrimental outcomes in neurodegenerative diseases. In gel gelatin zymography is a highly sensitive methodology commonly used in revealing levels of gelatinase activity and in separating the proform and active form of gelatinases, based on their different molecular weights. However, this methodology is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity can be regulated at transcriptional and/or post-translational levels under in vivo conditions resulting in alternation of their isoelectric focusing (IEF) points. In this chapter, we describe an advanced methodology, termed two-dimensional zymography, combining IEF with zymographic electrophoresis under non-reducing conditions to achieve significant improvement in separation of the gelatinase isoforms in both cell-based and in vivo models for acute brain injuries and neuroinflammation.

  13. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

    2013-01-01

    When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2......-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from...... turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site...

  14. Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

    Science.gov (United States)

    Besalduch, Núria; Tomàs, Marta; Santafé, Manel M; Garcia, Neus; Tomàs, Josep; Lanuza, Maria Angel

    2010-01-10

    Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

  15. Mena invasive (Mena(INV)) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM.

    Science.gov (United States)

    Roussos, Evanthia T; Goswami, Sumanta; Balsamo, Michele; Wang, Yarong; Stobezki, Robert; Adler, Esther; Robinson, Brian D; Jones, Joan G; Gertler, Frank B; Condeelis, John S; Oktay, Maja H

    2011-08-01

    Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (Mena(INV)) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena(INV) expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena(INV) mRNA than early non-metastatic ones. Furthermore, Mena(INV) expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena(INV) is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena(INV) expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

  16. The predominant WT1 isoform (+KTS) encodes a DNA-binding protein targeting the planar cell polarity gene Scribble in renal podocytes.

    Science.gov (United States)

    Wells, Julie; Rivera, Miguel N; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A

    2010-07-01

    WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three-amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)-regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway.

  17. The predominant WT1 isoform (+KTS) encodes a DNA binding protein targeting the planar cell polarity gene Scribble in renal podocytes

    Science.gov (United States)

    Wells, Julie; Rivera, Miguel N.; Kim, Woo Jae; Starbuck, Kristen; Haber, Daniel A.

    2010-01-01

    WT1 encodes a tumor suppressor, first identified by its inactivation in Wilms Tumor. While one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three amino acid (KTS) insertion. Using cells which conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning (ChIP-cloning) analysis to identify candidate WT1(+KTS) regulated promoters. We identified the planar cell polarity (PCP) gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33 nucleotide region within the Scribble promoter in both mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway. PMID:20571064

  18. Unique in vitro and in vivo thrombopoietic activities of ingenol 3,20 dibenzoate, a Ca(++-independent protein kinase C isoform agonist.

    Directory of Open Access Journals (Sweden)

    Frederick K Racke

    Full Text Available Thrombopoiesis following severe bone marrow injury frequently is delayed, thereby resulting in life-threatening thrombocytopenia for which there are limited treatment options. The reasons for these delays in recovery are not well understood. Protein kinase C (PKC agonists promote megakaryocyte differentiation in leukemia cell lines and primary cells. However, little is known about the megakaryopoietic effects of PKC agonists on primary CD34+ cells grown in culture or in vivo. Here we present evidence that the novel PKC isoform-selective agonist 3,20 ingenol dibenzoate (IDB potently stimulates early megakaryopoiesis of human CD34+ cells. In contrast, broad spectrum PKC agonists failed to do so. In vivo, a single intraperitoneal injection of IDB selectively increased platelets in mice without affecting hemoglobin or white counts. Finally, IDB strongly mitigated radiation-induced thrombocytopenia, even when administered 24 hours after irradiation. Our data demonstrate that novel PKC isoform agonists such as IDB may represent a unique therapeutic strategy for accelerating the recovery of platelet counts following severe marrow injury.

  19. Revealing the functions of the transketolase enzyme isoforms in Rhodopseudomonas palustris using a systems biology approach.

    Directory of Open Access Journals (Sweden)

    Chia-Wei Hu

    Full Text Available BACKGROUND: Rhodopseudomonas palustris (R. palustris is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. METHODOLOGY/PRINCIPAL FINDINGS: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth

  20. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    Directory of Open Access Journals (Sweden)

    Wouter Eilers

    2014-01-01

    Full Text Available We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis and slow-type muscle (soleus for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02. In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII.

  1. Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Riviello, Lea; Coniglio, Maria Gabriella; Vandooren, Jennifer; Liuzzi, Grazia Maria; Opdenakker, Ghislain; Riccio, Paolo

    2014-01-01

    The matrix metalloproteinases (MMPs) gelatinase A (MMP-2) and gelatinase B (MMP-9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2-DZ) as an extension of classic monodimensional zymography (1-DZ) to analyse the complete pattern of isoforms and post-translational modifications of both MMP-9 and MMP-2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP-2 isoforms, and at least three different isoforms and two different states of organization of MMP-9 (the multimeric MMP-9 and the N-GAL-MMP-9 complex) were observed. In addition, 2-DZ revealed for the first time that all MMP-9 and MMP-2 isoforms actually exist in the form of charge variants: four or five variants in the N-GAL complex, more charge variants in the case of MMP-9; and five to seven charge variants for MMP-2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP-9) and phosphorylation (MMP-2) contributed to molecular heterogeneity. The detection of charge variants of MMP-9 and MMP-2 in MS serum samples illustrates the power of 2-DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases. PMID:24616914

  2. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    OpenAIRE

    Fabian V Filipp

    2013-01-01

    Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2) is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor acti...

  3. Characterization of Alien isoforms in vertebrates : Caracterización de isoformas de Alien en vertebrados

    OpenAIRE

    Tenbaum, Stephan

    2002-01-01

    Alien protein isoforms have been described to be involved in a number of biological processes. Alienalpha is a corepressor of the thyroid hormone receptor mediating transcriptional repression in a ligand-sensitive manner. Furthermore, Alienalpha is a corepressor for the orphan receptor DAX1 and the vitamin-D3 receptor. Alienbetta/CSN2 is part of the COP9-signalosome complex that acts in protein phosphorylation, protein degradation and cell cycle regulation. The major goal of this...

  4. Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

    OpenAIRE

    Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

    1991-01-01

    Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an i...

  5. Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear.

    Science.gov (United States)

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J; Striessnig, Jörg; Singewald, Nicolas

    2008-05-01

    Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.

  6. Heterogeneous effects of M-CSF isoforms on the progression of MLL-AF9 leukemia.

    Science.gov (United States)

    Wang, Rong; Feng, Wenli; Yang, Feifei; Yang, Xiao; Wang, Lina; Chen, Chong; Hu, Yuting; Ren, Qian; Zheng, Guoguang

    2018-02-01

    Macrophage colony-stimulating factor (M-CSF) regulates both malignant cells and microenvironmental cells. Its splicing isoforms show functional heterogeneity. However, their roles on leukemia have not been well established. Here, the expression of total M-CSF in patients with hematopoietic malignancies was analyzed. The roles of M-CSF isoforms on the progression of acute myeloid leukemia (AML) were studied by establishing MLL-AF9-induced mouse AML models with high level membrane-bound M-CSF (mM-CSF) or soluble M-CSF (sM-CSF). Total M-CSF was highly expressed in myeloid leukemia patients. Furthermore, mM-CSF but not sM-CSF prolonged the survival of leukemia mice. While sM-CSF was more potent to promote proliferation and self-renew, mM-CSF was more potent to promote differentiation. Moreover, isoforms had different effects on leukemia-associated macrophages (LAMs) though they both increase monocytes/macrophages by growth-promoting and recruitment effects. In addition, mM-CSF promoted specific phagocytosis of leukemia cells by LAMs. RNA-seq analysis revealed that mM-CSF enhanced phagocytosis-associated genes and activated oxidative phosphorylation and metabolism pathway. These results highlight heterogeneous effects of M-CSF isoforms on AML progression and the mechanisms of mM-CSF, that is, intrinsically promoting AML cell differentiation and extrinsically enhancing infiltration of macrophages and phagocytosis by macrophages, which may provide potential clues for clinical diagnosis and therapy. © 2017 Australasian Society for Immunology Inc.

  7. Isoform-specific regulation of osteogenic factors by polypeptide N-Acetylgalactosaminyltransferases 1 and 4

    International Nuclear Information System (INIS)

    Tang, Juan; Zheng, Hanxi; Chen, Ling; Gao, Shangshang; Shi, Xiaorui; Liu, Jingjing; Xu, Lan

    2017-01-01

    The family of UDP-GalNAc polypeptide: N-Acetylgalactosaminlytransfersases (ppGalNAcTs) catalyzes the initial step of O-linked protein glycosylation. Mucin-type O-glycoproteins are abundant in the bone and may play an important role in osteogenesis. Herein, we examined the effects of ppGalNAc-T isoforms on osteogenesis of MC3T3-E1 pre-osteoblasts. We found that ppGalNAc-T1 and -T4 isoforms were highly expressed during osteogenesis of MC3T3-E1 and their knockdown by short hairpin RNA (shRNA) decreased osteoblast formation and bone mineralization. Knockdown of ppGalNAc-T1 or -T4 decreased mRNA and protein levels of bone sialoprotein (BSP). Knockdown of ppGalNAc-T1decreased mRNA levels of osteocalcin (OC), osteoprotegerin (OPG). Knockdown ofppGalNAc-T4 isoform decreased mRNA levels of OC, OPG and vitamin D receptor (VDR). While knockdown of T1 or T4 isoforms did not change the expression of osteopontin (OPN), COLLI, receptor activator for nuclear factor-κB ligand (RANKL) and transforming growth factor-β (TGF-β). Our results demonstrated that the ppGalNAc-T4 was highly expressed in MC3T3-E1 cells during osteogenesis for the first time. We also found that ppGalNAc-T1 and -T4 affected the expression of different osteogenic factors, suggesting distinct roles ppGalNAc-T isoformsplay in regulating osteogenesis in vitro. - Highlights: • ppGalNAc-T1 and T4 are highly expressed during MC3T3 cell osteogenesis. • Knockdown of ppGalNAc-T1 and -T4 decreases osteogenic differentiation and mineralization. • Expression of osteogenic factors are differentially affected by decreased ppGalNAc-T1 and -T4 expression.

  8. The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

    Science.gov (United States)

    2013-10-01

    2013 Accepted 14 February 2013 Available online 21 February 2013 Keywords: Myosin IC Isoforms Nucleolar localization signal Nucleolus Nucleus RNA...polymerase I Fibrillarinnt matter & 2013 Elsevier 1016/j.yexcr.2013.02.008 S, nucleolar localization ; No, nucleolus ; N, nucle bovine serum albumin; S...the nucleus, and the nucleolus . In the cytoplasm, myosin IC associ- ates with membranes and is involved in vesicle transport of membrane proteins [2

  9. Deregulation of the endogenous C/EBPβ LIP isoform predisposes to tumorigenesis.

    Science.gov (United States)

    Bégay, Valérie; Smink, Jeske J; Loddenkemper, Christoph; Zimmermann, Karin; Rudolph, Cornelia; Scheller, Marina; Steinemann, Doris; Leser, Ulf; Schlegelberger, Brigitte; Stein, Harald; Leutz, Achim

    2015-01-01

    Two long and one truncated isoforms (termed LAP*, LAP, and LIP, respectively) of the transcription factor CCAAT enhancer binding protein beta (C/EBPβ) are expressed from a single intronless Cebpb gene by alternative translation initiation. Isoform expression is sensitive to mammalian target of rapamycin (mTOR)-mediated activation of the translation initiation machinery and relayed through an upstream open reading frame (uORF) on the C/EBPβ mRNA. The truncated C/EBPβ LIP, initiated by high mTOR activity, has been implied in neoplasia, but it was never shown whether endogenous C/EBPβ LIP may function as an oncogene. In this study, we examined spontaneous tumor formation in C/EBPβ knockin mice that constitutively express only the C/EBPβ LIP isoform from its own locus. Our data show that deregulated C/EBPβ LIP predisposes to oncogenesis in many tissues. Gene expression profiling suggests that C/EBPβ LIP supports a pro-tumorigenic microenvironment, resistance to apoptosis, and alteration of cytokine/chemokine expression. The results imply that enhanced translation reinitiation of C/EBPβ LIP promotes tumorigenesis. Accordingly, pharmacological restriction of mTOR function might be a therapeutic option in tumorigenesis that involves enhanced expression of the truncated C/EBPβ LIP isoform. Elevated C/EBPβ LIP promotes cancer in mice. C/EBPβ LIP is upregulated in B-NHL. Deregulated C/EBPβ LIP alters apoptosis and cytokine/chemokine networks. Deregulated C/EBPβ LIP may support a pro-tumorigenic microenvironment.

  10. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Fang; Li, Xiuli [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China); Kong, Jian [Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing (China); Pan, Bing [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Sun, Min [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xian (China); Zheng, Lemin, E-mail: zhengl@bjmu.edu.cn [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Yao, Yuanqing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China)

    2013-11-08

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

  11. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    International Nuclear Information System (INIS)

    Gu, Fang; Li, Xiuli; Kong, Jian; Pan, Bing; Sun, Min; Zheng, Lemin; Yao, Yuanqing

    2013-01-01

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA 3.1 empty vector, pcDNA 3.1 -VEGF111b or pcDNA 3.1 -VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth

  12. Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform.

    Directory of Open Access Journals (Sweden)

    Coby M M Laarakkers

    Full Text Available Mass spectrometry (MS-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF platforms. As expected, implementation of hepcidin-25(+40 as internal standard in our weak cation exchange (WCX TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9 with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and

  13. Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

    International Nuclear Information System (INIS)

    Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

    2004-01-01

    The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

  14. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    El Eter, E. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Physiology Department, Faculty of Medicine, Alexandria University, Alexandria (Egypt); Al-Masri, A.A. [Physiology Department, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia); Cardiovascular Research Group, Faculty of Medicine, King Saud University, Riyadh (Saudi Arabia)

    2015-03-03

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors.

  15. Differential expression of a new isoform of DLG2 in renal oncocytoma

    Directory of Open Access Journals (Sweden)

    Kovacs Gyula

    2006-04-01

    Full Text Available Abstract Background Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type. Methods In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis. Results We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour. Conclusion The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma.

  16. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

    Science.gov (United States)

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.

  17. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    International Nuclear Information System (INIS)

    Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

    2010-01-01

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  18. Peroxiredoxin isoforms are associated with cardiovascular risk factors in type 2 diabetes mellitus

    International Nuclear Information System (INIS)

    El Eter, E.; Al-Masri, A.A.

    2015-01-01

    The production of oxygen free radicals in type 2 diabetes mellitus contributes to the development of complications, especially the cardiovascular-related ones. Peroxiredoxins (PRDXs) are antioxidant enzymes that combat oxidative stress. The aim of this study was to investigate the associations between the levels of PRDX isoforms (1, 2, 4, and 6) and cardiovascular risk factors in type 2 diabetes mellitus. Fifty-three patients with type 2 diabetes mellitus (28F/25M) and 25 healthy control subjects (7F/18M) were enrolled. We measured the plasma levels of each PRDX isoform and analyzed their correlations with cardiovascular risk factors. The plasma PRDX1, -2, -4, and -6 levels were higher in the diabetic patients than in the healthy control subjects. PRDX2 and -6 levels were negatively correlated with diastolic blood pressure, fasting blood sugar, and hemoglobin A1c. In contrast, PRDX1 levels were positively correlated with low-density lipoprotein and C-reactive protein levels. PRDX4 levels were negatively correlated with triglycerides. In conclusion, PRDX1, -2, -4, and -6 showed differential correlations with a variety of traditional cardiovascular risk factors. These results should encourage further research into the crosstalk between PRDX isoforms and cardiovascular risk factors

  19. Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms

    Science.gov (United States)

    Caiozzo, V. J.; Herrick, R. E.; Baldwin, K. M.

    1992-01-01

    This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.

  20. Circadian rhythmicity of active GSK3 isoforms modulates molecular clock gene rhythms in the suprachiasmatic nucleus.

    Science.gov (United States)

    Besing, Rachel C; Paul, Jodi R; Hablitz, Lauren M; Rogers, Courtney O; Johnson, Russell L; Young, Martin E; Gamble, Karen L

    2015-04-01

    The suprachiasmatic nucleus (SCN) drives and synchronizes daily rhythms at the cellular level via transcriptional-translational feedback loops comprising clock genes such as Bmal1 and Period (Per). Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, phosphorylates at least 5 core clock proteins and shows diurnal variation in phosphorylation state (inactivation) of the GSK3β isoform. Whether phosphorylation of the other primary isoform (GSK3α) varies across the subjective day-night cycle is unknown. The purpose of this study was to determine if the endogenous rhythm of GSK3 (α and β) phosphorylation is critical for rhythmic BMAL1 expression and normal amplitude and periodicity of the molecular clock in the SCN. Significant circadian rhythmicity of phosphorylated GSK3 (α and β) was observed in the SCN from wild-type mice housed in constant darkness for 2 weeks. Importantly, chronic activation of both GSK3 isoforms impaired rhythmicity of the GSK3 target BMAL1. Furthermore, chronic pharmacological inhibition of GSK3 with 20 µM CHIR-99021 enhanced the amplitude and shortened the period of PER2::luciferase rhythms in organotypic SCN slice cultures. These results support the model that GSK3 activity status is regulated by the circadian clock and that GSK3 feeds back to regulate the molecular clock amplitude in the SCN. © 2015 The Author(s).

  1. Role of p73 Dinucleotide Polymorphism in Prostate Cancer and p73 Protein Isoform Balance

    Directory of Open Access Journals (Sweden)

    L. Michael Carastro

    2014-01-01

    Full Text Available Background. Molecular markers for prostate cancer (PCa risks are currently lacking. Here we address the potential association of a dinucleotide polymorphism (DNP in exon 2 of the p73 gene with PCa risk/progression and discern any disruption of p73 protein isoforms levels in cells harboring a p73 DNP allele. Methods. We investigated the association between p73 DNP genotype and PCa risk/aggressiveness and survival by fitting logistic regression models in 1,292 incident cases and 682 controls. Results. Although we detected no association between p73 DNP and PCa risk, a significant inverse relationship between p73 DNP and PCa aggressiveness (AT/AT + GC/AT versus GC/GC, OR = 0.55, 95%Cl = 0.31–0.99 was detected. Also, p73 DNP is marginally associated with overall death (dominant model, HR = 0.76, 95%Cl = 0.57–1.00, P=0.053 as well as PCa specific death (HR = 0.69, 95%Cl = 0.45–1.06, P=0.09. Western blot analyses for p73 protein isoforms indicate that cells heterozygous for the p73 DNP have lower levels of ∆Np73 relative to TAp73 (P<0.001. Conclusions. Our findings are consistent with an association between p73 DNP and low risk for PCa aggressiveness by increasing the expressed TAp73/∆Np73 protein isoform ratio.

  2. Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

    Directory of Open Access Journals (Sweden)

    Rachel S. Lee

    2011-01-01

    Full Text Available The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

  3. Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

    DEFF Research Database (Denmark)

    Kjelgaard-Hansen, Mads Jens; Christensen, Michelle B.; Lee, Marcel Huisung

    2007-01-01

    Serum amyloid A (SAA) is a major acute phase protein in dogs. However, knowledge of qualitative properties of canine SAA and extent of its synthesis in extrahepatic tissues is limited. The aim of the study was to investigate expression of different SAA isoforms in serum and synovial fluid...... in samples obtained from dogs (n = 16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs...... with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local...

  4. Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

    National Research Council Canada - National Science Library

    VanHouten, Joshua N

    2008-01-01

    The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

  5. Expression of smooth muscle and non-muscle myosin heavy chain isoforms in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Rovner, A.S.; Murphy, R.A.; Owens, G.K.

    1986-01-01

    Immunocytochemical studies of cultured smooth muscle cells (SMCs) have disagreed on the nature of myosin expression. This investigation was undertaken to test for the presence of heterogeneous myosin heavy chain (MHC) isoforms in cell culture as a possible explanation for these results. Previously, Rovner et al. detected two MHCs in intact smooth muscles which differed in molecular weight by ca. 4000 daltons (SM1 and SM2) using a 3-4% acrylamide gradient SDS gel system. When sub-confluent primary cultures of rat aorta SMCs were assayed by this system, SM1 and SM2 were seen, along with large amounts of a third, unique MHC, NM, which closely resembled the MHC from human platelet in size and antigenicity. Data from 35 S-methionine autoradiograms showed that the log growth phase SMC cultures were producing almost exclusively NM, but the growth arrest, post-confluent cultures synthesized increased relative amounts of the SM MHC forms and contained comparable amounts of SM1, SM2, and NM. The same patterns of MHC synthesis were seen in sub-passaged SMCs. The expression of the SM-specific forms of myosin in quiescent, post-confluent cultures parallels that of smooth muscle actin suggesting that density induced growth arrest promotes cytodifferentiation in cultured vascular SMCs

  6. Comparative evaluation of 12 immature citrus fruit extracts for the inhibition of cytochrome P450 isoform activities.

    Science.gov (United States)

    Fujita, Tadashi; Kawase, Atsushi; Niwa, Toshiro; Tomohiro, Norimichi; Masuda, Megumi; Matsuda, Hideaki; Iwaki, Masahiro

    2008-05-01

    In a previous study we found that 50% ethanol extracts of immature fruits of Citrus unshiu (satsuma mandarin) have anti-allergic effects against the Type I, II and IV allergic reactions. However, many adverse interactions between citrus fruit, especially grapefruit juice, and drugs have been reported due to the inhibition of cytochrome P450 (CYP) activities. The purpose of this study was to examine the competitive inhibitory effects of extracts from immature citrus fruit on CYP activity. Extracts were prepared from 12 citrus species or cultivars, and were tested against three kinds of major CYPs, CYP2C9, CYP2D6 and CYP3A4, in human liver microsomes. We also estimated the amounts of flavonoids (narirutin, hesperidin, naringin and neohesperidin) and furanocoumarins (bergapten, 6',7'-dihydroxybergamottin and bergamottin) in each extract using HPLC. Citrus paradisi (grapefruit) showed the greatest inhibition of CYP activities, while Citrus unshiu which has an antiallergic effect, showed relatively weak inhibitory effects. Extracts having relatively strong inhibitory effects for CYP3A4 tended to contain higher amounts of naringin, bergamottin and 6',7'-dihydroxybergamottin. These results, providing comparative information on the inhibitory effects of citrus extracts on CYP isoforms, suggest that citrus extracts containing high levels of narirutin and hesperidin and lower levels of furanocoumarins such as C. unshiu are favorable as antiallergic functional ingredients.

  7. A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

    Directory of Open Access Journals (Sweden)

    Lawrence Kazak

    Full Text Available A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

  8. A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

    International Nuclear Information System (INIS)

    O'Donnell, Christopher D.; Tiwari, Vaibhav; Oh, Myung-Jin; Shukla, Deepak

    2006-01-01

    Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain

  9. Methamphetamine and inflammatory cytokines increase neuronal Na+/K+-ATPase isoform 3: relevance for HIV associated neurocognitive disorders.

    Directory of Open Access Journals (Sweden)

    Gurudutt Pendyala

    Full Text Available Methamphetamine (METH abuse in conjunction with human immunodeficiency virus (HIV exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS, collectively termed HIV Associated Neurocognitive Disorders (HAND. Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV differing by one regimen, METH treatment, we identified the neuron specific Na(+/K(+-ATPase alpha 1 isoform 3 (ATP1A3 to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse.

  10. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

    Science.gov (United States)

    Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

    2014-09-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

  11. The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

    Science.gov (United States)

    Voss, S C; Giraud, S; Alsayrafi, M; Bourdon, P C; Schumacher, Y O; Saugy, M; Robinson, N

    2013-08-01

    The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. The schizophrenia-associated Kv11.1-3.1 isoform results in reduced current accumulation during repetitive brief depolarizations.

    Directory of Open Access Journals (Sweden)

    Juliane Heide

    Full Text Available Recent genome wide association studies identified a brain and primate specific isoform of a voltage-gated potassium channel, referred to as Kv11.1-3.1, which is significantly associated with schizophrenia. The 3.1 isoform replaces the first 102 amino acids of the most abundant isoform (referred to as Kv11.1-1A with six unique amino acids. Here we show that the Kv11.1-3.1 isoform has faster rates of channel deactivation but a slowing of the rates of inactivation compared to the Kv11.1-1A isoform. The Kv11.1-3.1 isoform also has a significant depolarizing shift in the voltage-dependence of steady-state inactivation. The consequence of the altered gating kinetics is that there is lower current accumulation for Kv11.1-3.1 expressing cells during repetitive action potential firing compared to Kv11.1-1A expressing cells, which in turn will result in longer lasting trains of action potentials. Increased expression of Kv11.1-3.1 channels in the brain of schizophrenia patients might therefore contribute to disorganized neuronal firing.

  13. Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

    Science.gov (United States)

    Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

    2016-03-31

    Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

  14. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration.

    Science.gov (United States)

    Fearnley, Gareth W; Bruns, Alexander F; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2015-04-24

    Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response. © 2015. Published by The Company of Biologists Ltd.

  15. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2015-07-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

  16. Eff ect of vitamin E isoforms on the primary intention Eff ect of vitamin E isoforms on the primary intention skin wound healing of diabetic rats

    Directory of Open Access Journals (Sweden)

    Bijo Elsy

    2017-10-01

    Full Text Available Introduction: Impaired wound healing events is a common complication in diabetes. One of the effective nutritional antioxidant on skin wound healing is vitamin E which contains saturated tocopherol and unsaturated tocotrienol forms. This present study is designed to explore the effect of different vitamin E isoforms on stitched skin wound in both healthy and diabetic rats. Materials and Methods: Forty eight albino rats were divided into eight groups; healthy control, diabetic control, healthy treated (d-α-tocopherol, d-δ-TRF and co-administrated and diabetic treated (d-αtocopherol, d-δ-TRF and co-administrated. Diabetes was induced through single subcutaneous injection of alloxan at the dose of 100 mg/kg. Treated groups were administered d-a-tocopherol (200 mg/kg, d-δ-TRF (200 mg/kg and co-administration (100 mg/kg of these two compounds each orally and daily for three weeks. A horizontal skin incision was made on right mid-thigh region at 2.95 ± 0.17cm in length and wound was closed with an absorbable suture. Results: Histopathological and histomorphological results at the end of 3rd week revealed that the d-δ-TRF treated groups promote the regeneration and reorganization of epidermal and dermal components in healing of primary intention more effectively than the d-α-tocopherol and co-administrated groups. Conclusion: It is concluded that among different vitamin E isoforms the d-δ-TRF appears to be a more effective nutritional antioxidant on skin wound healing in both healthy and diabetics.

  17. Expression and Immunohistochemical Localisation of the G beta gamma activated and Calcineurin-inhibited Adenylyl Cyclase Isoforms in Rat Articular Chondrocytes

    International Nuclear Information System (INIS)

    Memon, I.; Khan, K.M.; Siddiqui, S.; Perveen, S.; Ishaq, M.

    2016-01-01

    Objective: To determine the expression and localisation of the Gβγ-activated adenylyl cyclase (AC) isoforms 2, 4, and 7 and calcineurin-inhibited AC isoform 9 in rat articular chondrocytes. Study Design: Experimental study. Place and Duration of Study: Jumma Research Laboratory and Histology Laboratory, The Aga Khan University, Karachi, from 2009 to 2011. Methodology: Fresh slices of articular cartilage were taken from various synovial joints of rats of different age groups. The expression of AC isoforms was determined by RT-PCR and immunohistochemistry was performed to localise these isoforms in articular chondrocytes. Tissue sections were processed for immunostaining with respective antibodies. The color was developed by diaminobenzidine. Results: All the studied AC isoforms were found to be differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms studied increased with age. The expression of the AC isoforms through PCR was almost consistent with the localisation of these isoforms by immunohistochemistry. Conclusion: These data add to the information about signalling cascades possibly involved in articular chondrocytes. Variable expression of AC isoforms 2, 4, 7, and 9 suggest a role for the signalling cascades regulated by the AC isoforms in articular chondrocytes. (author)

  18. CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

    International Nuclear Information System (INIS)

    Olsson, Eleonor; Lövgren, Kristina; Fernö, Mårten; Grabau, Dorthe; Borg, Åke; Hegardt, Cecilia; Honeth, Gabriella; Bendahl, Pär-Ola; Saal, Lao H; Gruvberger-Saal, Sofia; Ringnér, Markus; Vallon-Christersson, Johan; Jönsson, Göran; Holm, Karolina

    2011-01-01

    The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44 + /CD24 - phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to

  19. CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

    Directory of Open Access Journals (Sweden)

    Vallon-Christersson Johan

    2011-09-01

    Full Text Available Abstract Background The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. Methods We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Results Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. Conclusions We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in

  20. Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: the pathway and concentration dependence.

    Science.gov (United States)

    Kot, Marta; Daniel, Władysława A

    2008-04-01

    The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for pharmacokinetic interactions in pharmacological experiments. The results obtained using rat cDNA-expressed P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and substantially contributing to 3-N-demethylation (47%) and 1-N-demethylation (37.5%) at a caffeine concentration of 0.1mM (relevant to "the maximum therapeutic concentration in humans"). Furthermore, CYP2C11 considerably contributed to 3-N-demethylation (31