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Sample records for acsl6 isoforms role

  1. Activity of the acyl-CoA synthetase ACSL6 isoforms: role of the fatty acid Gate-domains

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    Siliakus Melvin

    2010-04-01

    Full Text Available Abstract Background Activation of fatty acids by acyl-CoA synthetase enzymes is required for de novo lipid synthesis, fatty acid catabolism, and remodeling of biological membranes. Human long-chain acyl-CoA synthetase member 6, ASCL6, is a form present in the plasma membrane of cells. Splicing events affecting the amino-terminus and alternative motifs near the ATP-binding site generate different isoforms of ACSL6. Results Isoforms with different fatty acid Gate-domain motifs have different activity and the form lacking this domain, isoform 3, showed no detectable activity. Enzymes truncated of the first 40 residues generate acyl-CoAs at a faster rate than the full-length protein. The gating residue, which prevents entry of the fatty acid substrate unless one molecule of ATP has already accessed the catalytic site, was identified as a tyrosine for isoform 1 and a phenylalanine for isoform 2 at position 319. All isoforms, with or without a fatty acid Gate-domain, as well as recombinant protein truncated of the N-terminus, can interact to form enzymatic complexes with identical or different isoforms. Conclusion The alternative fatty acid Gate-domain motifs are essential determinants for the activity of the human ACSL6 isoforms, which appear to act as homodimeric enzyme as well as in complex with other spliced forms. These findings provide evidence that the diversity of these enzyme species could produce the variety of acyl-CoA synthetase activities that are necessary to generate and repair the hundreds of lipid species present in membranes.

  2. ACSL6 is associated with the number of cigarettes smoked and its expression is altered by chronic nicotine exposure.

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    Jingchun Chen

    Full Text Available Individuals with schizophrenia tend to be heavy smokers and are at high risk for tobacco dependence. However, the nature of the comorbidity is not entirely clear. We previously reported evidence for association of schizophrenia with SNPs and SNP haplotypes in a region of chromosome 5q containing the SPEC2, PDZ-GEF2 and ACSL6 genes. In this current study, analysis of the control subjects of the Molecular Genetics of Schizophrenia (MGS sample showed similar pattern of association with number of cigarettes smoked per day (numCIG for the same region. To further test if this locus is associated with tobacco smoking as measured by numCIG and FTND, we conducted replication and meta-analysis in 12 independent samples (n>16,000 for two markers in ACSL6 reported in our previous schizophrenia study. In the meta-analysis of the replication samples, we found that rs667437 and rs477084 were significantly associated with numCIG (p = 0.00038 and 0.00136 respectively but not with FTND scores. We then used in vitro and in vivo techniques to test if nicotine exposure influences the expression of ACSL6 in brain. Primary cortical culture studies showed that chronic (5-day exposure to nicotine stimulated ACSL6 mRNA expression. Fourteen days of nicotine administration via osmotic mini pump also increased ACSL6 protein levels in the prefrontal cortex and hippocampus of mice. These increases were suppressed by injection of the nicotinic receptor antagonist mecamylamine, suggesting that elevated expression of ACSL6 requires nicotinic receptor activation. These findings suggest that variations in the ACSL6 gene may contribute to the quantity of cigarettes smoked. The independent associations of this locus with schizophrenia and with numCIG in non-schizophrenic subjects suggest that this locus may be a common liability to both conditions.

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

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    Jinxiong Wei

    2012-01-01

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

  4. The role of wild type RAS isoforms in cancer.

    Science.gov (United States)

    Zhou, Bingying; Der, Channing J; Cox, Adrienne D

    2016-10-01

    Mutationally activated RAS proteins are critical oncogenic drivers in nearly 30% of all human cancers. As with mutant RAS, the role of wild type RAS proteins in oncogenesis, tumour maintenance and metastasis is context-dependent. Complexity is introduced by the existence of multiple RAS genes (HRAS, KRAS, NRAS) and protein "isoforms" (KRAS4A, KRAS4B), by the ever more complicated network of RAS signaling, and by the increasing identification of numerous genetic aberrations in cancers that do and do not harbour mutant RAS. Numerous mouse model carcinogenesis studies and examination of patient tumours reveal that, in RAS-mutant cancers, wild type RAS proteins are likely to serve as tumour suppressors when the mutant RAS is of the same isoform. This evidence is particularly robust in KRAS mutant cancers, which often display suppression or loss of wild type KRAS, but is not as strong for NRAS. In contrast, although not yet fully elucidated, the preponderance of evidence indicates that wild type RAS proteins play a tumour promoting role when the mutant RAS is of a different isoform. In non-RAS mutant cancers, wild type RAS is recognized as a mediator of oncogenic signaling due to chronic activation of upstream receptor tyrosine kinases that feed through RAS. Additionally, in the absence of mutant RAS, activation of wild type RAS may drive cancer upon the loss of negative RAS regulators such as NF1 GAP or SPRY proteins. Here we explore the current state of knowledge with respect to the roles of wild type RAS proteins in human cancers.

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

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    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.

  6. Role of nuclear progesterone receptor isoforms in uterine pathophysiology.

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    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    Progesterone is a key hormonal regulator of the female reproductive system. It plays a major role to prepare the uterus for implantation and in the establishment and maintenance of pregnancy. Actions of progesterone on the uterine tissues (endometrium, myometrium and cervix) are mediated by the combined effects of two progesterone receptor (PR) isoforms, designated PR-A and PR-B. Both receptors function primarily as ligand-activated transcription factors. Progesterone action on the uterine tissues is qualitatively and quantitatively determined by the relative levels and transcriptional activities of PR-A and PR-B. The transcriptional activity of the PR isoforms is affected by specific transcriptional coregulators and by PR post-translational modifications that affect gene promoter targeting. In this context, appropriate temporal and cell-specific expression and function of PR-A and PR-B are critical for normal uterine function. Relevant studies describing the role of PRs in uterine physiology and pathology (endometriosis, uterine leiomyoma, endometrial cancer, cervical cancer and recurrent pregnancy loss) were comprehensively searched using PubMed, Cochrane Library, Web of Science, and Google Scholar and critically reviewed. Progesterone, acting through PR-A and PR-B, regulates the development and function of the endometrium and induces changes in cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs promotes myometrial relaxation and cervical closure. Withdrawal of PR-mediated progesterone signaling triggers menstruation and parturition. PR-mediated progesterone signaling is anti-mitogenic in endometrial epithelial cells, and as such, mitigates the tropic effects of estrogen on eutopic normal endometrium, and on ectopic implants in endometriosis. Similarly, ligand-activated PRs function as tumor suppressors in endometrial cancer cells through inhibition of key cellular signaling pathways

  7. Role of ROCK Isoforms in Regulation of Stiffness Induced Myofibroblast Differentiation in Lung Fibrosis.

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    Htwe, Su S; Cha, Byung H; Yue, Kan; Khademhosseini, Ali; Knox, Alan J; Ghaemmaghami, Amir M

    2017-02-22

    Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

  8. Divergent roles for thyroid hormone receptor β isoforms in the endocrine axis and auditory system

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    Abel, E. Dale; Boers, Mary-Ellen; Pazos-Moura, Carmen; Moura, Egberto; Kaulbach, Helen; Zakaria, Marjorie; Lowell, Bradford; Radovick, Sally; Liberman, M. Charles; Wondisford, Fredric

    1999-01-01

    Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TRα and TRβ isoforms are products of 2 distinct genes, and the β1 and β2 isoforms are splice variants of the same gene. Whereas TRα1 and TRβ1 are widely expressed, expression of the TRβ2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TRβ locus (TRβ-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TRβ2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TRβ2 isoform. TRβ2-null mice have preserved expression of the TRα and TRβ1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TRβ-null mice, indicating the important role of TRβ2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TRβ2-null mice exhibit no evidence of hearing impairment, indicating that TRβ1 and TRβ2 subserve divergent roles in the regulation of auditory function. PMID:10430610

  9. 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

    Skeletal muscle glucose transport is regulated via the canonical insulin-signaling cascade as well as by energy-sensing signals. 5'-AMP-activated protein kinase (AMPK) has been implicated in the energy status regulation of glucose transport. We determined the role of the AMPKgamma3 isoform...

  10. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

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    Jean-Simon eFortin

    2013-12-01

    Full Text Available The peptide repertoire (peptidome associated with MHC class II molecules (MHCIIs is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  11. Exposing the Specific Roles of the Invariant Chain Isoforms in Shaping the MHC Class II Peptidome.

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    Fortin, Jean-Simon; Cloutier, Maryse; Thibodeau, Jacques

    2013-12-13

    The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

  12. 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 we...... further aimed to investigate the role of CD97 small isoform in gastric cancer progression in vivo by employing the cells with a stable CD97 small isoform knock-down and an orthotopic gastric cancer mouse model. We could demonstrate that the knock down of CD97/EGF1,2,5, led to a significant decrease...... in the number of cells penetrating the gelatin coated membrane as compared with control cells. In the gastric cancer mouse model, both the hypodermic and the orthotopic yielded tumor masses of the CD97/EGF1,2,5kd group and were significantly smaller than the control. Metastatic tumor cell number in early...

  13. Numerous isoforms of Fgf8 reflect its multiple roles in the developing brain.

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    Sunmonu, N Abimbola; Li, Kairong; Li, James Y H

    2011-07-01

    Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination, and morphogenesis during development of multicellular organisms. Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces eight splice variants, which encode isoform proteins with different N-termini and distinct receptor-binding affinity and biological activity. In this article, we review the roles of Fgf8 in vertebrate development and summarize the recent findings on the in vivo function of different Fgf8 splice variants. We propose that multiple Fgf8 isoform proteins act in concert to regulate the overall function of Fgf8 and account for the diverse and essential role of Fgf8 during vertebrate development.

  14. ROCK1 isoform-specific deletion reveals a role for diet-induced insulin resistance

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    Lee, Seung-Hwan; Huang, Hu; Choi, Kangduk; Lee, Dae Ho; Shi, Jianjian; Liu, Tiemin; Chun, Kwang Hoon; Seo, Ji A.; Lima, Ines S.; Zabolotny, Janice M.; Wei, Lei

    2013-01-01

    Rho kinase (ROCK) isoforms regulate insulin signaling and glucose metabolism negatively or positively in cultured cell lines and skeletal muscle. However, the in vivo function of the ROCK1 isoform in adipose tissue has not been addressed. To determine the specific role of the adipose ROCK1 isoform in the development of insulin resistance and obesity, mice lacking ROCK1 in adipose tissue globally or selectively were studied. Here, we show that insulin's ability to activate IRS-1/PI3K/Akt signaling was greatly enhanced in adipose tissue of ROCK1−/− mice compared with wild-type mice. These effects resulted from the inhibitory effect of ROCK1 on insulin receptor action, as evidenced by the fact that IR tyrosine phosphorylation was abolished in ROCK1−/− MEF cells when ROCK1 was reexpressed. Consistently, adipose-specific disruption of ROCK1 increased IR tyrosine phosphorylation in adipose tissue and modestly improved sensitivity to insulin in obese mice induced by high-fat feeding. This effect is independent of any changes in adiposity, number or size of adipocytes, and metabolic parameters, including glucose, insulin, leptin, and triglyceride levels, demonstrating a minimal effect of adipose ROCK1 on whole body metabolism. Enzymatic activity of ROCK1 in adipose tissue remained ∼50%, which likely originated from the fraction of stromal vascular cells, suggesting involvement of these cells for adipose metabolic regulation. Moreover, ROCK isoform activities were increased in adipose tissue of diet-induced or genetically obese mice. These data suggest that adipose ROCK1 isoform plays an inhibtory role for the regulation of insulin sensitivity in diet-induced obesity in vivo. PMID:24326423

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

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

    2006-09-01

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

  16. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

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    Pedro Fernandez-Funez

    2009-06-01

    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover

  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 immuni...tyfunctions. Poli V. J Biol Chem. 1998 Nov 6;273(45):29279-82. (.png) (.svg) (.html) (.csml) Show The role o...f C/EBP isoforms in the control of inflammatory and native immunityfunctions. PubmedID 9792624 Title The role

  18. The Role of a Novel Myosin Isoform in Prostate Cancer Metastasis

    Science.gov (United States)

    2013-10-01

    specific N-terminal peptides. Despite the high sequence homology , the isoforms show differences in cellular distribution, in localization to nuclear...Interestingly, despite the sequence homology between the isoforms and the commonality of the NLS, the isoforms show differences in their nucleus to cytoplasm...Goeres J, Sixt KM, Bekes M, Zhang XD, Salvesen GS, Matunis MJ (2009). Protection from isopeptidase-mediated deconjugation regulates paralog -selective

  19. Role of L-Type Ca[superscript 2+] Channel Isoforms in the Extinction of Conditioned Fear

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    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jorg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca[superscript 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…

  20. Roles of the troponin isoforms during indirect flight muscle development in Drosophila

    Indian Academy of Sciences (India)

    Salam Herojeet Singh; Prabodh Kumar; Nallur B. Ramachandra; Upendra Nongthomba

    2014-08-01

    Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (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 exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

  1. Roles of the troponin isoforms during indirect flight muscle development in Drosophila.

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    Singh, Salam Herojeet; Kumar, Prabodh; Ramachandra, Nallur B; Nongthomba, Upendra

    2014-08-01

    Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (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 exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

  2. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

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    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  3. Role of JNK isoforms in the development of neuropathic pain following sciatic nerve transection in the mouse

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    Manassero Giusi

    2012-05-01

    Full Text Available Abstract Background Current tools for analgesia are often only partially successful, thus investigations of new targets for pain therapy stimulate great interest. Consequent to peripheral nerve injury, c-Jun N-terminal kinase (JNK activity in cells of the dorsal root ganglia (DRGs and spinal cord is involved in triggering neuropathic pain. However, the relative contribution of distinct JNK isoforms is unclear. Using knockout mice for single isoforms, and blockade of JNK activity by a peptide inhibitor, we have used behavioral tests to analyze the contribution of JNK in the development of neuropathic pain after unilateral sciatic nerve transection. In addition, immunohistochemical labelling for the growth associated protein (GAP-43 and Calcitonin Gene Related Peptide (CGRP in DRGs was used to relate injury related compensatory growth to altered sensory function. Results Peripheral nerve injury produced pain–related behavior on the ipsilateral hindpaw, accompanied by an increase in the percentage of GAP43-immunoreactive (IR neurons and a decrease in the percentage of CGRP-IR neurons in the lumbar DRGs. The JNK inhibitor, D-JNKI-1, successfully modulated the effects of the sciatic nerve transection. The onset of neuropathic pain was not prevented by the deletion of a single JNK isoform, leading us to conclude that all JNK isoforms collectively contribute to maintain neuropathy. Autotomy behavior, typically induced by sciatic nerve axotomy, was absent in both the JNK1 and JNK3 knockout mice. Conclusions JNK signaling plays an important role in regulating pain threshold: the inhibition of all of the JNK isoforms prevents the onset of neuropathic pain, while the deletion of a single splice JNK isoform mitigates established sensory abnormalities. JNK inactivation also has an effect on axonal sprouting following peripheral nerve injury.

  4. The three isoforms of the light-harvesting complex II Spectroscopic features, trimer formation, and functional roles

    CERN Document Server

    Standfuss, Jorg

    2004-01-01

    The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isofo...

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

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    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.

  6. Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression.

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    Ibrahim, Safaa A; Katara, Gajendra K; Kulshrestha, Arpita; Jaiswal, Mukesh K; Amin, Magdy A; Beaman, Kenneth D

    2015-10-20

    In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.

  7. Dissecting the roles of ROCK isoforms in stress-induced cell detachment.

    Science.gov (United States)

    Shi, Jianjian; Surma, Michelle; Zhang, Lumin; Wei, Lei

    2013-05-15

    The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1(-/-) and ROCK2(-/-) mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1(-/-) MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2(-/-) MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1(-/-) MEFs, but not ROCK2(-/-) MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.

  8. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

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

    Directory of Open Access Journals (Sweden)

    Kuypers Frans A

    2006-07-01

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

  10. Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

    Directory of Open Access Journals (Sweden)

    Brian Roland eLarsen

    2016-04-01

    Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

  11. 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.

  12. 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.

  13. 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

  14. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    Science.gov (United States)

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

  15. Role of PRMTs in cancer: Could minor isoforms be leaving a mark?

    Institute of Scientific and Technical Information of China (English)

    R; Mitchell; Baldwin; Alan; Morettin; Jocelyn; C?té

    2014-01-01

    Protein arginine methyltransferases(PRMTs) catalyze the methylation of a variety of protein substrates, many of which have been linked to the development, progression and aggressiveness of different types of cancer. Moreover, aberrant expression of PRMTs has been observed in several cancer types. While the link between PRMTs and cancer is a relatively new area of interest, the functional implications documented thus far warrant further investigations into its therapeutic potential. However, the expression of these enzymes and the regulation of their activity in cancer are still significantly understudied. Currently there are nine main members of the PRMT family. Further, the existence of alternatively spliced isoforms for several of these family members provides an additional layer of complexity. Specifically, PRMT1, PRMT2, CARM1 and PRMT7 have been shown to have alternative isoforms and others may be currently unrealized. Our knowledge with respect to the relative expression and the specific functions of these isoforms is largely lacking and needs attention. Here we present a review of the current knowledge of theknown alternative PRMT isoforms and provide a rationale for how they may impact on cancer and represent potentially useful targets for the development of novel therapeutic strategies.

  16. Exposing the specific roles of the invariant chain isoforms in shaping the MHC class II peptidome

    OpenAIRE

    Jean-Simon eFortin; Maryse eCloutier; Jacques eThibodeau

    2013-01-01

    The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common...

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

    Science.gov (United States)

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

    2009-06-01

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

  18. Specific roles of the p110alpha isoform of phosphatidylinsositol 3-kinase in hepatic insulin signaling and metabolic regulation.

    Science.gov (United States)

    Sopasakis, Victoria Rotter; Liu, Pixu; Suzuki, Ryo; Kondo, Tatsuya; Winnay, Jonathon; Tran, Thien T; Asano, Tomoichiro; Smyth, Graham; Sajan, Mini P; Farese, Robert V; Kahn, C Ronald; Zhao, Jean J

    2010-03-03

    The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism. 2010 Elsevier Inc. All rights reserved.

  19. Mutants, Overexpressors, and Interactors of Arabidopsis Plastocyanin Isoforms: Revised Roles of Plastocyanin in Photosynthetic Electron Flow and Thylakoid Redox State

    Institute of Scientific and Technical Information of China (English)

    Paolo Pesaresi; Michael Scharfenberg; Martin Weigel; Irene Granlund; Wolfgang P. Schr(o)der; Giovanni Finazzi; Fabrice Rappaport; Simona Masiero; Antonella Furini; Peter Jahns; Dario Leister

    2009-01-01

    Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis thaliana. The PETE2 transcript is expressed at considerably higher levels and the PETE2 protein is the more abundant isoform. Null mutations in the PETE genes resulted in plants, designated pete1 and pete2, with decreased plas-tocyanin contents. However, despite reducing plastocyanin levels by over~90%, a pete2 null mutation on its own affects rates of photosynthesis and growth only slightly, whereas pete1 knockout plants, with about 60-80% of the wild-type plastocyanin level, did not show any alteration. Hence, plastocyanin concentration is not limiting for photosynthetic elec-tron flow under optimal growth conditions, perhaps implying other possible physiological roles for the protein. Indeed, plastocyanin has been proposed previously to cooperate with cytochrome C6A (Cyt C6A) in thylakoid redox reactions, but we find no evidence for a physical interaction between the two proteins, using interaction assays in yeast. We observed homodimerization of Cyt C6A in yeast interaction assays, but also Cyt C6A homodimers failed to interact with plastocyanin. Moreover, phenotypic analysis of atc6-1 pete1 and atc6-1 pete2 double mutants, each lacking Cyt C6A and one of the two plastocyanin-encoding genes, failed to reveal any genetic interaction. Overexpression of either PETE1 or PETE2 in the pete1 pete2 double knockout mutant background results in essentially wild-type photosynthetic performance, excluding the possibility that the two plastocyanin isoforms could have distinct functions in thylakoid electron flow.

  20. ROCK in CNS: Different Roles of Isoforms and Therapeutic Target for Neurodegenerative Disorders.

    Science.gov (United States)

    Chong, Cheong-Meng; Ai, Nana; Lee, Simon Ming-Yuen

    2017-01-01

    Rho-associated protein kinase (ROCK) is a serine-threonine kinase originally identified as a crucial regulator of actin cytoskeleton. Recent studies have defined new functions of ROCK as a critical component of diverse signaling pathways in neurons. In addition, inhibition of ROCK causes several biological events such as increase of neurite outgrowth, axonal regeneration, and activation of prosurvival Akt. Thus, it has attracted scientist's strong attentions and considered ROCK as a promising therapeutic target for the treatment of neurodegenerative disorders including Alzheimer disease, Parkinson's disease, Huntington';s disease, multiple sclerosis, and amyotrophic lateral sclerosis. However, ROCK has two highly homologous isoforms, ROCK1 and ROCK2. Accumulated evidences indicate that ROCK1 and ROCK2 might involve in distinct cellular functions in central nervous system (CNS) and neurodegenerative processes. This review summarizes recent updates regarding ROCK isoformspecific functions in CNS and the progress of ROCK inhibitors in preclinical studies for neurodegenerative diseases.

  1. Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms.

    Science.gov (United States)

    Marín-Hernández, Alvaro; López-Ramírez, Sayra Y; Del Mazo-Monsalvo, Isis; Gallardo-Pérez, Juan C; Rodríguez-Enríquez, Sara; Moreno-Sánchez, Rafael; Saavedra, Emma

    2014-08-01

    The effect of hypoglycemia on the contents of glycolytic proteins, activities of enzymes/transporters and flux of HeLa and MCF-7 tumor cells was experimentally analyzed and modeled in silico. After 24 h hypoglycemia (2.5 mm initial glucose), significant increases in the protein levels of glucose transporters 1 and 3 (GLUT 1 and 3) (3.4 and 2.1-fold, respectively) and hexokinase I (HKI) (2.3-fold) were observed compared to the hyperglycemic standard cell culture condition (25 mm initial glucose). However, these changes did not bring about a significant increase in the total activities (Vmax ) of GLUT and HK; instead, the affinity of these proteins for glucose increased, which may explain the twofold increased glycolytic flux under hypoglycemia. Thus, an increase in more catalytically efficient isoforms for two of the main controlling steps was sufficient to induce increased flux. Further, a previous kinetic model of tumor glycolysis was updated by including the ratios of GLUT and HK isoforms, modified pyruvate kinase kinetics and an oxidative phosphorylation reaction. The updated model was robust in terms of simulating most of the metabolite levels and fluxes of the cells exposed to various glycemic conditions. Model simulations indicated that the main controlling steps were glycogen degradation > HK > hexosephosphate isomerase under hyper- and normoglycemia, and GLUT > HK > glycogen degradation under hypoglycemia. These predictions were experimentally evaluated: the glycolytic flux of hypoglycemic cells was more sensitive to cytochalasin B (a GLUT inhibitor) than that of hyperglycemic cells. The results indicated that cancer glycolysis should be inhibited at multiple controlling sites, regardless of external glucose levels, to effectively block the pathway. The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/database/achcar/index.html. [Database section added 21

  2. Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

    Science.gov (United States)

    Yoffe, Yael; Zuberek, Joanna; Lerer, Asaf; Lewdorowicz, Magdalena; Stepinski, Janusz; Altmann, Michael; Darzynkiewicz, Edward; Shapira, Michal

    2006-12-01

    The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

  3. Peroxisome proliferator-activated receptors: role of isoform gamma in the antineoplastic effect of iodine in mammary cancer.

    Science.gov (United States)

    Nunez-Anita, R E; Cajero-Juarez, M; Aceves, C

    2011-09-01

    Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors. Three subtypes--PPAR alpha, PPAR beta, and PPAR gamma--have been identified and are differentially expressed in tissues. Originally, they were described as molecular regulators of lipid metabolism; recently, it has been shown that they are also involved in regulating the cell cycle and apoptosis in both normal and tumoral cells. In fact, some synthetic PPAR ligands are used to treat dyslipidemia, metabolic diseases, and type 2 diabetes. Here, we review the role of PPAR gamma (PPARγ) in tumor initiation and progression, emphasizing the relationship between this isoform and the cellular and molecular mechanisms involved in the antineoplastic effect of iodine on mammary cancer.

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

    Directory of Open Access Journals (Sweden)

    Evonne eChin-Smith

    2014-05-01

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

  5. Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease.

    Science.gov (United States)

    Boczonadi, Veronika; Giunta, Michele; Lane, Maria; Tulinius, Mar; Schara, Ulrike; Horvath, Rita

    2015-06-01

    Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues. We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  6. Protective roles of grass carp Ctenopharyngodon idella Mx isoforms against grass carp reovirus.

    Directory of Open Access Journals (Sweden)

    Limin Peng

    Full Text Available BACKGROUND: Myxovirus resistance (Mx proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, two novel Mx genes (CiMx1 and CiMx3 were identified in grass carp (Ctenopharyngodon idella. CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%, and low identity with CiMx2 (49.2% and 49.5%, respectively from the GenBank database. The predicted three-dimensional (3D structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR following infection with grass carp reovirus (GCRV in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored. CONCLUSIONS/SIGNIFICANCE: The results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN

  7. Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

    Science.gov (United States)

    Teodoro, Bruno G; Sampaio, Igor H; Bomfim, Lucas H M; Queiroz, André L; Silveira, Leonardo R; Souza, Anderson O; Fernandes, Anna M A P; Eberlin, Marcos N; Huang, Tai-Yu; Zheng, Donghai; Neufer, P Darrell; Cortright, Ronald N; Alberici, Luciane C

    2017-02-01

    Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or β-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid

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

    Science.gov (United States)

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

    2008-02-01

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

  9. Numerous isoforms of Fgf8 reflect its multiple roles in the developing brain

    OpenAIRE

    Sunmonu, N. Abimbola; Li, Kairong; Li, James Y.H.

    2011-01-01

    Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination and morphogenesis during development of multicellular organisms. Fibroblast Growth Factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces...

  10. Roles of Na(+)/Ca(2+) exchanger isoforms NCX1 and NCX2 in motility in mouse ileum.

    Science.gov (United States)

    Nishiyama, Kazuhiro; Azuma, Yasu-Taka; Morioka, Ai; Yoshida, Natsuho; Teramoto, Midori; Tanioka, Kohta; Kita, Satomi; Hayashi, Satomi; Nakajima, Hidemitsu; Iwamoto, Takahiro; Takeuchi, Tadayoshi

    2016-10-01

    The Na(+)/Ca(2+) exchanger (NCX) is a plasma membrane transporter that is involved in regulating intracellular Ca(2+) concentrations in various tissues. The physiological roles by which NCX influences gastrointestinal motility are incompletely understood, although its role in the heart, brain, and kidney has been widely investigated. In this study, we focused on the functions of the NCX isoforms, NCX1 and NCX2, in the motility of the ileum in the gastrointestinal tract. We investigated the response to electric field stimulation (EFS) in the longitudinal smooth muscle of the ileum obtained from wild-type mice (WT), NCX1-heterozygote knockout mice (NCX1 HET), NCX2 HET and smooth muscle-specific NCX1.3 transgenic mice (NCX1.3 Tg). EFS induced a phasic contraction that persisted during EFS and a tonic contraction that occurred after the end of EFS. We found that the amplitudes of the phasic and tonic contractions were significantly smaller in NCX2 HET, but not in NCX1 HET, compared to WT. Moreover, the magnitudes of acetylcholine (ACh)- and substance P (SP)-induced contractions of NCX2 HET, but not of NCX1 HET, were smaller compared to WT. In contrast, the amplitudes of the phasic and tonic contractions were greater in NCX1.3 Tg compared to WT. Similar to EFS, the magnitude of ACh-induced contraction was greater in NCX1.3 Tg than in WT. Taken together, our findings indicated that NCX1 and NCX2 play important roles in ileal motility and suggest that NCX1 and NCX2 regulate the motility in the ileum by controlling the sensitivity of smooth muscles to ACh and SP.

  11. Dissecting the roles of ROCK isoforms in stress-induced cell detachment

    OpenAIRE

    Shi, Jianjian; Surma, Michelle; Zhang, Lumin; Wei, Lei

    2013-01-01

    The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1 −/− and ROCK2−/− mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin...

  12. Osteopontin (OPN) isoforms, diabetes, obesity, and cancer; what is one got to do with the other? A new role for OPN.

    Science.gov (United States)

    Sarosiek, Konrad; Jones, Elizabeth; Chipitsyna, Galina; Al-Zoubi, Mazhar; Kang, Christopher; Saxena, Shivam; Gandhi, Ankit V; Sendiky, Jocelyn; Yeo, Charles J; Arafat, Hwyda A

    2015-04-01

    Alternative splicing of osteopontin (OPN) produces three isoforms: OPNa, OPNb, and OPNc. The aims of this study were to examine the expression profile of OPN isoforms in sera from patients with pancreatic lesions and to determine their correlation with the presence of comorbid systemic inflammatory conditions, such as diabetes and/or obesity. Sera from 90 patients undergoing pancreatic surgery and 29 healthy volunteers were analyzed. Seventeen patients were diabetics, 17 were obese, and 6 had both diabetes and obesity. In patients with pancreatic lesions, OPNb was expressed in 48% of the patients' sera, OPNc in 34%, and both in 5%. The presence of diabetes and/or obesity was associated with complete disappearance of OPNb and expression of only OPNc. OPNc presence was significantly associated with diabetes and obesity (OR = 7.06 [95% CI 1.97-23.3]; p = 0.003). No OPNb or OPNc was detected in the normal sera. Overexpression of OPNb and OPNc isoforms in PDA cells significantly (p obesity. Unraveling the functional role of OPN isoforms in systemic inflammation is essential to understanding their significance as therapeutic targets in diabetes and obesity, and during metastasis development in PDA.

  13. SOD isoforms play no role in lifespan in ad lib or dietary restricted conditions, but mutational inactivation of SOD-1 reduces life extension by cold.

    Science.gov (United States)

    Yen, Kelvin; Patel, Harshil B; Lublin, Alex L; Mobbs, Charles V

    2009-03-01

    The free radical theory of aging is one of the most prominent theories of aging and senescence, but has yet to be definitively proven. If free radicals are the cause of senescence, then the cellular anti-oxidant system should play a large role in lifespan determination. Because superoxide dismutase (SOD) plays a central role in detoxifying superoxide radicals, we have examined the effects of mutational inactivation of each isoform of sod on normal lifespan and lifespan extension by dietary restriction (DR) or cold-/hypothermic-induced longevity (CHIL). We find no significant decrease in lifespan for control worms or worms undergoing DR when sod isoforms are knocked-out even though sod mutational inactivation produces hypersensitivity to paraquat. In contrast, sod-1 inactivation significantly reduces lifespan extension by CHIL, suggesting that CHIL requires a specific genetic program beyond simple reduction in metabolic rate. Furthermore, CHIL paradoxically increases lifespan while reducing resistance to oxidative stress, further disassociating oxidative stress resistance and lifespan.

  14. Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.

    Science.gov (United States)

    Fujiwara, Yuya; Hiraoka, Yuri; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2015-06-30

    To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

  15. Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

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    Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2012-04-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated, followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.

  16. The effects of salt stress cause a diversion of basal metabolism in barley roots: possible different roles for glucose-6-phosphate dehydrogenase isoforms.

    Science.gov (United States)

    Cardi, Manuela; Castiglia, Daniela; Ferrara, Myriam; Guerriero, Gea; Chiurazzi, Maurizio; Esposito, Sergio

    2015-01-01

    In this study the effects of salt stress and nitrogen assimilation have been investigated in roots of hydroponically-grown barley plants exposed to 150 mM NaCl, in presence or absence of ammonium as the sole nitrogen source. Salt stress determines a diversion of root metabolism towards the synthesis of osmolytes, such as glycine betaine and proline, and increased levels of reduced glutathione. The metabolic changes triggered by salt stress result in a decrease in both activities and protein abundance of key enzymes, namely GOGAT and PEP carboxylase, and in a slight increase in HSP70. These variations would enhance the requirement for reductants supplied by the OPPP, consistently with the observed increase in total G6PDH activity. The involvement and occurrence of the different G6PDH isoforms have been investigated, and the kinetic properties of partially purified cytosolic and plastidial G6PDHs determined. Bioinformatic analyses examining co-expression profiles of G6PDHs in Arabidopsis and barley corroborate the data presented. Moreover, the gene coding for the root P2-G6PDH isoform was fully sequenced; the biochemical properties of the corresponding protein were examined experimentally. The results are discussed in the light of the possible distinct roles and regulation of the different G6PDH isoforms during salt stress in barley roots.

  17. The two different isoforms of the RSC chromatin remodeling complex play distinct roles in DNA damage responses.

    Science.gov (United States)

    Chambers, Anna L; Brownlee, Peter M; Durley, Samuel C; Beacham, Tracey; Kent, Nicholas A; Downs, Jessica A

    2012-01-01

    The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains.

  18. Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.

    Science.gov (United States)

    Dvorák, Jan; Delcroix, Melaine; Rossi, Andrea; Vopálenský, Václav; Pospísek, Martin; Sedinová, Miroslava; Mikes, Libor; Sajid, Mohammed; Sali, Andrej; McKerrow, James H; Horák, Petr; Caffrey, Conor R

    2005-07-01

    Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous

  19. Histamine H3-receptor isoforms.

    Science.gov (United States)

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  20. AQP2 exocytosis in the renal collecting duct -- involvement of SNARE isoforms and the regulatory role of Munc18b.

    NARCIS (Netherlands)

    Procino, G.; Barbieri, C.; Tamma, G.; Benedictis, L De; Pessin, J.E.; Svelto, M.; Valenti, G.

    2008-01-01

    Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of

  1. Two cytosolic glutamine synthetase isoforms play specific roles for seed germination and seed yield structure in Arabidopsis.

    Science.gov (United States)

    Guan, M; Møller, I S; Schjoerring, J K

    2015-01-01

    Nitrogen (N) remobilization from reserves to sinks is essential for seedling establishment and seed production. Cytosolic glutamine synthetase (GS1) is up-regulated during both seed germination and seed filling in plants. However, the specific roles of the individual GS1 isogenes with respect to N remobilization, early seedling vigour, and final seed productivity are not known. In this study, impairment of seed germination and seedling establishment is demonstrated in the single knockout mutant gln1;2, and the double knockout mutant gln1;1:gln1;2. The negative effect of Gln1;2 deficiency was associated with reduced N remobilization from the cotyledons and could be fully alleviated by exogenous N supply. Following reproductive growth, both the single and double Gln1;2-knockout mutants showed decreased seed yield due to fewer siliques, less seeds per silique, and lower dry weight per seed. The gln1;1 single mutant had normal seed yield structure but primary root development during seed germination was reduced in the presence of external N. Gln1;2 promoter-green fluorescent protein constructs showed that Gln1;2 localizes to the vascular cells of roots, petals, and stamens. It is concluded that Gln1;2 plays an important role in N remobilization for both seedling establishment and seed production in Arabidopsis.

  2. AQP2 exocytosis in the renal collecting duct -- involvement of SNARE isoforms and the regulatory role of Munc18b.

    Science.gov (United States)

    Procino, Giuseppe; Barbieri, Claudia; Tamma, Grazia; De Benedictis, Leonarda; Pessin, Jeffrey E; Svelto, Maria; Valenti, Giovanna

    2008-06-15

    Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.

  3. An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation.

    Science.gov (United States)

    Nguyen, Nhu-Y N; Maxwell, Mhairi J; Ooms, Lisa M; Davies, Elizabeth M; Hilton, Adrienne A; Collinge, Janelle E; Hilton, Douglas J; Kile, Benjamin T; Mitchell, Christina A; Hibbs, Margaret L; Jane, Stephen M; Curtis, David J

    2011-05-19

    In a recessive ENU mutagenesis screen for embryonic lethality, we identified a mouse pedigree with a missense mutation of SHIP1 (SHIP1(el20)) leading to an amino acid substitution I641T in the inositol-5'-phosphatase domain that represses phosphatidylinositol-3-kinase signaling. Despite detectable expression of functional SHIP1 protein, the phenotype of homozygous SHIP1(el20/el20) mice was more severe than gene-targeted SHIP1-null (SHIP1(-/-)) mice. Compared with age-matched SHIP1(-/-) mice, 5-week-old SHIP1(el20/el20) mice had increased myeloid cells, serum IL-6 levels, marked reductions in lymphoid cells, and died by 7 weeks of age with infiltration of the lungs by activated macrophages. Bone marrow transplantation demonstrated that these defects were hematopoietic-cell-autonomous. We show that the el20 mutation reduces expression in SHIP1(el20/el20) macrophages of both SHIP1 and s-SHIP, an isoform of SHIP1 generated by an internal promoter. In contrast, SHIP1(-/-) macrophages express normal levels of s-SHIP. Compound heterozygous mice (SHIP1(-/el20)) had the same phenotype as SHIP1(-/-) mice, thus providing genetic proof that the more severe phenotype of SHIP1(el20/el20) mice is probably the result of concomitant loss of SHIP1 and s-SHIP. Our results suggest that s-SHIP synergizes with SHIP1 for suppression of macrophage activation, thus providing the first evidence for a role of s-SHIP in adult hematopoiesis.

  4. Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs.

    Directory of Open Access Journals (Sweden)

    Mansoor A Syed

    Full Text Available The role of vascular endothelial growth factor (VEGF-induced 3 different nitric oxide synthase (NOS isoforms in lung development and injury in the newborn (NB lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.We studied NB wild type (WT, lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG, VEGFTG treated with a NOS1 inhibitor (L-NIO, VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA for 7 postnatal (PN days. Lung morphometry (chord length, vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin, cell proliferation (Ki67, vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG were evaluated.VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2-/- and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.

  5. Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1β treatment.

    Science.gov (United States)

    Schwarz, Alexander P; Trofimov, Alexander N; Zubareva, Olga E; Lioudyno, Victoria I; Kosheverova, Vera V; Ischenko, Alexander M; Klimenko, Victor M

    2017-08-30

    Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1β (1μg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1β. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1β-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

    Directory of Open Access Journals (Sweden)

    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

  7. The ζ isoform of diacylglycerol kinase plays a predominant role in regulatory T cell development and TCR-mediated ras signaling.

    Science.gov (United States)

    Joshi, Rohan P; Schmidt, Amanda M; Das, Jayajit; Pytel, Dariusz; Riese, Matthew J; Lester, Melissa; Diehl, J Alan; Behrens, Edward M; Kambayashi, Taku; Koretzky, Gary A

    2013-11-26

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)-stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (T(reg)) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating T(reg) cell development.

  8. ApoE isoform-dependent changes in hippocampal synaptic function

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    Sullivan Patrick M

    2009-05-01

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

  9. Isoforms of receptors of fibroblast growth factors.

    Science.gov (United States)

    Gong, Siew-Ging

    2014-12-01

    The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

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

    Science.gov (United States)

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

    2010-06-01

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

  11. Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L. and its role in PGE2 induced immunomodulation in vitro.

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    Tz Chun Guo

    Full Text Available PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4 are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line, we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

  12. Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

    Science.gov (United States)

    Guo, Tz Chun; Gamil, Amr Ahmed Abdelrahim; Koenig, Melanie; Evensen, Øystein

    2015-01-01

    PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

  13. Characterization of Acyl-CoA synthetase isoforms in pancreatic beta cells: Gene silencing shows participation of ACSL3 and ACSL4 in insulin secretion.

    Science.gov (United States)

    Ansari, Israr-Ul H; Longacre, Melissa J; Stoker, Scott W; Kendrick, Mindy A; O'Neill, Lucas M; Zitur, Laura J; Fernandez, Luis A; Ntambi, James M; MacDonald, Michael J

    2017-03-15

    Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ∼50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Na+/K+-ATPase β1-subunit is recruited in Na-K-2Cl co-transporter isoform 2 multiprotein complexes in rat kidneys: possible role in blood pressure regulation.

    Science.gov (United States)

    Carmosino, Monica; Torretta, Silvia; Procino, Giuseppe; Timperio, Annamaria; Zolla, Lello; Svelto, Maria

    2014-09-01

    The progression from prehypertensive to hypertensive state in spontaneous hypertensive rats (SHRs) is accompanied by a significant increase in membrane expression of Na-K-2Cl co-transporter isoform 2 (NKCC2), suggesting that the altered NKCC2 trafficking and activity are directly related with the development of hypertension in this strain. The aim of this work is to gain insights on the molecular mechanism that underlies this phenomenon. We performed a comparative analysis of NKCC2 multiprotein complexes (MPCs) in the kidney of SHRs versus Wistar Kyoto rats by Blue Native difference gel electrophoresis combined with mass spectrometry. We found that the recruitment of the β-subunit isoform 1 of the Na(+)-K(+)-ATPase (β1NK) in NKCC2 MPCs was significantly increased in the kidneys of SHR compared with Wistar Kyoto rat control strain. Co-immunoprecipitation experiments showed that β1NK actually interacts with NKCC2 in the native tissue. The analysis of the physiological role of β1NK-NKCC2 interaction in human embryonic kidney cells showed that β1NK increased the steady-state membrane expression and activity of NKCC2 enhancing NKCC2 trafficking toward the plasma membrane. We identify a new NKCC2-interacting partner involved in the modulation of NKCC2 intracellular trafficking and possibly involved in the regulation of blood pressure.

  15. The Role of Dermcidin Isoform 2: A Two-Faceted Atherosclerotic Risk Factor for Coronary Artery Disease and the Effect of Acetyl Salicylic Acid on It

    Directory of Open Access Journals (Sweden)

    Rajeshwary Ghosh

    2012-01-01

    Full Text Available Hypertension and diabetes mellitus are considered to be two major atherosclerotic risk factors for coronary artery disease (CAD. A stress-induced protein identified to be dermcidin isoform 2 of Mr. 11 kDa from blood plasma of hypertensive persons when injected (0.1 μM in rabbits increased the systolic pressure by 77% and diastolic pressure by 45% over the controls within 2 h. Ingestion of acetyl salicylic acid (150 mg/70 kg by these subjects reduced systolic (130 mm Hg and diastolic pressures (80 mm Hg with reduction of plasma dermcidin level to normal ranges (9 nM. The protein was found to be a potent activator of platelet cyclooxygenase and inhibited insulin synthesis. Aspirin was found to reduce hypertension by reduction of plasma dermcidin level, neutralized the effect of cyclooxygenase, and restored the pancreatic insulin synthesis through NO synthesis. These results indicated that dermcidin could be a novel atherosclerotic risk factor for its hypertensive and diabetogenic effects.

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

    Science.gov (United States)

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

    2012-10-01

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

  17. Sitagliptin reduces cardiac apoptosis, hypertrophy and fibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms.

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    Belén Picatoste

    Full Text Available BACKGROUND: Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1 enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart. METHODS: Goto-Kakizaki (GK rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin or vehicle (n=10, each. After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays. RESULTS: Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36, alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells. CONCLUSIONS: Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36 promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actions.

  18. A common role for various human truncated adenomatous polyposis coli isoforms in the control of beta-catenin activity and cell proliferation.

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    Shree Harsha Vijaya Chandra

    Full Text Available The tumour suppressor gene adenomatous polyposis coli (APC is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR. The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5 with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the "just right" level.

  19. Roles of three branchial Na(+)-K(+)-ATPase α-subunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.

    Science.gov (United States)

    Ip, Yuen K; Loong, Ai M; Kuah, Jie S; Sim, Eugene W L; Chen, Xiu L; Wong, Wai P; Lam, Siew H; Delgado, Inês L S; Wilson, Jonathan M; Chew, Shit F

    2012-07-01

    Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.

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

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-11-17

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

  1. Implication of insulin receptor A isoform and IRA/IGF-IR hybrid receptors in the aortic vascular smooth muscle cell proliferation: role of TNF-α and IGF-II.

    Science.gov (United States)

    Gómez-Hernández, Almudena; Escribano, Óscar; Perdomo, Liliana; Otero, Yolanda F; García-Gómez, Gema; Fernández, Silvia; Beneit, Nuria; Benito, Manuel

    2013-07-01

    To assess the role of insulin receptor (IR) isoforms (IRA and IRB) in the proliferation of vascular smooth muscle cells (VSMCs) involved in the atherosclerotic process, we generated new VSMC lines bearing IR (wild-type VSMCs; IRLoxP(+/+) VSMCs), lacking IR (IR(-/-) VSMCs) or expressing IRA (IRA VSMCs) or IRB (IRB VSMCs). Insulin and different proatherogenic stimuli induced a significant increase of IRA expression in IRLoxP(+/+) VSMCs. Moreover, insulin, through ERK signaling, and the proatherogenic stimuli, through ERK and p38 signaling, induced a higher proliferation in IRA than IRB VSMCs. The latter effect might be due to IRA cells showing a higher expression of angiotensin II, endothelin 1, and thromboxane 2 receptors and basal association between IRA and these receptors. Furthermore, TNF-α induced in a ligand-dependent manner a higher association between IRA and TNF-α receptor 1 (TNF-R1). On the other hand, IRA overexpression might favor the atherogenic actions of IGF-II. Thereby, IGF-II or TNF-α induced IRA and IGF-I receptor (IGF-IR) overexpression as well as an increase of IRA/IGF-IR hybrid receptors in VSMCs. More importantly, we observed a significant increase of IRA, TNF-R1, and IGF-IR expression as well as higher association of IRA with TNF-R1 or IGF-IR in the aorta from ApoE(-/-) and BATIRKO mice, 2 models showing vascular damage. In addition, anti-TNF-α treatment prevented those effects in BATIRKO mice. Finally, our data suggest that the IRA isoform and its association with TNF-R1 or IGF-IR confers proliferative advantage to VSMCs, mainly in response to TNF-α or IGF-II, which might be of significance in the early atherosclerotic process.

  2. Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

    Science.gov (United States)

    Ostrakhovitch, E A; Song, Y P; Cherian, M G

    2016-05-01

    Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

  3. 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.

  4. Neuronal profilin isoforms are addressed by different signalling pathways.

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    Kai Murk

    Full Text Available Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF, on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

  5. Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development

    OpenAIRE

    Sims-Lucas, Sunder; Cusack, Brian; Baust, Jeffrey; Eswarakumar, Veraragavan P.; Masatoshi, Hagiwara; Takeuchi, Akihide; Bates, Carlton M.

    2011-01-01

    Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc−/−) had normal kidneys. Combination of Fgfr2IIIc−/− with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1Mes−/−Fgfr2IIIc−/−) had small but identifiable MM at E10.5, expressing mesenchymal markers includi...

  6. CD44 variant isoforms in non-Hodgkin's lymphoma : a new independent prognostic factor

    OpenAIRE

    Stauder, R.; Eisterer, W.; Thaler, J; Günthert, U

    1995-01-01

    Isoforms of the transmembrane glycoprotein CD44, generated by alternative RNA splicing, have been correlated to tumor dissemination. For evaluation of the potential role of CD44 variant isoforms in non-Hodgkin's lymphoma (NHL), the presence of CD44 isoforms was analyzed in a large panel of reactive and neoplastic lymphoid tissues by immunohistochemical staining, as well as detection of CD44 variant RNAs by the reverse transcriptase-polymerase chain reaction. Whereas the CD44 standard or hemat...

  7. Amyloid-beta Isoform Metabolism Quantitation by Stable Isotope Labeled Kinetics

    OpenAIRE

    Mawuenyega, Kwasi G.; Kasten, Tom; Sigurdson, Wendy; Bateman, Randall J.

    2013-01-01

    Abundant evidence suggests a central role for the amyloid-β (Aβ) peptide in Alzheimer’s disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate th...

  8. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    Science.gov (United States)

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  9. Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

    Science.gov (United States)

    Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

    2016-08-01

    GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. 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

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

    Directory of Open Access Journals (Sweden)

    Antonella Borrelli

    2014-01-01

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

  12. AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b

    Science.gov (United States)

    Procino, Giuseppe; Barbieri, Claudia; Tamma, Grazia; De Benedictis, Leonarda; Pessin, Jeffrey E.; Svelto, Maria; Valenti, Giovanna

    2015-01-01

    Summary Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells. PMID:18505797

  13. Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin.

    Science.gov (United States)

    Kawataki, Tomoyuki; Yamane, Tetsu; Naganuma, Hirofumi; Rousselle, Patricia; Andurén, Ingegerd; Tryggvason, Karl; Patarroyo, Manuel

    2007-11-01

    Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.

  14. Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Sixt, M; Engelhardt, B; Pausch, F; Hallmann, R; Wendler, O; Sorokin, L M

    2001-05-28

    An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE). Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.

  15. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

    Directory of Open Access Journals (Sweden)

    Fahad Benthani

    2015-05-01

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

  16. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  17. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  18. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease.

    Science.gov (United States)

    Kamphuis, Willem; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-03-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. 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.

  20. 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

  1. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  2. Identification and evolutionary analysis of tissue-specific isoforms of mitochondrial complex I subunit NDUFV3.

    Science.gov (United States)

    Guerrero-Castillo, Sergio; Cabrera-Orefice, Alfredo; Huynen, Martijn A; Arnold, Susanne

    2017-03-01

    Mitochondrial complex I is the largest respiratory chain complex. Despite the enormous progress made studying its structure and function in recent years, potential regulatory roles of its accessory subunits remained largely unresolved. Complex I gene NDUFV3, which occurs in metazoa, contains an extra exon that is only present in vertebrates and thereby evolutionary even younger than the rest of the gene. Alternative splicing of this extra exon gives rise to a short NDUFV3-S and a long NDUFV3-L protein isoform. Complexome profiling revealed that the two NDUFV3 isoforms are constituents of the multi-subunit complex I. Further mass spectrometric analyses of complex I from different murine and bovine tissues showed a tissue-specific expression pattern of NDUFV3-S and NDUFV3-L. Hence, NDUFV3-S was identified as the only isoform in heart and skeletal muscle, whereas in liver, brain, and lung NDUFV3-L was expressed as the dominant isoform, together with NDUFV3-S present in all tissues analyzed. Thus, we identified NDUFV3 as the first out of 30 accessory subunits of complex I present in vertebrate- and tissue-specific isoforms. Interestingly, the tissue-specific expression pattern of NDUFV3-S and NDUFV3-L isoforms was paralleled by changes in kinetic parameters, especially the substrate affinity of complex I. This may indicate a regulatory role of the NDUFV3 isoforms in different vertebrate tissues.

  3. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  4. CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients

    NARCIS (Netherlands)

    Birzele, F.; Voss, E.; Nopora, A.; Honold, K.; Heil, F.; Lohmann, S.; Verheul, H.; Tourneau, C. Le; Delord, J.P.; Herpen, C.M. van; Mahalingam, D.; Coveler, A.L.; Meresse, V.; Weigand, S.; Runza, V.; Cannarile, M.

    2015-01-01

    PURPOSE: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform

  5. Distinct and shared transcriptomes are regulated by microphthalmia-associated transcription factor isoforms in mast cells.

    Science.gov (United States)

    Shahlaee, Amir H; Brandal, Stephanie; Lee, Youl-Nam; Jie, Chunfa; Takemoto, Clifford M

    2007-01-01

    The Microphthalmia-associated transcription factor (Mitf) is an essential basic helix-loop-helix leucine zipper transcription factor for mast cell development. Mice deficient in Mitf harbor a severe mast cell deficiency, and Mitf-mutant mast cells cultured ex vivo display a number of functional defects. Therefore, an understanding of the genetic program regulated by Mitf may provide important insights into mast cell differentiation. Multiple, distinct isoforms of Mitf have been identified in a variety of cell types; we found that Mitf-a, Mitf-e, and Mitf-mc were the major isoforms expressed in mast cells. To determine the physiologic function of Mitf in mast cells, we restored expression of these isoforms in primary mast cells from Mitf(-/-) mice. We found that these isoforms restored granular morphology and integrin-mediated migration. By microarray analysis, proteases, signaling molecules, cell surface receptor, and transporters comprised the largest groups of genes up-regulated by all isoforms. Furthermore, we found that isoforms also regulated distinct genes sets, suggesting separable biological activities. This work defines the transcriptome regulated by Mitf in mast cells and supports its role as master regulator of mast cell differentiation. Expression of multiple isoforms of this transcription factor may provide for redundancy of biological activities while also allowing diversity of function.

  6. 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.

  7. Functional differences between L- and T-plastin isoforms.

    Science.gov (United States)

    Arpin, M; Friederich, E; Algrain, M; Vernel, F; Louvard, D

    1994-12-01

    Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin, C.-S., R. H. Aebersold, and J. Leavitt. 1990. Mol. Cell. Biol. 10: 1818-1821). To learn more about the biological significance of their tissue specificity, we have overproduced the T- and L-plastin isoforms in a fibroblast-like cell line, CV-1, and in a polarized epithelial cell line, LLC-PK1. In CV-1 cells, overproduction of T- and L-plastins induces cell rounding and a concomitant reorganization of actin stress fibers into geodesic structures. L-plastin remains associated with microfilaments while T-plastin is almost completely extracted after treatment of the cells with non-ionic detergent. In LLC-PK1 cells, T-plastin induces shape changes in microvilli and remains associated with microvillar actin filaments after detergent extraction while L-plastin has no effect on these structures and is completely extracted. The effect of T-plastin on the organization of microvilli differs from that of villin, another actin-bundling protein. Our experiments indicate that these two isoforms play differing roles in actin filament organization, and do so in a cell type-specific fashion. Thus it is likely that these plastin isoforms play fundamentally different roles in cell function.

  8. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells.

    Science.gov (United States)

    Müller-Ostermeyer, F; Claus, P; Grothe, C

    2001-01-01

    Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

  9. Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

    Directory of Open Access Journals (Sweden)

    Stefanie K Schweinhuber

    Full Text Available The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as

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

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    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.

  11. VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues

    Science.gov (United States)

    1996-01-01

    VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle- associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells. PMID:8567721

  12. FSH isoform pattern in classic galactosemia

    OpenAIRE

    Gubbels, C.S.; Thomas, C. M.; Wodzig, K.W.H.; Olthaar, A. J.; Jaeken, J.; Sweep, F.C.; Rubio-Gozalbo, M.E.

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  13. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. 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

    2015-04-01

    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.

  15. RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFAxxxb isoforms.

    Science.gov (United States)

    Bridgett, Stephen; Dellett, Margaret; Simpson, David A

    2017-12-01

    Vascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA121, VEGFA165 and VEGFA189. A second set of anti-angiogenic isoforms termed VEGFAxxxb that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFAxxxb isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFAxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7-8 junction in 10 tissues did not identify any VEGFAxxxb transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFAxxxb isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA145 and VEGFA148 isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.

  16. Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

    DEFF Research Database (Denmark)

    Clausen, Johannes D; Vandecaetsbeek, Ilse; Wuytack, Frank

    2012-01-01

    The molecular mechanism underlying the characteristic high apparent Ca2+ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11 amino-acid luminal extension (LE). The effects...... from Ca2E1. Addition of the SERCA2b tail to SERCA1a slowed Ca2+ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca2E1. The interaction of LE with L7/8 is also important for the low rate...

  17. Regulation of NADPH oxidase 5 by protein kinase C isoforms.

    Directory of Open Access Journals (Sweden)

    Feng Chen

    Full Text Available NADPH oxidase5 (Nox5 is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular

  18. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    Science.gov (United States)

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  19. 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.

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

    Science.gov (United States)

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

    2010-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Prudovsky Igor

    2010-12-01

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

  2. 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

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

    Science.gov (United States)

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

    2009-01-01

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

  4. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

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    Nila Volpi

    2013-01-01

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

  5. Expression of gene, protein and immunohistochemical localization of the estrogen receptor isoform ERα1 in male rainbow trout lymphoid organs; indication of the role of estrogens in the regulation of immune mechanisms.

    Science.gov (United States)

    Massart, Sophie; Milla, Sylvain; Kestemont, Patrick

    2014-08-01

    In vertebrates, estrogens act on the reproductive system but also affect the functioning of non-reproductive tissues such as the immune system. In teleost fish, effects of estrogens and xenoestrogens have been reported extensively, but the available information on targeted tissues and cells is still scarce. Moreover, a better knowledge of the distinct ER subtypes is required to find out the mechanistic pathways by which estrogen compounds are able to disrupt endogenous estrogen signaling in fish immunity. The present study aimed at characterizing, in male rainbow trout juveniles, multi-tissue gene expression pattern of one isoform of estrogen receptor (ER), ERα1, at the mRNA and protein levels. The mRNA levels for ERα1 were measured in various lymphoid organs by real-time RT-PCR and ERα1 protein level by Western blot. Furthermore, this protein was located by immunohistochemistry in the same organs. The transcripts were ubiquitously expressed, but at a higher level in testis and liver, while the protein was more abundant in testis and skin. Moreover, the ERα1 was detected in endothelial, Kupffer, mucous and chloride cells, hematopoietic tissues, proximal tubule, epithelia of the skin and intestine, in the lamina propria and in the stratum granulosum. This distribution backs the idea that, in male rainbow trout, estrogeno-mimetic compounds could be involved in different immune mechanisms such as inflammatory response, transport of Ig, mucus production, regulation of cellular immunity and development and maturation of lymphoid and myeloid cells.

  6. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  7. 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.

  8. 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.

  9. Differential expression of the interleukin 5 receptor alpha isoforms in blood and tissue eosinophils of nasal polyp patients

    OpenAIRE

    Gevaert, Philippe; Hellman, C.; Lundblad, L.; J. Lundahl; Holtappels, Gabriële; Van Cauwenberge, Paul; Tavernier, Jan; Bachert, Claus

    2009-01-01

    Given the key role of interleukin-5 (IL-5) in eosinophil function, we investigated the regulated expression of the membrane-anchored (TM-IL-5R alpha) isoform, or a secreted (SOL IL-5R alpha) isoform, on both protein and transcript level in vitro and in vivo. A real-time PCR, FACS and ELISA were established to determine IL-5R alpha isoform expression in peripheral blood and nasal tissue from control subjects and nasal polyp (NP) patients with or without asthma. Human peripheral blood eosino...

  10. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  11. p53 isoforms change p53 paradigm

    OpenAIRE

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  12. Absolute quantitation of protein posttranslational modification isoform.

    Science.gov (United States)

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

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

    African Journals Online (AJOL)

    Apolipoprotein (A) Isoform Distribution and Plasma Lipoprotein (a) Levels In Nigerian Subjects With and Without Coronary Heart Disease. ... Plasma lipoprotein (a) Concentrations and apo(a) isoforms were determined in 101 ... Article Metrics.

  14. Relative contributions of NOS isoforms during experimental colitis : endothelial-derived NOS maintains mucosal integrity

    NARCIS (Netherlands)

    Vallance, BA; Dijkstra, G; Qiu, BS; van der Waaij, LA; van Goor, H; Jansen, PLM; Mashimo, H; Collins, SM

    2004-01-01

    The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating

  15. Translational control of SCL-isoform expression in hematopoietic lineage choice

    NARCIS (Netherlands)

    Calkhoven, Cornelis F; Muller, Christine; Martin, Richard; Krosl, Goradz; Pietsch, Hubertus; Hoang, Trang; Leutz, Achim

    2003-01-01

    We investigated the translational regulation of SCL protein expression and its role in hematopoietic lineage choice. We show that the expression of different SCL protein isoforms is regulated by signal transduction pathways that modulate translation initiation factor (eIF) function. A conserved smal

  16. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  17. Epidermal growth-factor-induced transcript isoform variation drives mammary cell migration.

    Directory of Open Access Journals (Sweden)

    Wolfgang J Köstler

    Full Text Available Signal-induced transcript isoform variation (TIV includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF. We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.

  18. New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents

    Directory of Open Access Journals (Sweden)

    Hanna Szaefer

    2013-08-01

    Full Text Available Cytochromes P450 (P450 are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms’ genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1 that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

  19. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

    Science.gov (United States)

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J; Vacic, Vladimir; Calderwood, Michael A; Roth, Frederick P; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M

    2014-04-11

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

  20. The FU gene and its possible protein isoforms

    Directory of Open Access Journals (Sweden)

    Nöthen Markus M

    2004-07-01

    Full Text Available Abstract Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci. Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1 maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing

  1. 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

  2. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B

    1998-01-01

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

  3. [Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

    Science.gov (United States)

    Okuneva, A D; Vikhliantsev, I M; Shpagina, M D; Rogachevskiĭ, V V; Khutsian, S S; Poddubnaia, Z A; Grigor'ev, A I

    2012-01-01

    Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

  4. 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.

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

    Science.gov (United States)

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

    2004-04-01

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

  6. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have u...... synthesized cohorts of pumps from the Golgi apparatus to the plasma membrane....

  7. FSH isoform pattern in classic galactosemia.

    Science.gov (United States)

    Gubbels, Cynthia S; Thomas, Chris M G; Wodzig, Will K W H; Olthaar, André J; Jaeken, Jaak; Sweep, Fred C G J; Rubio-Gozalbo, M Estela

    2011-04-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

  8. Signalling by Transforming Growth Factor Beta Isoforms in Wound Healing and Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Richard W.D. Gilbert

    2016-06-01

    Full Text Available Transforming growth factor beta (TGFβ signalling is essential for wound healing, including both non-specific scar formation and tissue-specific regeneration. Specific TGFβ isoforms and downstream mediators of canonical and non-canonical signalling play different roles in each of these processes. Here we review the role of TGFβ signalling during tissue repair, with a particular focus on the prototypic isoforms TGFβ1, TGFβ2, and TGFβ3. We begin by introducing TGFβ signalling and then discuss the role of these growth factors and their key downstream signalling mediators in determining the balance between scar formation and tissue regeneration. Next we discuss examples of the pleiotropic roles of TGFβ ligands during cutaneous wound healing and blastema-mediated regeneration, and how inhibition of the canonical signalling pathway (using small molecule inhibitors blocks regeneration. Finally, we review various TGFβ-targeting therapeutic strategies that hold promise for enhancing tissue repair.

  9. 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.

  10. Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

    Science.gov (United States)

    Pires, Susana S; Hughes, Steven; Turton, Michael; Melyan, Zare; Peirson, Stuart N; Zheng, Lei; Kosmaoglou, Maria; Bellingham, James; Cheetham, Michael E; Lucas, Robert J; Foster, Russell G; Hankins, Mark W; Halford, Stephanie

    2009-09-30

    Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

  11. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  12. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-10-10

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

  14. Muscle Specific Fragile X Related Protein 1 Isoforms are Sequestered in the Nucleus of Undifferentiated Myoblast

    Directory of Open Access Journals (Sweden)

    Khandjian Edouard W

    2000-12-01

    Full Text Available Abstract Background The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied. Results Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism. Conclusions The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

  15. Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain [v1; ref status: indexed, http://f1000r.es/1mg

    Directory of Open Access Journals (Sweden)

    Lara Kaddoum

    2013-10-01

    Full Text Available Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2 that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.

  16. Expression of Contractile Protein Isoforms in Microgravity

    Science.gov (United States)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  17. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.

    Science.gov (United States)

    Gilbreth, Ryan N; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B; Li, Nan-Sheng; Piccirilli, Joseph A; Chen, Yuan; Koide, Shohei

    2011-05-10

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  18. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

    Energy Technology Data Exchange (ETDEWEB)

    Gilbreth, Ryan N.; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B.; Li, Nan-Sheng; Piccirilli, Joseph A.; Chen, Yuan; Koide, Shohei (UC); (CHNMC)

    2011-07-25

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a 'monobody,' a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K{sub d} values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  19. NHE-1 isoform of the Na+/H+ antiport is expressed in the rat and rabbit esophagus.

    Science.gov (United States)

    Shallat, S; Schmidt, L; Reaka, A; Rao, D; Chang, E B; Rao, M C; Ramaswamy, K; Layden, T J

    1995-11-01

    Rabbit esophageal cells show an amiloride-sensitive Na+/H+ antiporter activity. Several distinct molecular isoforms of the Na+/H+ exchanger family (NHE) are reported to be present in the gastrointestinal tract of rats and rabbits. The aim of this study was to examine which isoforms are present in rabbit and rat esophageal cells and whether this isoform could be up-regulated by serum factors. Specific primers designed from the rat NHE-1-4 and the rabbit NHE-1-3 isoform sequences were used for reverse-transcription polymerase chain reaction analysis with RNA from rabbit and rat esophageal cells. Ribonuclease protection assay was used to determine the serum-induced up-regulation of NHE-1. Antibodies raised against the NHE-1 C-terminal fragment were used for Western blotting with rabbit esophageal membranes. In both the rat and rabbit esophagus, only the NHE-1 isoform messenger RNA could be detected. The NHE-1 messenger RNA, detected in rabbit esophageal cells grown from tissue explants, was up-regulated by serum factors. The antibody detected a 95-kilodalton protein in esophageal cell membranes. The rabbit and rat esophagus exclusively express the NHE-1 isoform, hypothesized to be involved in cytoplasmic pH regulation. Therefore, the results of this study suggest a role for NHE-1 in protecting cells against gastric acid that is refluxed into the esophagus.

  20. 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.

  1. 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.

  2. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

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

  3. The landscape of isoform switches in human cancers

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Sandelin, Albin Gustav

    2017-01-01

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

  4. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    Science.gov (United States)

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

  5. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    Science.gov (United States)

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  6. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Directory of Open Access Journals (Sweden)

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  7. Regulation of the viability of Nf1 deficient cells by PKC isoforms.

    Science.gov (United States)

    Zhou, Xiaodong; Shen, Ling; Parris, Toshima; Huang, Junchi; Yi, Bo; Helou, Khalil; Chen, Changyan

    2014-11-15

    Suppression of protein kinase C (PKC) is known to be synthetically lethal with ras mutations in various types of cancer cells. The studies also showed that blockade of PKC affected the viability of Nf1 deficient cells. Since PKC family consists of more than 10 isoforms, our study aimed at identifying which isoform(s) played the crucial role in sensitizing Nf1 deficient cells to apoptosis. Using genetic and chemical PKC inhibitors, we demonstrated that the concurrent inhibition of PKC α and β induced Nf1 deficient ST or 96.2 cells, but not SNF02.2 cells with a normal Nf1 or ST cells ectopically expressing Nf1 effective domain gene, to apoptosis. In this process, PKC δ in Nf1 deficient cells, but not in ST/Nf1 cells, was upregulated and translocated to the nucleus. Furthermore, caspase 3 was cleaved and cytochrome c was released to the cytosol. Thus, it appeared that PKC δ and α/β are the crucial components for sustaining the aberrant Ras signaling and further viability of Nf1 deficient cells. The abrogation of these two isoforms activated their opponent PKC δ for switching on the caspase 3-governed apoptotic machinery.

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

    Science.gov (United States)

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

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Zorka Milićević

    2014-01-01

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

  10. Progesterone receptor isoforms in the mammary gland of cats and dogs.

    Science.gov (United States)

    Gracanin, A; de Gier, J; Zegers, K; Bominaar, M; Rutteman, G R; Schaefers-Okkens, A C; Kooistra, H S; Mol, J A

    2012-12-01

    Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

  11. Functional Cooperativity between ABCG4 and ABCG1 Isoforms.

    Directory of Open Access Journals (Sweden)

    Zoltán Hegyi

    Full Text Available ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system.

  12. Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

    Science.gov (United States)

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

    This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

  13. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    Science.gov (United States)

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  14. Tocopherol isoforms in parenteral lipid emulsions and neutrophil activation.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Beunk, J.; Naber, A.H.J.; Swinkels, D.W.

    2002-01-01

    BACKGROUND AND AIMS: Tocopherol is a lipid-soluble anti-oxidant that exists in several isoforms. Patients on total parenteral nutrition depend on lipid emulsions for their tocopherol intake. In the present study, we analysed the content of tocopherol isoforms in various lipid emulsions. We also

  15. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2000-12-01

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

  17. 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.

  18. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

    Institute of Scientific and Technical Information of China (English)

    Anastasia Pavlidou; Maria Dalamaga; Christos Kroupis; George Konstantoudakis; Maria Belimezi; George Athanasas; Kleanthi Dimas

    2011-01-01

    AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler- Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.

  19. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    Science.gov (United States)

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 m

  20. eIF5A isoforms and cancer: two brothers for two functions?

    Science.gov (United States)

    Caraglia, M; Park, M H; Wolff, E C; Marra, M; Abbruzzese, A

    2013-01-01

    Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. The role of hypusine formation in the eIF5A protein in the regulation of cell proliferation and apoptosis is addressed in the present review. Moreover, vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. However, the biological functions of these two isoforms may be significantly different. In fact, eIF5A-1 is demonstrable in most cells of different histogenesis, whereas eIF5A-2 protein is detectable only in certain human cancer cells or tissues, suggesting its role as a potential oncogene. In this review we focus our attention on the involvement of eIF5A-1 in the triggering of an apoptotic program and in the regulation of cell proliferation. In addition, the potential oncogenic role and prognostic significance of eIF5A-2 in the prediction of the survival of cancer patients is described. eIF5A-1 and/or the eIF5A-2 isoform may serve as a new molecular diagnostic or prognostic marker or as a molecular target for anti-cancer therapy.

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

    National Research Council Canada - National Science Library

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

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

  2. The Role of Akt Isoforms in Colorectal Cancer

    Science.gov (United States)

    2015-09-01

    tumors derive from somatic modification of genes known to be involved in CRC; 3) the somatic mutations involve the colonic epithelium ; 4) tumors... lung cancer. Mol. Cell 2014;53:577–590. 4. Hung KE, Maricevich MA, Richard LG, et al. Development of a mouse model for sporadic and metastatic colon

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

    Directory of Open Access Journals (Sweden)

    Tine Glendorf

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

  4. 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

  5. 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

  6. Nonmuscle myosin II isoforms coassemble in living cells.

    Science.gov (United States)

    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

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

    Science.gov (United States)

    Wang, Xiaoxing; Yang, Yali; Dai, Wei

    2006-03-01

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

  8. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    Science.gov (United States)

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  9. Fruitless isoforms and target genes specify the sexually dimorphic nervous system underlying Drosophila reproductive behavior.

    Science.gov (United States)

    Nojima, Tetsuya; Neville, Megan C; Goodwin, Stephen F

    2014-01-01

    Courtship is pivotal to successful reproduction throughout the animal kingdom. Sexual differences in the nervous system are thought to underlie courtship behavior. Male courtship behavior in Drosophila is in large part regulated by the gene fruitless (fru). fru has been reported to encode at least three putative BTB-zinc-finger transcription factors predicted to have different DNA-binding specificities. Although a large number of previous studies have demonstrated that fru plays essential roles in male courtship behavior, we know little about the function of Fru isoforms at the molecular level. Our recent study revealed that male-specific Fru isoforms are expressed in highly overlapping subsets of neurons in the male brain and ventral nerve cord. Fru isoforms play both distinct and redundant roles in male courtship behavior. Importantly, we have identified for the first time, by means of the DamID technique, direct Fru transcriptional target genes. Fru target genes overwhelmingly represent genes previously reported to be involved in the nervous system development, such as CadN, lola and pdm2. Our study provides important insight into how the sexually dimorphic neural circuits underlying reproductive behavior are established.

  10. Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

    Directory of Open Access Journals (Sweden)

    Lee June-Hyuk

    2004-06-01

    Full Text Available Abstract Background The functional role of nitric oxide (NO and various nitric oxide synthase (NOS isoforms in asthma remains unclear. Objective This study investigated the effects of ozone and ovalbumin (OVA exposure on NOS isoforms. Methods The expression of inducible NOS (iNOS, neuronal NOS (nNOS, and endothelial NOS (eNOS in lung tissue was measured. Enhanced pause (Penh was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL fluid were measured using a modified Griess reaction. Results The nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced Penh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group. Conclusion In mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.

  11. Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy.

    Directory of Open Access Journals (Sweden)

    Shaon Naskar

    Full Text Available A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy or detrimental (pathological hypertrophy. This study was undertaken to establish the role of different protein kinase-C (PKC isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

  12. Differential and Conditional Activation of PKC-Isoforms Dictates Cardiac Adaptation during Physiological to Pathological Hypertrophy

    Science.gov (United States)

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation. PMID:25116170

  13. Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma

    Science.gov (United States)

    Vicari, Luisa; La Rosa, Cristina; Forte, Stefano; Calabrese, Giovanna; Colarossi, Cristina; Aiello, Eleonora; Salluzzo, Salvatore; Memeo, Lorenzo

    2016-01-01

    Background Activating transcription factor 5 (ATF5) is a member of the activating transcription/cAMP response element-binding protein family of basic leucine zipper proteins that plays an important role in cell survival, differentiation, proliferation, and apoptosis. The ATF5 gene generates two transcripts: ATF5 isoform 1 and ATF5 isoform 2. A number of studies indicate that ATF5 could be an attractive target for therapeutic intervention in several tumor types; however, so far, the role of ATF5 has not been investigated in papillary thyroid carcinoma (PTC). Methods Quantitative real-time reverse transcription polymerase chain reaction and immuno-histochemical staining were used to study ATF5 mRNA and protein expression in PTC. Results We report here that ATF5 is expressed more in PTC tissue than in normal thyroid tissue. Furthermore, this is the first study that describes the presence of both ATF5 isoforms in PTC. Conclusion These findings could provide potential applications in PTC cancer treatment.

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

    Science.gov (United States)

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

    2013-06-01

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

  15. Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy.

    Science.gov (United States)

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

  16. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent.

    Science.gov (United States)

    Kim, Hak Rim; Gallant, Cynthia; Leavis, Paul C; Gunst, Susan J; Morgan, Kathleen G

    2008-09-01

    Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

  17. Effects of contraction on localization of GLUT4 and v-SNARE isoforms in rat skeletal muscle

    DEFF Research Database (Denmark)

    Rose, Adam John; Jeppesen, Jacob; Kiens, Bente

    2009-01-01

    In skeletal muscle, contractions increase glucose uptake due to a translocation of GLUT4 glucose transporters from intracellular storage sites to the surface membrane. Vesicle associated membrane proteins (VAMPs) are believed to play an important role in docking and fusion of the GLUT4 transporters...... at the surface membrane. However, knowledge about which VAMP isoforms in fact co-localize with GLUT4 vesicles in mature skeletal muscle and whether they translocate during muscle contractions is incomplete. The aim of the present study was to further identify VAMP isoforms which are associated with GLUT4...... vesicles and examine which VAMP isoforms translocate to surface membranes in skeletal muscles undergoing contractions. VAMP2, VAMP3, VAMP5 and VAMP7 were enriched in immuno-precipitated GLUT4 vesicles. In response to 20 min of in situ contractions, there was a redistribution of GLUT4 (+64 +/- 13...

  18. 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

  19. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna

    2015-01-01

    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 compare to tissue. This revealed unexpectedly...... complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.......Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass...

  20. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    Science.gov (United States)

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  1. Development of a new model system to dissect isoform specific Akt signalling in adipocytes

    Science.gov (United States)

    Kajno, Esi; McGraw, Timothy E.; Gonzalez, Eva

    2015-01-01

    Protein kinase B (Akt) kinases are critical signal transducers mediating insulin action. Genetic studies revealed that Akt1 and Akt2 signalling differentially contribute to sustain lipid and glucose homoeostasis; however Akt isoform-specific effectors remain elusive due to the lack of a suitable model system to mechanistically interrogate Akt isoform-specific signalling. To overcome those technical limitations we developed a novel model system that provides acute and specific control of signalling by Akt isoforms. We generated mutants of Akt1 and Akt2 resistant to the allosteric Akt inhibitor MK-2206. We then developed adipocyte cell lines, in which endogenous Akt1 or Akt2 has been replaced by their corresponding drug-resistant Akt mutant. Treatment of those cells with MK-2206 allowed for acute and specific control of either Akt1 or Akt2 function. Our data showed that Akt1W80A and Akt2W80A mutants are resistant to MK-2206, dynamically regulated by insulin and able to signal to Akt downstream effectors. Analyses of insulin action in this cellular system showed that Akt1 and Akt2 are both able to mediate insulin regulation of the transcription factor forkhead box O1 (FoxO1) and the glucose transporter 4 (GLUT4), revealing a redundant role for these Akt kinases in the control of glucose transport into fat cells. In contrast, Akt1 signalling is uniquely required for adipogenesis, by controlling the mitotic clonal expansion (MCE) of pre-adipocytes that precedes white adipose cell differentiation. Our data provide new insights into the role of Akt kinases in glucose transport and adipogenesis and support our model system as a valuable tool for the biochemical characterization of signalling by specific Akt isoforms. PMID:25856301

  2. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M

    2015-11-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

  3. Formation of VEGF isoform-specific spatial distributions governing angiogenesis: computational analysis

    Directory of Open Access Journals (Sweden)

    Mac Gabhann Feilim

    2011-05-01

    Full Text Available Abstract Background The spatial distribution of vascular endothelial growth factor A (VEGF is an important mediator of vascular patterning. Previous experimental studies in the mouse hindbrain and retina have suggested that VEGF alternative splicing, which controls the ability of VEGF to bind to heparan sulfate proteoglycans (HSPGs in the extracellular matrix (ECM, plays a key role in controlling VEGF diffusion and gradients in tissues. Conversely, proteolysis notably by matrix metalloproteinases (MMPs, plays a critical role in pathological situations by releasing matrix-sequestered VEGF and modulating angiogenesis. However, computational models have predicted that HSPG binding alone does not affect VEGF localization or gradients at steady state. Results Using a 3D molecular-detailed reaction-diffusion model of VEGF ligand-receptor kinetics and transport, we test alternate models of VEGF transport in the extracellular environment surrounding an endothelial sprout. We show that differences in localization between VEGF isoforms, as observed experimentally in the mouse hindbrain, as well as the ability of proteases to redistribute VEGF in pathological situations, are consistent with a model where VEGF is endogenously cleared or degraded in an isoform-specific manner. We use our predictions of the VEGF distribution to quantify a tip cell's receptor binding and gradient sensing capacity. A novel prediction is that neuropilin-1, despite functioning as a coreceptor to VEGF165-VEGFR2 binding, reduces the ability of a cell to gauge the relative steepness of the VEGF distribution. Comparing our model to available in vivo vascular patterning data suggests that vascular phenotypes are most consistently predicted at short range by the soluble fraction of the VEGF distributions, or at longer range by matrix-bound VEGF detected in a filopodia-dependent manner. Conclusions Isoform-specific VEGF degradation provides a possible explanation for numerous examples

  4. Mdm2 Splice isoforms regulate the p53/Mdm2/Mdm4 regulatory circuit via RING domain-mediated ubiquitination of p53 and Mdm4.

    Science.gov (United States)

    Fan, Chuandong; Wang, Xinjiang

    2017-02-06

    p53 is regulated by heterodimer E3 ligase Mdm2-Mdm4 via RING domain interaction. Mdm2 transcripts undergo alternative splicing, and Mdm2 splice isoforms are increased in cancer and induced by DNA damage. Although two major Mdm2 splice isoforms that do not bind to p53 were reported to impact the p53 pathway, the underlying biochemical mechanisms were not understood. Here, we show that these Mdm2 splice isoforms ubiquitinate Mdm2 and Mdm4 in vitro and regulate the activity of Mdm2-Mdm4 E3 complex in cells. The Mdm2 isoforms are capable of promoting p53 ubiquitination in the absence of Mdm2 or Mdm4. The two isoforms stimulate Mdm2 or Mdm4 activity for p53 ubiquitination in vitro and promote degradation of p53 and Mdm4 in cells. However, the Mdm2 isoforms have opposing effects on the steady-state p53 levels depending on the stoichiometric ratios of Mdm2, Mdm4 and the isoforms, causing either decreased or increased p53 levels in cells. Our data indicate that the Mdm2 splice isoforms can act as independent E3 ligases for p53 when Mdm2 and Mdm4 are absent, form potent heterodimer E3 ligases with either Mdm2 or Mdm4 for targeting p53 degradation, or act as inhibitory regulators of Mdm2-Mdm4 E3 ligase activity by downregulating Mdm4. These findings suggest that Mdm2 splice isoforms may play critical roles in the regulatory loop of p53/Mdm2-Mdm4 via a RING domain-mediated biochemical mechanism.

  5. 人类成纤维细胞生长因子受体1的Ⅲb亚型在胰腺癌细胞中的作用%The role of human fibroblast growth factor receptor 1-Ⅲb isoform in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    刘占兵; 杨尹默; 乔岐禄; 赵建勋; 黄莚庭; Marko Kornmann

    2007-01-01

    Objective To study the role of Ⅲb isoform of human fibroblast growth factor receptor 1 (FGFR1-Ⅲb) in PANC-1 pancreatic cancer cells. Methods The plasmid of human full-length FGFR1-Ⅲb isoform,pSVK4/FGFR1-Ⅲb, was stable transfected into cultured PNAC-1 pancreatic cancer cell lines facilitated by lipofectamine. The function of FGFR1-Ⅲb in transfected pancreatic cancer cells were examined by MTT assay, soft agar assay, cell migration assay, single cell movement assay, In vivo tumorigenicity assay. Results The basal anchorage-dependent and -independent cell growth was significantly inhibited. Additionally, FGFR1-Ⅲb expression inhibited single cell movement and in vitro invasion as determined by time-lapse microscopy and boyden chamber assay as well as in vivo tumor formation and growth in nude mice. Microscopic analysis of the xenograft tumors revealed a reduced Ki-67 labelling, lower amount of tumor necrosis and higher grade of differentiation in FGFR1-Ⅲb expressing tumors. Conclusion We identified a functional human FGFR mRNA splice variant that inhibits the transforming potential of pancreatic cancer cells.%目的 探讨人类成纤维细胞生长因子受体1的Ⅲb亚型(FGFR1-Ⅲb)在胰腺癌细胞中的作用. 方法 以脂质体法将人类全长FGFR1-Ⅲb表达质粒pSVK4/FGFR1-Ⅲb稳定转染入PANC-1胰腺癌细胞,通过MTT试验、软琼脂试验、细胞迁移试验、单个细胞运动试验和裸鼠移植瘤试验检测FGFR1-Ⅲb在转染的胰腺癌细胞中的功能. 结果 转染FGFR1-Ⅲb受体的胰腺癌细胞,在细胞生长、细胞迁移、细胞运动、移植瘤形成及生长方面均受抑,且移植瘤Ki-67表达减低、肿瘤组织中坏死灶减少. 结论 人类FGFR1-Ⅲb受体为胰腺癌细胞的功能性受体,对胰腺癌细胞具有抑制作用.

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

    Directory of Open Access Journals (Sweden)

    Shinobu Tsuzuki

    2007-05-01

    Full Text Available AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.These data demonstrate that the "a" isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood

  7. Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1

    Science.gov (United States)

    Tsuzuki, Shinobu; Hong, Dengli; Gupta, Rajeev; Matsuo, Keitaro; Seto, Masao; Enver, Tariq

    2007-01-01

    Background AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo. Methods and Findings The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo. Conclusions These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of

  8. Isoform-specific targeting of ROCK proteins in immune cells

    OpenAIRE

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform ...

  9. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  10. Yeast ADP/ATP Carrier Isoform 2

    Science.gov (United States)

    Clémençon, Benjamin; Rey, Martial; Trézéguet, Véronique; Forest, Eric; Pelosi, Ludovic

    2011-01-01

    The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family responsible for exchanging ADP and ATP across the mitochondrial inner membrane. ADP/ATP transport involves Ancp switching between two conformational states. These can be analyzed using specific inhibitors, carboxyatractyloside (CATR) and bongkrekic acid (BA). The high resolution three-dimensional structure of bovine Anc1p (bAnc1p), as a CATR-carrier complex, has been solved. However, because the structure of the BA-carrier complex has not yet been determined, the detailed mechanism of transport remains unknown. Recently, sample processing for hydrogen/deuterium exchange experiments coupled to mass spectrometry was improved, providing novel insights into bAnc1p conformational transitions due to inhibitor binding. In this work we performed both hydrogen/deuterium exchange-mass spectrometry experiments and genetic manipulations. Because these are very difficult to apply with bovine Anc1p, we used Saccharomyces cerevisiae Anc isoform 2 (ScAnc2p). Significant differences in solvent accessibility were observed throughout the amino acid sequence for ScAnc2p complexed to either CATR or BA. Interestingly, in detergent solution, the conformational dynamics of ScAnc2p were dissimilar to those of bAnc1p, in particular for the upper half of the cavity, toward the intermembrane space, and the m2 loop, which is thought to be easily accessible to the solvent from the matrix in bAnc1p. Our study then focused on the methionyl residues of the Ancp signature sequence, RRRMMM. All our results indicate that the methionine cluster is involved in the ADP/ATP transport mechanism and confirm that the Ancp cavity is a highly dynamic structure. PMID:21868387

  11. Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells

    Directory of Open Access Journals (Sweden)

    Zhang Yumin

    2011-04-01

    Full Text Available Abstract Background Excessive production of nitric oxide (NO by inducible nitric oxide synthase (iNOS in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia. Methods Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF-κB in activated microglia was examined by determining NF-κB transcriptional response element- (TRE- driven, promoter-mediated luciferase activity. Results Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC β attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF-κB activation that leads to the expression of iNOS in reactive microglia. Conclusions Our results identify PKC δ and β as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF-κB. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.

  12. 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...

  13. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    OpenAIRE

    Fagotti, A; Gabbiani, G.; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a stre...

  14. Alternative splicing during Arabidopsis flower development results in constitutive and stage-regulated isoforms

    Directory of Open Access Journals (Sweden)

    Haifeng eWang

    2014-02-01

    Full Text Available Alternative splicing (AS is a process in eukaryotic gene expression, in which the primary transcript of a multi-exon gene is spliced into two or more different mature transcripts, thereby increasing proteome diversity. AS is often regulated differentially between different tissues or developmental stages. Recent studies suggested that up to 60% of intron-containing genes in Arabidopsis thaliana undergo AS. Yet little is known about this complicated and important process during floral development. To investigate the preferential expression of different isoforms of individual alternatively spliced genes, we used high throughput RNA-Seq technology to explore the transcriptomes of three floral development stages of Arabidopsis thaliana and obtained information of various alternative splicing events. We identified approximately 24,000 genes that were expressed at one or more of these stages, and found that nearly 25% of multi-exon genes had two or more spliced variants. This is less frequent than the previously reported 40%~60% for multiple organs and stages of A. thaliana, indicating that many genes expressed in floral development function with a single predominant isoform. On the other hand, 1,716 isoforms were differentially expressed between the three stages, suggesting that AS might still play important roles in stage transition during floral development. Moreover, 337 novel transcribed regions were identified and most of them have a single exon. In addition, our analyses provide a comprehensive survey of alternative splicing in floral development and facilitate further genomic and genetic studies.

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

    Directory of Open Access Journals (Sweden)

    Katherine J D A Excoffon

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

  16. Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals.

    Science.gov (United States)

    Lakowski, Jörn; Majumder, Anirban; Lauderdale, James D

    2007-07-15

    The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

  17. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.

  18. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    ZHOU YanXia; TAN HongWei; YANG ZuoYin; JIA ZongChao; LIU RuoZhuang; CHEN GuangJu

    2007-01-01

    The insect spruce budworm (Choristoneura fumiferana) produces antifreeze protein (AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms, known as CfAFP-501 and CfAFP-337, show that both possess similar left-handed β-helical structure, although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to, and cannot be simply accounted for, by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In order to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general, we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs, a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337, strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the antifreeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implication for antifreeze activity are reported for β-helical antifreeze proteins.

  19. Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

    Science.gov (United States)

    Wong, Marty Kwok-Shing; Pipil, Supriya; Kato, Akira; Takei, Yoshio

    2016-09-01

    Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney.

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

    Directory of Open Access Journals (Sweden)

    M. Aprile

    2014-01-01

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

  1. Prokaryotic expression and characterization of a pea actin isoform (PEAcl) fused to GFP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shaobin; REN Dongtao; XU Xiaojing; LIU Guoqin

    2004-01-01

    Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells, it is difficult to purify each actin isoform in sufficient quantities for analyzing its physicochemical properties. In the present study, a pea(Pisum Sativum L.) actin isoform (PEAc1) fused to His-tag at its amino terminus and GFP (green fluorescent protein) at its Carboxyl terminus were expressed in E. Coli in inclusion bodies. The fusion protein (PEAc1-GFP) was highly purified with the yield of above 2 mg/L culture by dissolving inclusions in 8 mol/L urea, renaturing by dialysis in a gradient of urea, and affinity binding to Ni-resin. The purified mono meric PEAc1-GFP could efficiently bind on Dnase Ⅰ and inhibit the latter's enzyme activity. PEAc1-GFP could polymerize into green fluorescent filamentous structures (F-PEAc1-GFP), which could be labeled by TRITC-phalloidin, a specific agent for observing microfilaments. The PEAc1-GFP polymerization curve was identical with that of chicken skeletal muscle actin. The critical concentration for PEAc1-GFP to polymerize into filaments is 0.24 μmol/L. The F-PEAc1-GFP could stimulate myosin Mg-ATPase activity in a protein concentration dependant manner (about 4 folds at1 mg/mL F-PEAc1-GFP). The results above show that the PEAcl fused to GFP retained the assembly characteristic of actin, indicating that gene fusion, prokaryotic expression,denaturation and renaturation, and affinity chromatography is a useful strategy for obtaining plant actin isoform proteins in a large amount.

  2. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Science.gov (United States)

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  3. Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid.

    Science.gov (United States)

    Silva, Alexandra; Sampaio-Marques, Belém; Fernandes, Angela; Carreto, Laura; Rodrigues, Fernando; Holcik, Martin; Santos, Manuel A S; Ludovico, Paula

    2013-01-01

    Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.

  4. Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid

    Science.gov (United States)

    Silva, Alexandra; Sampaio-Marques, Belém; Fernandes, Ângela; Carreto, Laura; Rodrigues, Fernando; Holcik, Martin; Santos, Manuel A. S.; Ludovico, Paula

    2013-01-01

    Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress. PMID:23967187

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

    Directory of Open Access Journals (Sweden)

    Francesca Rappa

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

  7. Research resource: new and diverse substrates for the insulin receptor isoform a revealed by quantitative proteomics after stimulation with igf-ii or insulin

    DEFF Research Database (Denmark)

    Morcavallo, Alaide; Gaspari, Marco; Pandini, Giuseppe;

    2011-01-01

    The isoform A of the insulin receptor (IR) (IR-A) is a bifunctional receptor, because it binds both insulin and IGF-II. IR-A activation by IGF-II plays a role in development, but its physiological role in adults is unknown. IGF-II signaling through IR-A is deregulated in cancer and favors tumor p...

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

    Science.gov (United States)

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

    2017-03-01

    during human brain development provides vital insight into gene function and identifies critical sensitive periods whereby genetic factors may influence risk for psychiatric disease. Here the authors provide comprehensive insight into the transcriptional landscape of the psychiatric risk gene, NRG3, in human neocortical development and expand on previous findings in schizophrenia to identify increased expression of developmentally and genetically regulated isoforms in the brain of patients with mood disorders. Principally, the finding that NRG3 classes II and III are brain-specific isoforms predicted by rs10748842 risk genotype and are increased in mood disorders further implicates a molecular mechanism of psychiatric risk at the NRG3 locus and identifies a potential developmental role for NRG3 in bipolar disorder and major depression. These observations encourage investigation of the neurobiology of NRG3 isoforms and highlight inhibition of NRG3 signaling as a potential target for psychiatric treatment development.

  9. The differences in binding 12-carbon aliphatic ligands by bovine β-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography.

    Science.gov (United States)

    Loch, Joanna I; Bonarek, Piotr; Polit, Agnieszka; Swiątek, Śylwia; Dziedzicka-Wasylewska, Marta; Lewiński, Krzysztof

    2013-08-01

    Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance.

  10. Different expression patterns of renal Na(+)/K(+)-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

    Science.gov (United States)

    Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-12-01

    Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na(+)/K(+)-ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences.

  11. Chemical origins of isoform selectivity in histone deacetylase inhibitors.

    Science.gov (United States)

    Butler, Kyle V; Kozikowski, Alan P

    2008-01-01

    Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.

  12. Isoform-targeted regulation of cardiac adenylyl cyclase.

    Science.gov (United States)

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  13. p53 isoform profiling in glioblastoma and injured brain.

    Science.gov (United States)

    Takahashi, R; Giannini, C; Sarkaria, J N; Schroeder, M; Rogers, J; Mastroeni, D; Scrable, H

    2013-06-27

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10 to 70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, some of which can modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry and reverse transcription-PCR. We found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared with tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions.

  14. Tolerance to the antinociceptive and hypothermic effects of morphine is mediated by multiple isoforms of c-Jun N-terminal kinase.

    Science.gov (United States)

    Yuill, Matthew B; Zee, Michael L; Marcus, David; Morgan, Daniel J

    2016-04-13

    The abuse and overdose of opioid drugs are growing public health problems worldwide. Although progress has been made toward understanding the mechanisms governing tolerance to opioids, the exact cellular machinery involved remains unclear. However, there is growing evidence to suggest that c-Jun N-terminal kinases (JNKs) play a major role in mu-opioid receptor regulation and morphine tolerance. In this study, we aimed to determine the potential roles of different JNK isoforms in the development of tolerance to the antinociceptive and hypothermic effects of morphine. We used the hot-plate and tail-flick tests for thermal pain to measure tolerance to the antinociceptive effects of once-daily subcutaneous injections with 10 mg/kg morphine. Body temperature was also measured to determine tolerance to the hypothermic effects of morphine. Tolerance to morphine was assessed in wild-type mice and compared with single knockout mice each lacking the JNK isoforms (JNK1, JNK2, or JNK3). We found that loss of each individual JNK isoform causes impairment in tolerance for the antinociceptive and hypothermic effects of daily morphine. However, disruption of JNK2 seems to have the most profound effect on morphine tolerance. These results indicate a clear role for JNK signaling pathways in morphine tolerance. This complements previous studies suggesting that the JNK2 isoform is required for morphine tolerance, but additionally presents novel data suggesting that additional JNK isoforms also contribute toward this process.

  15. Structure- and isoform-specific glucuronidation of six curcumin analogs.

    Science.gov (United States)

    Lu, Danyi; Liu, Hui; Ye, Wencai; Wang, Ying; Wu, Baojian

    2017-04-01

    1. In the present study, we aimed to characterize the glucuronidation of six curcumin analogs (i.e. RAO-3, RAO-8, RAO-9, RAO-18, RAO-19, and RAO-23) derived from galangal using human liver microsomes (HLM) and twelve expressed UGT enzymes. 2. Formation of glucuronide was confirmed using high-resolution mass spectrometry. Single glucuronide metabolite was generated from each of six curcumin analogs. The fragmentation patterns were analyzed and were found to differ significantly between alcoholic and phenolic glucuronides. 3. All six curcumin analogs except one (RAO-23) underwent significant glucuronidation in HLM and expressed UGT enzymes. In general, the methoxy group (close to the phenolic hydroxyl group) enhanced the glucuronidation liability of the curcumin analogs. 4. UGT1A9 and UGT2B7 were primarily responsible for the glucuronidation of two alcoholic analogs (RAO-3 and RAO-18). By contrast, UGT1A9 and four UGT2Bs (UGT2B4, 2B7, 2B15 and 2B17) played important roles in conjugating three phenolic analogs (RAO-8, RAO-9, and RAO-19). Interestingly, the conjugated double bonds system (in the aliphatic chain) was crucial to the substrate selectivity of gastrointestinal UGTs (i.e. UGT1A7, 1A8 and 1A10). 5. In conclusion, glucuronidation of six curcumin analogs from galangal were structure- and isoform-specific. The knowledge should be useful in identifying a curcumin analog with improved metabolic property.

  16. The in vivo phosphorylation sites in multiple isoforms of amphiphysin I from rat brain nerve terminals

    DEFF Research Database (Denmark)

    Craft, George E; Graham, Mark E; Bache, Nicolai;

    2008-01-01

    -proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple......, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non...

  17. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Laminin isoforms in endothelial and perivascular basement membranes.

    Science.gov (United States)

    Yousif, Lema F; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.

  20. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Expression of multiple cbb3 cytochrome c oxidase isoforms by combinations of multiple isosubunits in Pseudomonas aeruginosa.

    Science.gov (United States)

    Hirai, Takehiro; Osamura, Tatsuya; Ishii, Masaharu; Arai, Hiroyuki

    2016-10-24

    The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa has five terminal oxidases for aerobic respiration and uses them under different growth conditions. Two of them are cbb3-type cytochrome c oxidases encoded by the gene clusters ccoN1O1Q1P1 and ccoN2O2Q2P2, which are the main terminal oxidases under high- and low-oxygen conditions, respectively. P. aeruginosa also has two orphan gene clusters, ccoN3Q3 and ccoN4Q4, encoding the core catalytic CcoN isosubunits, but the roles of these genes have not been clarified. We found that 16 active cbb3 isoforms could be produced by combinations of four CcoN, two CcoO, and two CcoP isosubunits. The CcoN3- or CcoN4-containing isoforms were produced in the WT cell membrane in response to nitrite and cyanide, respectively. The strains carrying these isoforms were more resistant to nitrite or cyanide under low-oxygen conditions. These results indicate that P. aeruginosa gains resistance to respiratory inhibitors using multiple cbb3 isoforms with different features, which are produced through exchanges of multiple core catalytic isosubunits.

  3. Membrane-impermeable cross-linking provides evidence for homophilic, isoform-specific binding of desmosomal cadherins in epithelial cells.

    Science.gov (United States)

    Nie, Zhuxiang; Merritt, Anita; Rouhi-Parkouhi, Mansour; Tabernero, Lydia; Garrod, David

    2011-01-21

    Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.

  4. Murine fibroblast growth factor receptor 1alpha isoforms mediate node regression and are essential for posterior mesoderm development.

    Science.gov (United States)

    Xu, X; Li, C; Takahashi, K; Slavkin, H C; Shum, L; Deng, C X

    1999-04-15

    Alternative splicing in the fibroblast growth factor receptor 1 (Fgfr1) locus generates a variety of splicing isoforms, including FGFR1alpha isoforms, which contain three immunoglobulin-like loops in the extracellular domain of the receptor. It has been previously shown that embryos carrying targeted disruptions of all major isoforms die during gastrulation, displaying severe growth retardation and defective mesodermal structures. Here we selectively disrupted the FGFR1alpha isoforms and found that they play an essential role in posterior mesoderm formation during gastrulation. We show that the mutant embryos lack caudal somites, develop spina bifida, and die at 9.5-12.5 days of embryonic development because they are unable to establish embryonic circulation. The primary defect is a failure of axial mesoderm cell migration toward the posterior portions of the embryos during gastrulation, as revealed by regional marker analysis and DiI labeling. In contrast, the anterior migration of the notochord is unaffected and the embryonic structures rostral to the forelimb are relatively normal. These data demonstrate that FGF/FGFR1alpha signals are posteriorizing factors that control node regression and posterior embryonic development.

  5. A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation.

    Science.gov (United States)

    Thomas, Jean-Claude; Ughy, Bettina; Lagoutte, Bernard; Ajlani, Ghada

    2006-11-28

    Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNR(S) approximately 34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNR(L) approximately 46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNR(S), is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNR(L) is an NADP(+) reductase, whereas FNR(S) is an NADPH oxidase.

  6. Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

    Science.gov (United States)

    He, Ying; Wang, Zaijie Jim

    2015-03-18

    As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

  7. Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hongyu LIU; Zhuomin WANG; Yun BAI; Min WANG; Ying LI; Sen WEI; Qinghua ZHOU; Jun CHEN

    2009-01-01

    Aim:BAG-1 is a multifunctional anti-apoptotic gene with four isoforms,and different BAG-1 isoforms have different anti-apoptotic functions.In this study,we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER nega-tive) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens.Methods: The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive).After stable cell lines were made,a variety of apoptosis-inducing agents,including doxorubicin,docetaxel,and 5-FU,was used to treat these cell lines with or without estrogen to test the role of BAG-1.The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay.Results: The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis.BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis.The cell viability assay showed that only BAG-1 p50,but not p46,p33,or p29,increased estrogen-dependent function in ER-positive cell line MCF-7.Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen,while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms.In the detection of the expression of K-ras,Hsp70,cytochrome c,Raf-1,ER-α,and Bcl-2 in MCF-7 cells by Western blot,only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46,respectively.Furthermore,the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells.Conclusion: Distinct isoforrns of BAG-1 have different anti-apoptotic functions in breast cancer cells,and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.

  8. 14-3-3 phosphoprotein interaction networks – does isoform diversity present functional interaction specification?

    Directory of Open Access Journals (Sweden)

    Anna-Lisa ePaul

    2012-08-01

    Full Text Available The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organisms might carry isoform specific functions. However there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ specific expression. This situation has led to a currently unresolved question – does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory that sequence diversity does lead to functional diversity among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in experiments designed to address 14-3-3 functional roles. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis.

  9. Two isoforms of ferredoxin:NADP(+) oxidoreductase from wheat leaves: purification and initial biochemical characterization.

    Science.gov (United States)

    Grzyb, Joanna; Malec, Przemysław; Rumak, Izabela; Garstka, Maciej; Strzałka, Kazimierz

    2008-04-01

    Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

  10. Clinical chemistry of common apolipoprotein E isoforms

    NARCIS (Netherlands)

    Brouwer, DAJ; vanDoormaal, JJ; Muskiet, FAJ

    1996-01-01

    Apolipoprotein E plays a central role in clearance of lipoprotein remnants by serving as a ligand for low-density lipoprotein and apolipoprotein E receptors. Three common alleles (apolipoprotein E(2), E(3) and E(4)) give rise to six phenotypes. Apolipoprotein E(3) is the ancestral form. Common apoli

  11. Clinical chemistry of common apolipoprotein E isoforms

    NARCIS (Netherlands)

    Brouwer, DAJ; vanDoormaal, JJ; Muskiet, FAJ

    1996-01-01

    Apolipoprotein E plays a central role in clearance of lipoprotein remnants by serving as a ligand for low-density lipoprotein and apolipoprotein E receptors. Three common alleles (apolipoprotein E(2), E(3) and E(4)) give rise to six phenotypes. Apolipoprotein E(3) is the ancestral form. Common

  12. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.

    Science.gov (United States)

    Harauz, George; Boggs, Joan M

    2013-05-01

    The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP's protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.

  13. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Science.gov (United States)

    Eksi, Ridvan; Li, Hong-Dong; Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S; Kretzler, Matthias; Guan, Yuanfang

    2013-01-01

    Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

  14. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Directory of Open Access Journals (Sweden)

    Ridvan Eksi

    Full Text Available Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

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

    OpenAIRE

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

    2012-01-01

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

  16. Identifying the role of pre-and postsynaptic GABAB receptors in behavior

    Science.gov (United States)

    Kasten, Chelsea R.; Boehm, Stephen L.

    2015-01-01

    Although many reviews exist characterizing the molecular differences of GABAB receptor isoforms, there is no current review of the in vivo effects of these isoforms. The current review focuses on whether the GABAB1a and GABAB1b isoforms contribute differentially to behaviors in isoform knockout mice. The roles of these receptors have primarily been characterized in cognitive, anxiety, and depressive phenotypes. Currently, the field supports a role of GABAB1a in memory maintenance and protection against an anhedonic phenotype, whereas GABAB1b appears to be involved in memory formation and a susceptibility to developing an anhedonic phenotype. Although GABAB receptors have been strongly implicated in drug abuse phenotypes, no isoform-specific work has been done in this field. Future directions include developing site-specific isoform knockdown to identify the role of different brain regions in behavior, as well as identifying how these isoforms are involved in development of behavioral phenotypes. PMID:26283074

  17. Conformational Flexibility Differentiates Naturally Occurring Bet v 1 Isoforms.

    Science.gov (United States)

    Grutsch, Sarina; Fuchs, Julian E; Ahammer, Linda; Kamenik, Anna S; Liedl, Klaus R; Tollinger, Martin

    2017-06-03

    The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform's higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

  18. Protein tyrosine phosphatase PTPRR isoforms in cellular signaling and trafficking

    NARCIS (Netherlands)

    Dilaver, Gönül

    2005-01-01

    Previous work has revealed the existence of two Protein Tyrosine Phosphatases in mouse, PTPBR7 and PTP-SL, that were in part identical, suggesting that they originated from the same gene, termed Ptprr (1,5,6). In this thesis, I report on the characterization of the various PTPRR isoforms in neuronal

  19. Murine Sirt3 protein isoforms have variable half-lives

    Science.gov (United States)

    Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3...

  20. 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 2-sial

  1. Differential water permeability and regulation of three aquaporin 4 isoforms

    DEFF Research Database (Denmark)

    Fenton, Robert A.; Moeller, Hanne B; Zelenina, Marina

    2010-01-01

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the b...

  2. Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

    Directory of Open Access Journals (Sweden)

    Apiruck Watthanasurorot

    2011-06-01

    Full Text Available The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam, encodes 9(Ig-4(FNIII-(Ig-2(FNIII-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

  3. 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.

  4. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    Science.gov (United States)

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons.

  5. A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin

    Directory of Open Access Journals (Sweden)

    Hebert Michael D

    2005-04-01

    Full Text Available Abstract Background Cajal bodies (CBs are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus. Results In this report, we identify a novel protein isoform of EB-1/AIDA-1, termed AIDA-1c, that interacts with the CB marker protein, coilin. Northern and nested PCR experiments reveal that the AIDA-1c isoform is expressed in brain and several cancer cell lines. Competition binding experiments demonstrate that AIDA-1c competes with SmB' for coilin binding sites, but does not bind SMN. When ectopically expressed, AIDA-1c is predominantly nuclear with no obvious accumulations in CBs. Interestingly, another EB-1/AIDA-1 nuclear isoform, AIDA-1a, does not bind coilin in vivo as efficiently as AIDA-1c. Knockdown of EB-1/AIDA-1 isoforms by siRNA altered Cajal body organization and reduced cell viability. Conclusion These data suggest that specific EB-1/AIDA-1 isoforms, such as AIDA-1c, may participate in the regulation of nucleoplasmic coilin protein interactions in neuronal and transformed cells.

  6. Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin Ⅰ

    Institute of Scientific and Technical Information of China (English)

    Qi Tian; Ji-Feng Zhang; Jinjin Fan; Zhihong Song; Yuan Chen

    2012-01-01

    Objective Formation of the endophilin Ⅱ-Ca2+ channel complex is Ca2+-dependent in clathrin-mediated endocytosis.However,little is known about whether the other two endophilin isoforms have the same features.The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca2+ channels and dynamin Ⅰ.Methods N-type Ca2+ channel C-terminal fragments (NCFs) synthesized with a 3H-leucine-labeled kit,were incubated with endophilin-GST fusion proteins,followed by pull-down assay.Results were counted on a scintillation counter.In addition,the different endophilin isoforms were each co-transfected with dynamin Ⅰ into 293T cells,followed by flow cytometry and co-immunoprecipitation assay.Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin Ⅰ.Results All three isoforms interacted with NCF.Endophilins Ⅰ and Ⅱ demonstrated clear Ca2+-dependent interactions with NCF,whereas endophilin Ⅲ did not.Co-immunoprecipitation showed that,compared to endophilin Ⅰ/Ⅱ,the interaction between endophilin Ⅲ and dynamin Ⅰ was significantly increased.Similar results were obtained from flow cytometry.Furthermore,endophilin Ⅲ had a higher overlap coefficient with dynamin Ⅰ in co-transfected 293T cells.Conclusion Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin Ⅰ.Endophilin Ⅲ binding to NCF is Ca2+-independent,implying that it plays a different role in clathrin-mediated endocytosis.

  7. DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells.

    Science.gov (United States)

    Duymich, Christopher E; Charlet, Jessica; Yang, Xiaojing; Jones, Peter A; Liang, Gangning

    2016-04-28

    Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells.

  8. Identification of two frataxin isoforms in Zea mays: Structural and functional studies.

    Science.gov (United States)

    Buchensky, Celeste; Sánchez, Manuel; Carrillo, Martin; Palacios, Oscar; Capdevila, Mercè; Domínguez-Vera, Jose M; Busi, Maria V; Atrian, Sílvia; Pagani, Maria A; Gomez-Casati, Diego F

    2017-09-01

    Frataxin is a ubiquitous protein that plays a role in Fe-S cluster biosynthesis and iron and heme metabolism, although its molecular functions are not entirely clear. In non-photosynthetic eukaryotes, frataxin is encoded by a single gene, and the protein localizes to mitochondria. Here we report the presence of two functional frataxin isoforms in Zea mays, ZmFH-1 and ZmFH-2. We confirmed our previous findings regarding plant frataxins: both proteins have dual localization in mitochondria and chloroplasts. Physiological, biochemical and biophysical studies show some differences in the expression pattern, protection against oxidants and in the aggregation state of both isoforms, suggesting that the two frataxin homologs would play similar but not identical roles in plant cell metabolism. In addition, two specific features of plant frataxins were evidenced: their ability to form dimers and their tendency to undergo conformational change under oxygen exposure. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  9. Short-term control of maize cell and root water permeability through plasma membrane aquaporin isoforms.

    Science.gov (United States)

    Hachez, Charles; Veselov, Dmitry; Ye, Qing; Reinhardt, Hagen; Knipfer, Thorsten; Fricke, Wieland; Chaumont, François

    2012-01-01

    Although it is widely accepted that aquaporins are involved in the regulation of root water uptake, the role of specific isoforms in this process is poorly understood. The mRNA expression and protein level of specific plasma membrane intrinsic proteins (PIPs) were analysed in Zea mays in relation to cell and root hydraulic conductivity. Plants were analysed during the day/night period, under different growth conditions (aeroponics/hydroponics) and in response to short-term osmotic stress applied through polyethylene glycol (PEG). Higher protein levels of ZmPIP1;2, ZmPIP2;1/2;2, ZmPIP2;5 and ZmPIP2;6 during the day coincided with a higher water permeability of root cortex cells during the day compared with night period. Similarly, plants which were grown under aeroponic conditions and which developed a hypodermis ('exodermis') with Casparian bands, effectively forcing more water along a membranous uptake path across roots, showed increased levels of ZmPIP2;5 and ZmPIP1;2 in the rhizodermis and exodermis. When PEG was added to the root medium (2-8 h), expression of PIPs and cell water permeability in roots increased. These data support a role of specific PIP isoforms, in particular ZmPIP1;2 and ZmPIP2;5, in regulating root water uptake and cortex cell hydraulic conductivity in maize.

  10. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing;

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  11. Mast cells express novel functional IL-15 receptor alpha isoforms.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  12. Growth hormone isoforms in a girl with gigantism.

    Science.gov (United States)

    Ng, L L; Chasalow, F I; Escobar, O; Blethen, S L

    1999-01-01

    Several previous investigations have suggested that there may be different growth hormone isoforms in patients with acromegaly. We used three different site-specific monoclonal antibodies (MAbs) to investigate growth hormone (GH) isoforms in serum from an 8 year-old girl with a GH and prolactin secreting adenoma. The pattern of GH-immunoreactivity was dependent on the circumstances of collection. Serum obtained after oral glucose had very little cross reactivity with MAb 352 although concentrations of up to 15 micrograms/l were found with two other MAbs, 033 and 665. MAb 352 does not recognize the 20,000 dalton isoform of GH (20K) while both MAb 033 and 665 do. The same pattern of GH immunoreactivity (low MAb 352, equal and higher MAb 033 and 665) was seen in other baseline samples. In contrast, samples obtained after TRH/GnRH showed immunoreactivity patterns expected for a mixture of 22,000 dalton isoform of GH (22K) with only a small amount of 20K. GH samples obtained during sleep showed both patterns with episodic peaks with equal immunoreactivity superimposed on the basal pattern (decreased activity with MAb 352). Affinity chromatography of basal samples showed that a portion of the GH immunoreactivity was neither 22K nor 20K, although in stimulated samples, over 70% of GH was 22K or 20K GH. In conclusion, the nature of GH isoforms present in serum varies with GH concentration. These differences may contribute to the known difficulty in correlating disease activity and random GH measurements in patients with GH secreting adenomas.

  13. Parallel subfunctionalisation of PsbO protein isoforms in angiosperms revealed by phylogenetic analysis and mapping of sequence variability onto protein structure.

    Science.gov (United States)

    Duchoslav, Miloš; Fischer, Lukáš

    2015-06-09

    PsbO, the manganese-stabilising protein, is an indispensable extrinsic subunit of photosystem II. It plays a crucial role in the stabilisation of the water-splitting Mn4CaO5 cluster, which catalyses the oxidation of water to molecular oxygen by using light energy. PsbO was also demonstrated to have a weak GTPase activity that could be involved in regulation of D1 protein turnover. Our analysis of psbO sequences showed that many angiosperm species express two psbO paralogs, but the pairs of isoforms in one species were not orthologous to pairs of isoforms in distant species. Phylogenetic analysis of 91 psbO sequences from 49 land plant species revealed that psbO duplication occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the level of isoform divergence was similar in different species. Moreover, mapping of the differences on the protein tertiary structure showed that the isoforms in individual species differ from each other on similar positions, mostly on the luminally exposed end of the β-barrel structure. Comparison of these differences with the location of differences between PsbOs from diverse angiosperm families indicated various selection pressures in PsbO evolution and potential interaction surfaces on the PsbO structure. The analyses suggest that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. We speculate that the presence of two PsbO isoforms helps the plants to finely adjust the photosynthetic apparatus in response to variable conditions. This might be mediated by diverse GTPase activity, since the isoform differences predominate near the predicted GTP-binding site.

  14. Secondary structures involving the poly(A tail and other 3’ sequences are major determinants of mRNA isoform stability in yeast

    Directory of Open Access Journals (Sweden)

    Zarmik Moqtaderi

    2014-04-01

    Full Text Available In Saccharomyces cerevisiae, previous measurements of mRNA stabilities have been determined on a per-gene basis. We and others have recently shown that yeast genes give rise to a highly heterogeneous population of mRNAs due to extensive alternative 3’ end formation. Typical genes can have fifty or more distinct mRNA isoforms with 3’ endpoints differing by as little as one and as many as hundreds of nucleotides. In our recent paper [Geisberg et al. Cell (2014 156: 812-824] we measured half-lives of individual mRNA isoforms in Saccharomyces cerevisiae by using the anchor away method for the rapid removal of Rpb1, the largest subunit of RNA Polymerase II, from the nucleus, followed by direct RNA sequencing of the cellular mRNA population over time. Combining these two methods allowed us to determine half-lives for more than 20,000 individual mRNA isoforms originating from nearly 5000 yeast genes. We discovered that different 3’ mRNA isoforms arising from the same gene can have widely different stabilities, and that such half-life variability across mRNA isoforms from a single gene is highly prevalent in yeast cells. Determining half-lives for many different mRNA isoforms from the same genes allowed us to identify hundreds of RNA sequence elements involved in the stabilization and destabilization of individual isoforms. In many cases, the poly(A tail is likely to participate in the formation of stability - enhancing secondary structures at mRNA 3’ ends. Our results point to an important role for mRNA structure at 3’ termini in governing transcript stability, likely by reducing the interaction of the mRNA with the degradation apparatus.

  15. Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing

    Science.gov (United States)

    Edelman, Theresa L. B.; McCulloch, Katherine A.; Barr, Angela; Frøkjær-Jensen, Christian; Jorgensen, Erik M.; Rougvie, Ann E.

    2016-01-01

    The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing. PMID:27729432

  16. Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing.

    Science.gov (United States)

    Edelman, Theresa L B; McCulloch, Katherine A; Barr, Angela; Frøkjær-Jensen, Christian; Jorgensen, Erik M; Rougvie, Ann E

    2016-12-07

    The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

  17. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Takahiro; Itoh, Kyoko, E-mail: kxi14@koto.kpu-m.ac.jp; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  18. CEACAM1 Long Cytoplasmic Domain Isoform is Associated with Invasion and Recurrence of Hepatocellular Carcinoma

    Science.gov (United States)

    Kiriyama, Shigehisa; Yokoyama, Shozo; Ueno, Masaki; Hayami, Shinya; Ieda, Junji; Yamamoto, Naoyuki; Yamaguchi, Shunsuke; Mitani, Yasuyuki; Nakamura, Yasushi; Tani, Masaji; Mishra, Lopa; Shively, John E.; Yamaue, Hiroki

    2014-01-01

    Background The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. β2-spectrin (β2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-β signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and β2SP. Methods Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and β2SP. Results In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p<0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of β2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with β2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to β2SP. Conclusions CEACAM1-L may enhance the HCC invasiveness through an interaction with β2SP and subsequent effects on TGF-β signaling. PMID:24390710

  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. Energy balance-dependent regulation of ovine glucose 6-phosphate dehydrogenase protein isoform expression.

    Science.gov (United States)

    Triantaphyllopoulos, Kostas A; Laliotis, George P; Bizelis, Iosif A

    2014-01-01

    G6PDH is the rate-limiting enzyme of the pentose phosphate pathway and one of the principal source of NADPH, a major cellular reductant. Importantly, in ruminant's metabolism the aforementioned NADPH provided, is utilized for de novo fatty acid synthesis. Previous work of cloning the ovine (Ovis aries) og6pdh gene has revealed the presence of two cDNA transcripts (og6pda and og6pdb), og6pdb being a product of alternative splicing not similar to any other previously reported.(1) In the current study the effect of energy balance in the ovine G6PDH protein expression was investigated, shedding light on the biochemical features and potential physiological role of the oG6PDB isoform. Changes in energy balance leads to protein expression changes in both transcripts, to the opposite direction and not in a proportional way. Negative energy balance was not in favor of the presence of any particular isoform, while both protein expression levels were not significantly different (P > 0.05). In contrast, at the transition point from negative to positive and on the positive energy balance, there is a significant increase of oG6PDA compared with oG6PDB protein expression (P < 0.001). Both oG6PDH protein isoforms changed significantly toward the positive energy balance. oG6PDA is escalating, while oG6PDB is falling, under the same stimulus (positive energy balance alteration). This change is also positively associated with increasing levels in enzyme activity, 4 weeks post-weaning in ewes' adipose tissue. Furthermore, regression analysis clearly demonstrated the linear correlation of both proteins in response to the WPW, while energy balance, enzyme activity, and oG6PDA relative protein expression follow the same escalating trend; in contrast, oG6PDB relative protein expression falls in time, similar to both transcripts accumulation pattern, as reported previously.(2.)

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  2. Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

    NARCIS (Netherlands)

    Kooij, V.; Venkatraman, V.; Kirk, J.A.; Ubaida-Mohien, C.; Graham, D.R.; Faber, M.J.; Eyk, J.E. Van

    2014-01-01

    PURPOSE: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. EXPER

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  4. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Science.gov (United States)

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

  5. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Directory of Open Access Journals (Sweden)

    Angélique Guillaudeau

    Full Text Available The EGFR (epidermal growth factor receptor is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a, normal and tumor cells produce soluble EGFR isoforms (sEGFR that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2, 3 (v3 and 4 (v4 mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab and intracellular domain targeted antibody (ICD-Ab. EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade, histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS. PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  7. Characterization of four hemocyanin isoforms in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    XU Jingxiang; RUAN Lingwei; LI Zhen; YU Xiaoman; LI Sedong; SHI Hong; XU Xun

    2015-01-01

    In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in inver-tebrate.

  8. Disulfide isoforms of recombinant glia maturation factor beta.

    Science.gov (United States)

    Zaheer, A; Lim, R

    1990-09-14

    Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.

  9. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  10. 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

    2015-04-01

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Consolación (A)lvarez; Irene García; Luis C.Romero; Cecilia Gotor

    2012-01-01

    In non-cyanogenic species,the main source of cyanide derives from ethylene and camalexin biosyntheses.In mitochondria,cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolized bythe β-cyanoalanine synthase CYS-C1,catalyzing the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine.The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform,OAS-C,which catalyzes the incorporation of sulfide to O-acetylserine to produce cysteine,thus generating a cyclic pathway in the mitochondria.The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme,showing defects in root hair formation.Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation,restoring the wild-type phenotype.The mitochondria compromise their capacity to properly detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant.Consequently,we observe an accumulation of sulfide and cyanide and of the alternative oxidase,which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules.Our results allow us to suggest that the significance of OAS-C is related to its role in the proper sulfide and cyanide detoxification in mitochondria.

  12. Glycation alter the process of Tau phosphorylation to change Tau isoforms aggregation property.

    Science.gov (United States)

    Liu, Kefu; Liu, Yutong; Li, Lingyun; Qin, Peibin; Iqbal, Javed; Deng, Yulin; Qing, Hong

    2016-02-01

    The risk of tauopathies depends in part on the levels and modified composition of six Tau isoforms in the human brain. Abnormal phosphorylation of the Tau protein and the shift of the ratio of 3R Tau to 4R Tau are presumed to result in neurofibrillary pathology and neurodegeneration. Glycation has recently been linked to dementia and metabolic syndrome. To determine the contribution of Tau protein glycation and phosphorylation on Tau aggregation propensity, the assembled kinetics were examined in vitro using Thioflavin T fluorescence assays. We found that glycation and phosphorylation have different effects on aggregation propensity in different Tau isoforms. Different Tau proteins play important parts in each tauopathies, but 3R0N, fetal Tau protein, has no effect on tauopathies. Conversely, 4R2N has more modified sites and a higher tendency to aggregate, playing the most important role in 4R tauopathies. Finally, Glycation, which could modulate Tau phosphorylation, may occur before any other modification. It also regulates the 3R to 4R ratio and promotes 4R2N Tau protein aggregation. Decreasing the sites of glycation, as well as shifting other Tau proteins to 3R0N Tau proteins has potential therapeutic implications for tauopathies.

  13. Remodeling of the Cervix and Parturition in Mice Lacking the Progesterone Receptor B Isoform1

    Science.gov (United States)

    Yellon, Steven M.; Oshiro, Bryan T.; Chhaya, Tejas Y.; Lechuga, Thomas J.; Dias, Rejane M.; Burns, Alexandra E.; Force, Lindsey; Apostolakis, Ede M.

    2011-01-01

    Withdrawal of progestational support for pregnancy is part of the final common pathways for parturition, but the role of nuclear progesterone receptor (PGR) isoforms in this process is not known. To determine if the PGR-B isoform participates in cervical remodeling at term, cervices were obtained from mice lacking PGR-B (PGR-BKO) and from wild-type (WT) controls before or after birth. PGR-BKO mice gave birth to viable pups at the same time as WT controls during the early morning of Day 19 postbreeding. Morphological analyses indicated that by the day before birth, cervices from PGR-BKO and WT mice had increased in size, with fewer cell nuclei/area as well as diminished collagen content and structure, as evidenced by optical density of picrosirius red-stained sections, compared to cervices from nonpregnant mice. Moreover, increased numbers of resident macrophages, but not neutrophils, were found in the prepartum cervix of PGR-BKO compared to nonpregnant mice, parallel to findings in WT mice. These results suggest that PGR-B does not contribute to the growth or degradation of the extracellular matrix or proinflammatory processes associated with recruitment of macrophages in the cervix leading up to birth. Rather, other receptors may contribute to the progesterone-dependent mechanism that promotes remodeling of the cervix during pregnancy and in the proinflammatory process associated with ripening before parturition. PMID:21613631

  14. Distinct Functions of Different scl Isoforms in Zebrafish Definitive Hematopoietic Stem Cell Initiation and Maintenance

    Science.gov (United States)

    Lan, Yahui

    2011-07-01

    The establishment of entire blood system relies on the multi-potent hematopoietic stem cells (HSCs), thus identifying the molecular mechanism in HSC generation is of importance for not only complementing the fundamental knowledge in stem cell biology, but also providing insights to the regenerative therapies. Recent researches have documented the formation of nascent HSCs through a direct transition from ventral aortic endothelium, named as endothelial hematopoietic transition (EHT) process. However, the precise genetic program engaged in this process remains largely elusive. The transcription factor scl plays pivotal and conserved roles in embryonic and adult hematopoiesis from teleosts to mammals. Our lab have previously identified a new truncated scl isoform, scl-beta, which is indispensible for the specification of HSCs in the ventral wall of dorsal aorta (VDA), the zebrafish equivalent of mammalian fetal hematopoietic organ. Here we observe that, by combining time-lapse confocal imaging of transgenic zebrafish and genetic epistasis analysis, scl-beta is expressed in a subset of ventral aortic endothelial cells and critical for their forthcoming transformation to hemogenic endothelium; in contrast, runx1 is required downstream to govern the successful egress of the hemogenic endothelial cells to become naive HSCs. In addition, the traditional known full-length scl-alpha isoform is firstly evidenced to be required for the maintenance or survival of newly formed HSCs in VDA. Collectively our data has established the genetic hierarchy controlling discrete steps in the consecutive process of HSC formation from endothelial cells and further development in VDA.

  15. Mesalamine suppresses the expression of TC22, a novel tropomyosin isoform associated with colonic neoplasia.

    Science.gov (United States)

    Das, Koushik K; Bajpai, Manisha; Kong, Yingxin; Liu, Jianying; Geng, Xin; Das, Kiron M

    2009-07-01

    Although a protective role for mesalamine against colon cancer in ulcerative colitis has been shown epidemiologically, its molecular mechanism is unknown. We cloned and sequenced a novel human tropomyosin (hTM) isoform, TC22, which is an alternatively spliced variant of normal epithelial hTM isoform 5 (hTM5), identical apart from 25 C-terminal amino acids. TC22 is expressed in 100% of colorectal carcinoma but is not expressed in normal colon epithelial cells. To explore a molecular mechanism of chemoprevention, we examined the effect of mesalamine on TC22 expression using LS180 colon cancer cells. Expression of hTM5 and TC22 was investigated at the protein and gene levels by fluorescence-activated cell sorting and real-time reverse transcription-polymerase chain reaction. Small interference RNA (siRNA) against the TC22 variant were transfected into LS180 colon cancer cells, reducing protein and transcript levels by 45 to 50%. Mesalamine or sulfasalazine (2 mM), but not sulfapyridine, significantly (p mesalamine, sulfasalazine, and rosiglitazone significantly reduced the cellular expression of TC22, implicating PPARgamma in this modulation. Similar suppression of TC22 by siRNA produced gene level changes on several critical carcinogenic pathways. These findings suggest a novel antineoplastic molecular effect of mesalamine.

  16. MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

    Science.gov (United States)

    Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

    2017-01-01

    An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

  17. HLA-G5 and G7 Isoforms in Pregnant Women.

    Directory of Open Access Journals (Sweden)

    Saeid Abediankenari

    2015-04-01

    Full Text Available Human leukocyte antigen-G which is an immune tolerance effecter molecule has an important role in the maintenance of fetus during pregnancy. Abortion is one of the complications of pregnancy period.  In this research, we have studied levels of HLA-G 5, HLA-G7 isoforms in the abortion-threatened pregnant women in comparison with controls. In a case-control study, 101 abortion-threatened women and 101 healthy pregnant women (healthy controls with age range 21-32 years were studied. Gene expression of HLA-5 and HLA-7 isoforms was analyzed by real-time polymerase chain reaction after mRNA extraction and cDNA synthesis. The results indicated that HLA-G5 was significantly lower in abortion-threatened women in comparison with the control group whereas HLA-G7 was not significantly differentbetween the 2 groups. HLA-G is a vital molecule during pregnancy that can be a key factor in prevention of abortion. It is concluded that determination of HLA-G5 can be of value in pregnancy.

  18. 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.

  19. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel

    2011-01-01

    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  20. [Clinical relevance of myosin isoforms in the diaphragm].

    Science.gov (United States)

    Gayan-Ramirez, G; Decramer, M

    2000-06-01

    The diaphragm as a striated muscle is characterized by the repetition of a single element arranged in series: the sarcomere containing two kinds of myofilaments: a thick one constituted by the myosin, and a thin one primarily composed of actin. The myosin molecule consists of two heads where two myosin heavy chains (MHC) are fixed, a flexible hinge with two light (MLC) chains, and long rod-shaped tails. The diaphragm contains 4 MHC isoforms (MHC-slow, MHC-2A, MHC-2B, MHC-2X) and 6 MLC isoforms (MLC-1f, MLC-3f, MLC-1sa, MLC-1sb, MLC-2f, MLC-2s/v). In humans, the diaphragm contains mainly fibers expressing the isoforms MHC-slow, MHC-2A, and MLC-2f, MLC-2s et MLC-1f. For the mechanical properties of the different isoforms, there is a gradient from the MHC-slow to the MHC-2A, MHC-2B and MHC-2X/2B. According to the circumstances, the diaphragm will adapt towards a slow profile (COPD, cardiac failure and in animals: Duchenne muscular dystrophy, denervation-1 week, age-female, corticosteroids, chronic stimulation), or a fast profile (in animals: chronic hypoxia, denervation-2 weeks, age-males) or a more oxidative profile (in animals: cachexia, obesity). The reasons why the diaphragm adapts towards a slower or a faster muscle are not known. In fact, for a given pathological situation, several factors are able to influence the fiber composition of the diaphragm. Therefore, the net result of the influence of these different factors in terms of MHC and MLC diaphragm adaptation is difficult to predict.

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

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M.

    2010-01-01

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

  2. 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.

  3. 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.

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

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    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

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

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    Mari Sepp

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

  8. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    Science.gov (United States)

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

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

    Science.gov (United States)

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

    2015-03-15

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

  10. Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

    Science.gov (United States)

    Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

    2017-07-01

    The transformer-2 ( tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c, and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages ( P≤0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

  11. Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

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    Insa Bultmann-Mellin

    2015-04-01

    Full Text Available Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C, which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−, the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/− mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM. Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.

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

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    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.

  13. Evaluation of Hepcidin Isoforms in Hemodialysis Patients by a Proteomic Approach Based on SELDI-TOF MS

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    Natascia Campostrini

    2010-01-01

    Full Text Available The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versus 4.69 nM, and 4.06 versus 1.76 nM, resp., P<.05 for both. The clearance effects of a single dialysis session by different dialysis techniques and membranes were also investigated, showing an average reduction by 51.3% ± 29.2% for hepcidin-25 and 34.2% ± 28.4% for hepcidin-20 but only minor differences among the different dialysis modalities. Measurement of hepcidin isoforms through MS-based techniques can be a useful tool for better understanding of their biological role in hemodialysis patients and other clinical conditions.

  14. Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

    Science.gov (United States)

    Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

    2016-09-01

    The transformer-2 (tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages (P≤ 0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

  15. High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

    Science.gov (United States)

    Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V; Hurley, Thomas D

    2010-01-01

    In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV. PMID:20509168

  16. High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

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    Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V.; Hurley, Thomas D. (Indiana-Med); (Indiana)

    2010-09-07

    In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.

  17. High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones.

    Science.gov (United States)

    Perez-Miller, Samantha; Zou, Qin; Novotny, Milos V; Hurley, Thomas D

    2010-08-01

    In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP-IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP-IV is highly specific for the male mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X-ray crystal structure of MUP-IV bound to three mouse pheromones: SBT, 2,5-dimethylpyrazine, and 2-heptanone. We also obtained the structure of MUP-IV with 2-ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water-mediated hydrogen bond network in the MUP-I and MUP-II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP-IV.

  18. Molecular identity, expression and functional analysis of interleukin-lα and its isoforms in rat testis

    Institute of Scientific and Technical Information of China (English)

    T.Sultana; K.V.Svechnikov; K.Gustafsson; A.Wahlgren; E.Tham; G.Weber; O.Soder

    2004-01-01

    Interleukin-1α(IL-1α) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1α in testicular physiology. Bioactive IL-1α isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in athymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1α are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL- 1α is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis.Pathophysiologically, testicular IL-1α may contribute to testicular relapse of acute lymphocytic leukemia in boys.

  19. RNA-Seq analysis of the parietal cortex in Alzheimer's disease reveals alternatively spliced isoforms related to lipid metabolism.

    Science.gov (United States)

    Mills, James D; Nalpathamkalam, Thomas; Jacobs, Heidi I L; Janitz, Caroline; Merico, Daniele; Hu, Pingzhao; Janitz, Michael

    2013-03-01

    The parietal cortex of the human brain plays a unique role in the coordination of movement and in the integration of signals from the other cortices. Because of its extensive connections and involvement in many higher-order cognitive functions, neurodegenerative changes in the parietal lobe are believed to be crucial in the early symptoms of Alzheimer's disease (AD). Little is known about the transcriptome of this part of the human brain or how it is perturbed by the neurodegenerative process. To that end, we performed mRNA sequencing using the Illumina RNA-Seq technique on samples derived from normal and AD parietal lobes. Gene expression analysis evaluating alternatively spliced isoform expression and promoter usage revealed surprisingly elevated transcriptome activity in the AD condition. This phenomenon was particularly apparent in the alternative usage of transcriptional start sites. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways related to lipid metabolism, thus highlighting the importance of astrocyte activity in the neurodegenerative process. We also identified an upregulation of the diazepam-binding inhibitor (DBI) gene in AD, as the result of a splicing switch toward shorter, intron-retaining isoforms driven by alternative promoters and was coupled with a simultaneous decrease in the abundance of protein-coding transcripts. These two DBI isoforms have not been described previously.

  20. Identification and characterization of the two isoforms of the vertebrate H2A.Z histone variant.

    Science.gov (United States)

    Matsuda, Ryo; Hori, Tetsuya; Kitamura, Hiroshi; Takeuchi, Kozo; Fukagawa, Tatsuo; Harata, Masahiko

    2010-07-01

    Histone variants play important roles in the epigenetic regulation of genome function. The histone variant H2A.Z is evolutionarily conserved from yeast to vertebrates, and it has been reported to have multiple effects upon gene expression and insulation, and chromosome segregation. Recently two genes encoding H2A.Z were identified in the vertebrate genome. However, it is not yet clear whether the proteins transcribed from these genes are functionally distinct. To address this issue, we knocked out each gene individually in chicken DT40 cells. We found that two distinct proteins, H2A.Z-1 and H2A.Z-2, were produced from these genes, and that these proteins could be separated on a long SDS-PAGE gel. The two isoforms were deposited to a similar extent by the SRCAP chromatin-remodeling complex, suggesting redundancy to their function. However, cells lacking either one of the two isoforms exhibited distinct alterations in cell growth and gene expression, suggesting that the two isoforms have differential effects upon nucleosome stability and chromatin structure. These findings provide insight into the molecular basis of the multiple functions of the H2A.Z gene products.

  1. Molecular cloning and characterization of a novel isoform of the non-canonical poly(A) polymerase PAPD7

    Energy Technology Data Exchange (ETDEWEB)

    Ogami, Koichi; Cho, Rihe [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Hoshino, Shin-ichi, E-mail: hoshino@phar.nagoya-cu.ac.jp [Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)

    2013-03-01

    Highlights: ► So far, only an enzymatically inactive isoform of PAPD7 was reported. ► The novel isoform: PAPD7 l shows robust nucleotidyl transferase activity. ► The newly identified amino terminal region is required for the activity. ► PAPD7 l localizes to the nucleoplasm. ► The N terminal region identified is also required for the nuclear localization. - Abstract: Non-canonical poly(A) polymerases (ncPAPs) catalyze the addition of poly(A) tail to the 3′ end of RNA to play pivotal roles in the regulation of gene expression and also in quality control. Here we identified a novel isoform of the 7th member of ncPAPs: PAPD7 (PAPD7 l), which contains 230 extra amino acids at the amino terminus of the previously identified PAPD7 (PAPD7 s). In sharp contrast to the inactive PAPD7 s, PAPD7 l showed robust nucleotidyl transferase activity when tethered to an RNA. A region required for the activity was localized to 187–219 aa, and this region was also required for the nuclear retention of PAPD7 l. Western blot analysis revealed that 94 kDa band (corresponding to PAPD7 l) but not 62 kDa band (corresponding to PAPD7 s) detected by PAPD7 antibody was specifically depleted by treatment with PAPD7 siRNA in both HeLa and U2OS cells. These results suggest that PAPD7 l is the major and active isoform of PAPD7 expressed in cells.

  2. Novel intracellular N-terminal truncated matrix metalloproteinase-2 isoform in skeletal muscle ischemia-reperfusion injury.

    Science.gov (United States)

    Joshi, Sunil K; Lee, Lawrence; Lovett, David H; Kang, Heejae; Kim, Hubert T; Delgado, Cynthia; Liu, Xuhui

    2016-03-01

    Ischemia-reperfusion injury (IRI) occurs when blood returns to tissues following a period of ischemia. Reintroduction of blood flow results in the production of free radicals and reactive oxygen species that damage cells. Skeletal muscle IRI is commonly seen in orthopedic trauma patients. Experimental studies in other organ systems have elucidated the importance of extracellular and intracellular matrix metalloproteinase-2 (MMP-2) isoforms in regulating tissue damage in the setting of oxidant stress resulting from IRI. Although the extracellular full-length isoform of MMP-2 (FL-MMP-2) has been previously studied in the setting of skeletal muscle IRI, studies investigating the role of the N-terminal truncated isoform (NTT-MMP-2) in this setting are lacking. In this study, we first demonstrated significant increases in FL- and NTT-MMP-2 gene expression in C2C12 myoblast cells responding to re-oxygenation following hypoxia in vitro. We then evaluated the expression of FL- and NTT-MMP-2 in modulating skeletal muscle IRI using a previously validated murine model. NTT-MMP-2, but not FL-MMP-2 expression was significantly increased in skeletal muscle following IRI. Moreover, the expression of toll-like receptors (TLRs) -2 and -4, IL-6, OAS-1A, and CXCL1 was also significantly up-regulated following IRI. Treatment with the potent anti-oxidant pyrrolidine dithiocarbamate (PDTC) significantly suppressed NTT-MMP-2, but not FL-MMP-2 expression and improved muscle viability following IRI. This data suggests that NTT-MMP-2, but not FL-MMP-2, is the major isoform of MMP-2 involved in skeletal muscle IRI.

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

    Directory of Open Access Journals (Sweden)

    Hartmann-Fatu Cristina

    2006-03-01

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

  4. Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

    Science.gov (United States)

    Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

    2016-09-15

    Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context.

  5. Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity.

    Science.gov (United States)

    Vinciguerra, Manlio; Santini, Maria Paola; Claycomb, William C; Ladurner, Andreas G; Rosenthal, Nadia

    2009-12-10

    Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD(+)-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic.

  6. Expression and functional analysis of two osmotin (PR5) isoforms with differential antifungal activity from Piper colubrinum: prediction of structure-function relationship by bioinformatics approach.

    Science.gov (United States)

    Mani, Tomson; Sivakumar, K C; Manjula, S

    2012-11-01

    Osmotin, a pathogenesis-related antifungal protein, is relevant in induced plant immunity and belongs to the thaumatin-like group of proteins (TLPs). This article describes comparative structural and functional analysis of the two osmotin isoforms cloned from Phytophthora-resistant wild Piper colubrinum. The two isoforms differ mainly by an internal deletion of 50 amino acid residues which separates them into two size categories (16.4 kDa-PcOSM1 and 21.5 kDa-PcOSM2) with pI values 5.6 and 8.3, respectively. Recombinant proteins were expressed in E. coli and antifungal activity assays of the purified proteins demonstrated significant inhibitory activity of the larger osmotin isoform (PcOSM2) on Phytophthora capsici and Fusarium oxysporum, and a markedly reduced antifungal potential of the smaller isoform (PcOSM1). Homology modelling of the proteins indicated structural alterations in their three-dimensional architecture. Tertiary structure of PcOSM2 conformed to the known structure of osmotin, with domain I comprising of 12 β-sheets, an α-helical domain II and a domain III composed of 2 β-sheets. PcOSM1 (smaller isoform) exhibited a distorted, indistinguishable domain III and loss of 4 β-sheets in domain I. Interestingly, an interdomain acidic cleft between domains I and II, containing an optimally placed endoglucanase catalytic pair composed of Glu-Asp residues, which is characteristic of antifungal PR5 proteins, was present in both isoforms. It is well accepted that the presence of an acidic cleft correlates with antifungal activity due to the presence of endoglucanase catalytic property, and hence the present observation of significantly reduced antifungal capacity of PcOSM1 despite the presence of a strong acidic cleft, is suggestive of the possible roles played by other structural features like domain I or/and III, in deciding the antifungal potential of osmotin.

  7. Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function.

    Science.gov (United States)

    Pradhan, Amynah A; Perroy, Julie; Walwyn, Wendy M; Smith, Monique L; Vicente-Sanchez, Ana; Segura, Laura; Bana, Alia; Kieffer, Brigitte L; Evans, Christopher J

    2016-03-23

    Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor-Ca(2+)channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit

  8. Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania.

    Science.gov (United States)

    Rastogi, Ruchir; Verma, Jitender Kumar; Kapoor, Anjali; Langsley, Gordon; Mukhopadhyay, Amitabha

    2016-07-08

    Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

  9. Isoforms of the nuclear envelope protein Nurim are differentially expressed during heart development in mice.

    Science.gov (United States)

    Zhang, Wan; Bai, Tianyu; Zhang, Shuai; Xu, Shiqiang; Chen, Hengling; Li, Chenhong

    2017-09-05

    To date, transcript variants of the nuclear envelope protein Nurim and their expression profiles in mice have never been elucidated. In this study, we determined that the primary Nurim variant a was abundantly expressed in mouse heart, liver, spleen and kidney. The protein level of isoform a is initiated at an early stage of heart formation and demonstrated a significant increase in expression throughout embryonic heart development. Interestingly, Nurim b is also up-regulated from E12.5 to E18.5 in different individuals. Our research represents the first report on alternative splicing variants of mouse Nurim and their differential expression profile during embryonic development. These studies suggest a potential role for Nurim in early heart morphogenesis and should help further elucidate the function of Nurim. Copyright © 2017. Published by Elsevier B.V.

  10. 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.

  11. Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

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    Vassiliki Lila Koumandou

    Full Text Available Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA, the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4, which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%. Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD, in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms

  12. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  13. PSA Isoforms' Velocities for Early Diagnosis of Prostate Cancer.

    Science.gov (United States)

    Heidegger, Isabel; Klocker, Helmut; Pichler, Renate; Horninger, Wolfgang; Bektic, Jasmin

    2015-06-01

    Free prostate-specific antigen (fPSA) and its molecular isoforms are suggested for enhancement of PSA testing in prostate cancer (PCa). In the present study we evaluated whether PSA isoforms' velocities might serve as a tool to improve early PCa diagnosis. Our study population included 381 men who had undergone at least one ultrasound-guided prostate biopsy whose pathologic examination yielded PCa or showed no evidence of prostatic malignancy. Serial PSA, fPSA, and proPSA measurements were performed on serum samples covering 7 years prior to biopsy using Beckmann Coulter Access immunoassays. Afterwards, velocities of PSA (PSAV), fPSA% (fPSA%V), proPSA% (proPSA%V) and the ratio proPSA/PSA/V were calculated and their ability to discriminate cancer from benign disease was evaluated. Among 381 men included in the study, 202 (53%) were diagnosed with PCa and underwent radical prostatectomy at our Department. PSAV, fPSA%V, proPSA%V as well as proPSA/PSA/V were able to differentiate significantly between PCa and non-cancerous prostate. The highest discriminatory power between cancer and benign disease has been observed two and one year prior to diagnosis with all measured parameters. Among all measured parameters, fPSA%V showed the best cancer specificity of 45.3% with 90% of sensitivity. In summary, our results highlight the value of PSA isoforms' velocity for early detection of PCa. Especially fPSA%V should be used in the clinical setting to increase cancer detection specificity.

  14. Identification of an alternative splicing isoform of chicken Lmbr1.

    Science.gov (United States)

    Huang, Yanqun; Chen, Wen; Li, Ning; Deng, Xuemei; Kang, Xiangtao; Liu, Xiaojun

    2011-10-01

    Lmbr1 is the key candidate gene for limb development. Until now, at least five and four alternative splicing isoforms of Lmbr1 gene have been found in human and mouse, respectively. However, only two alternative splicing isoforms of this homologous gene have been reported in chicken. In the present study, one novel chicken Lmbr1 transcript variant (designated Lmbr1-1) was identified by 5' RACE and RT-PCR. Chicken Lmbr1-1 possesses one novel transcription start site different from Lmbr1-N, and was predicted to encode one 192 amino acid protein with length variation in comparison with chicken LMBR1-N protein, which was produced by 5' spliced site variation of chicken Lmbr1-N exon 10. Comparing with Lmbr1-N transcript, chicken Lmbr1-1 exhibited restricted tissue distribution of the expression. Comparative sequence analysis revealed a highly conservative intron element between chicken and mammalians from the intron 9 of chicken Lmbr1-N, indicating their possible importance as intronic elements in the regulation of alternative splicing of Lmbr1 in vertebrates. By direct PCR sequencing the exon 10 and its flanking sequences in chicken Lmbr1-N, four variation sites/haplotypes were identified from six chicken breeds. One 797A/G nonsynonymous mutation (266Arg/Gln) locating in exon 10 of chicken Lmbr1-N was predicted to affect the exon splice enhancer motif for serine/arginine-rich protein recognition. These data demonstrated that chicken Lmbr1 was alternatively spliced to generate multiple splice forms, as was the case in mammals and each of the alternative splicing isoforms might function differentially.

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

    Directory of Open Access Journals (Sweden)

    Marina Ilicic

    2017-01-01

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

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

    Science.gov (United States)

    2017-01-01

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

  17. PKC isoforms interact with and phosphorylate DNMT1

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    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  18. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

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    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  19. Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy.

    Science.gov (United States)

    Iwatsubo, Kousaku; Tsunematsu, Takashi; Ishikawa, Yoshihiro

    2003-06-01

    Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoform dependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.

  20. Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

    Science.gov (United States)

    Pedrosa, M M; Legaz, M E

    1995-04-01

    Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

  1. 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.

  2. 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.

  3. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

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    Halsey Christina

    2012-08-01

    Full Text Available Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.

  4. Nuclear progesterone receptor isoforms and their functions in the female reproductive tract.

    Science.gov (United States)

    Rekawiecki, R; Kowalik, M K; Kotwica, J

    2011-01-01

    Progesterone (P4), which is produced by the corpus luteum (CL), creates proper conditions for the embryo implantation, its development, and ensures proper conditions for the duration of pregnancy. Besides the non-genomic activity of P4 on target cells, its main physiological effect is caused through genomic action by the progesterone nuclear receptor (PGR). This nuclear progesterone receptor occurs in two specific isoforms, PGRA and PGRB. PGRA isoform acts as an inhibitor of transcriptional action of PGRB. The inactive receptor is connected with chaperone proteins and attachment of P4 causes disconnection of chaperones and unveiling of DNA binding domain (DBD). After receptor dimerization in the cells' nucleus and interaction with hormone response element (HRE), the receptor coactivators are connected and transcription is initiated. The ratio of these isoforms changes during the estrous cycle and reflects the different levels of P4 effect on the reproductive system. Both isoforms, PGRA and PGRB, also show a different response to the P4 receptor antagonist activity. Connection of the antagonist to PGRA can block PGRB, but acting through the PGRB isoform, P4 receptor antagonist may undergo conversion to a strongly receptor agonist. A third isoform, PGRC, has also been revealed. This isoform is the shortest and does not have transcriptional activity. Alternative splicing and insertion of additional exons may lead to the formation of different PGR isoforms. This paper summarizes the available data on the progesterone receptor isoforms and its regulatory action within the female reproductive system.

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

    Science.gov (United States)

    Schwab, Ryan S; Ihnatovych, Ivanna; Yunus, Sharifah Z S A; Domaradzki, Tera; Hofmann, Wilma A

    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.

  6. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    Science.gov (United States)

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  7. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

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

    2009-03-01

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

  8. BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

    Science.gov (United States)

    Foltran, Rocío Beatriz; Diaz, Silvina Laura

    2016-07-01

    The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors. © 2016 International Society for Neurochemistry.

  9. Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

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    Aris Haryanto

    2015-10-01

    Full Text Available Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC and typically requires a specific nuclear localization signal (NLS. In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc, phosphorylated recombinant HBV core (rHBc and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

  10. Dopamine-induced apoptosis of lactotropes is mediated by the short isoform of D2 receptor.

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    Daniela Betiana Radl

    Full Text Available Dopamine, through D2 receptor (D2R, is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L and short (D2S, are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850. SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process.

  11. Avian cytochrome P450 (CYP 1-3 family genes: isoforms, evolutionary relationships, and mRNA expression in chicken liver.

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    Kensuke P Watanabe

    Full Text Available Cytochrome P450 (CYP of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.

  12. AU-RICH ELEMENTS IN THE 3′-UTR REGULATE THE STABILITY OF THE 141 AMINO ACID ISOFORM OF PARATHYROID HORMONE-RELATED PROTEIN mRNA

    Science.gov (United States)

    Luchin, Alexander I.; Nadella, Murali V.P.; Thudi, Nanda K.; Dirksen, Wessel P.; Gulati, Parul; Fernandez, Soledad A.; Rosol, Thomas J.

    2012-01-01

    We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-β. In order to understand how PTHrP mRNA is stabilized by TGF-β, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3′-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3′-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA. PMID:22960231

  13. AU-rich elements in the 3'-UTR regulate the stability of the 141 amino acid isoform of parathyroid hormone-related protein mRNA.

    Science.gov (United States)

    Luchin, Alexander I; Nadella, Murali V P; Thudi, Nanda K; Dirksen, Wessel P; Gulati, Parul; Fernandez, Soledad A; Rosol, Thomas J

    2012-11-25

    We demonstrated previously that parathyroid hormone-related protein (PTHrP) 1-141 mRNA is the least stable of three isoforms and is the only isoform that is stabilized by TGF-β. In order to understand how PTHrP mRNA is stabilized by TGF-β, we first sought to elucidate the mechanism(s) that are responsible for the instability of PTHrP isoform 1-141 mRNA. The 3'-UTR of isoform 1-141 contains four AU-rich elements (AREs), which are known to mediate mRNA degradation. We utilized a luciferase reporter system to test whether these four AREs are responsible for the short half-life of PTHrP 1-141 mRNA. Our results demonstrated that ARE elements in the 3'-UTR of PTHrP 1-141 mRNA play a significant role in regulation of the stability of the mRNA. It is known that AREs mediate their effects on mRNA stability through a number of ARE-binding proteins that recruit the exosome, a complex of exonucleases that degrades the mRNA. We identified tristetraproline (TTP) as an RNA-binding protein that may be involved in ARE-mediated degradation of PTHrP 1-141 mRNA. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  14. Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis, towards different stress stimulations.

    Science.gov (United States)

    Leung, Priscilla T Y; Park, T J; Wang, Yu; Che, C M; Leung, Kenneth M Y

    2014-08-01

    Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green-lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non-metal oxidant (hydrogen peroxide; H2 O2 ) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10-I and 72 for MT10-II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two-dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10-I and MT10-II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10-I responded promptly to Cd but had a lagged induction to H2 O2 treatments, while MT10-II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H2 O2 , compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Loss of glycogen synthase kinase 3 isoforms during murine oocyte growth induces offspring cardiac dysfunction.

    Science.gov (United States)

    Monteiro da Rocha, André; Ding, Jun; Slawny, Nicole; Wolf, Amber M; Smith, Gary D

    2015-05-01

    Glycogen synthase kinase-3 (GSK3) is a constitutively active serine threonine kinase with 1) two isoforms (GSK3A and GSK3B) that have unique and overlapping functions, 2) multiple molecular intracellular mechanisms that involve phosphorylation of diverse substrates, and 3) implications in pathogenesis of many diseases. Insulin causes phosphorylation and inactivation of GSK3 and mammalian oocytes have a functional insulin-signaling pathway whereby prolonged elevated insulin during follicle/oocyte development causes GSK3 hyperphosphorylation, reduced GSK3 activity, and altered oocyte chromatin remodeling. Periconceptional diabetes and chronic hyperinsulinemia are associated with congenital malformations and onset of adult diseases of cardiovascular origin. Objectives were to produce transgenic mice with individual or concomitant loss of GSK3A and/or GSK3B and investigate the in vivo role of oocyte GSK3 on fertility, fetal development, and offspring health. Wild-type males bred to females with individual or concomitant loss of oocyte GSK3 isoforms did not have reduced fertility. However, concomitant loss of GSK3A and GSK3B in the oocyte significantly increased neonatal death rate due to congestive heart failure secondary to ventricular hyperplasia. Individual loss of oocyte GSK3A or GSK3B did not induce this lethal phenotype. In conclusion, absence of oocyte GSK3 in the periconceptional period does not alter fertility yet causes offspring cardiac hyperplasia, cardiovascular defects, and significant neonatal death. These results support a developmental mechanism by which periconceptional hyperinsulinemia associated with maternal metabolic syndrome, obesity, and/or diabetes can act on the oocyte and affect offspring cardiovascular development, function, and congenital heart malformation.

  16. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Directory of Open Access Journals (Sweden)

    Naveen K Jha

    Full Text Available A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950 internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800. Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  17. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

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

    KAUST Repository

    Moritz, Tom

    2016-02-05

    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 with an important role in castration-resistant stage. In the present work, we identified its impact in mechanisms leading to neuroendocrine (NE) transdifferentiation. We established for long-term PC-1 overexpression an inducible expression system derived from the prostate carcinoma cell line LNCaP. We observed that PC-1 overexpression itself initiates characteristics of neuroendocrine cells, but the effect was much more pronounced in the presence of the cytokine interleukin-6 (IL-6). Moreover, to our knowledge, this is the first report that treatment with IL-6 leads to a significant upregulation of PC-1 in LNCaP cells. Other TPD52 isoforms were not affected. Proceeding from this result, we conclude that PC-1 overexpression enhances the IL-6-mediated differentiation of LNCaP cells into a NE-like phenotype, noticeable by morphological changes and increased expression of typical NE markers, like chromogranin A, synaptophysin or beta-3 tubulin. Immunofluorescent staining of IL-6-treated PC-1-overexpressing LNCaP cells indicates a considerable PC-1 accumulation at the end of the long-branched neuron-like cell processes, which are typically formed by NE cells. Additionally, the experimentally initiated NE transdifferentiation correlates with the androgen receptor status, which was upregulated additively. In summary, our data provide evidence for an involvement of PC-1 in NE transdifferentiation, frequently associated with castration resistance, which is a major therapeutic challenge in the treatment of advanced prostate cancer.

  19. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

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

    Science.gov (United States)

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

    2014-12-01

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

  1. Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears.

    Science.gov (United States)

    Nelson, O Lynne; Robbins, Charles T; Wu, Yiming; Granzier, Henk

    2008-07-01

    The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly

  2. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides.

    Science.gov (United States)

    Silver, Kristopher S; Soderlund, David M

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel alpha subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na(v)1.2a, Na(v)1.4, Na(v)1.5, and Na(v)1.8 sodium channel alpha subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat beta1 auxiliary subunit on the sensitivity of the Na(v)1.2a and Na(v)1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel alpha subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na(v)1.4 > Na(v)1.2a > Na(v)1.5 > Na(v)1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na(v)1.8 isoform was most sensitive, the Na(v)1.4 isoform was completely insensitive, and the sensitivities of the Na(v)1.5 and Na(v)1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na(v)1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na(v)1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na(v)1.2a or Na(v)1.4 isoforms with the beta1 subunit was the modest reduction in the sensitivity of the Na(v)1.2a isoform to RH 3421. These results demonstrate that mammalian sodium channel isoforms differ in their sensitivities to pyrazoline-type insecticides.

  3. Nesprins: tissue-specific expression of epsilon and other short isoforms.

    Directory of Open Access Journals (Sweden)

    Nguyen Thuy Duong

    Full Text Available Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These "muscle-specific" isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and

  4. Regulation of Eukaryotic Initiation Factor 4E and Its Isoform: Implications for Antiviral Strategy in Plants

    Institute of Scientific and Technical Information of China (English)

    Yu-Yang Zhang; Han-Xia Li; Bo Ou-yang; Zhi-Biao Ye

    2006-01-01

    In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomicaiiy important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotic initiation factor 4E (elF4E). elF4E is one of the most important translation initiation factors involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by elF4E and its isoform elF (iso)4E occurs in several plant-virus interactions, thus indicating a potential new role for elF4E/elL(iso)4E in resistance strategies against plant viruses. In this review, we briefly describe elF4E activity in plant translation, its potential role, and functions of the elF4E subfamily in plant-virus interactions. Other initiation factors such as elF4G could also play a role in plant resistance against viruses. Finally, the potential for developing elF4E-medlated resistance to plant viruses in the future is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by elF4E. Knowledge of a particular plant-virus interaction will help to deepen our understanding of elF4E and other eukaryotic initiation factors, and their involvement in virus disease control.

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

    Science.gov (United States)

    Schjoldager, Katrine T-B G; Vakhrushev, Sergey Y; Kong, Yun; Steentoft, Catharina; Nudelman, Aaron S; Pedersen, Nis B; Wandall, Hans H; Mandel, Ulla; Bennett, Eric P; Levery, Steven B; Clausen, Henrik

    2012-06-19

    Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by zinc finger nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept 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 function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.

  6. Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms

    DEFF Research Database (Denmark)

    Schjaerff, Mette; Keller, Stefan M; Fass, Joseph;

    2016-01-01

    CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific a......CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform......-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two...... recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our...

  7. Isolation and characterization of multiple abundant lipid transfer protein isoforms in developing sesame (Sesamum indicum L.) seeds.

    Science.gov (United States)

    Choi, Ah Mi; Lee, Saet Buyl; Cho, Sung Ho; Hwang, Inhwan; Hur, Cheol-Goo; Suh, Mi Chung

    2008-02-01

    Sesame (Sesamum indicum) is an important oilseed crop; approximately 50% of the seed dry weight is storage oil. In a previous report, developing sesame seed expressed sequence tags (ESTs) revealed that ESTs encoding lipid transfer protein (LTPs) were one of the most abundant groups of sesame ESTs. LTP functions in the transfer of wax or cutin monomers and in the defense response against pathogen attack. To study the biological role of the abundant LTP isoforms in developing seeds, 122 ESTs out of 3328 sesame ESTs were analyzed against Arabidopsis and rice proteome databases. LTP fraction, which was partially purified from developing sesame seeds, actively transferred fluorescent phospholipids and bound to fatty acids. Full-length cDNAs of five out of 21 LTP isoforms were isolated and named SiLTP1-SiLTP5. The predicted amino acid sequences of the five SiLTPs harbor typical characteristics of LTPs, including conserved arrangement of cysteine residues. Northern blot analysis revealed that the five SiLTP isoforms were most abundantly expressed in developing seeds, but were also detected in flower tissues. Also, SiLTP3 and SiLTP4 transcripts were expressed in leaves and seed-pot walls, respectively. In addition, SiLTP2 and SiLTP4 transcripts were significantly induced in 6-day-old sesame seedlings by application of NaCl, mannitol, and abscisic acid (ABA). Transient expression of green fluorescent protein (GFP)-fusion constructs in Arabidopsis protoplasts revealed that SiLTP1 and SiLTP2 were secreted by different pathways. Taken together, the abundant LTPs in developing sesame seeds are involved in lipid transfer into the extracellular matrix. Possible biological roles of SiLTPs related to organ-specific expression and abiotic stresses are discussed.

  8. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gloria; Spring, Frances A.; Parons, Stephen F.; Mankelow, Tosti J.; Peters, Luanne L.; Koury, Mark J.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2003-02-18

    ICAM-4, a newly characterized adhesion molecule, is expressed early in human erythropoiesis and functions as a ligand for binding a4b1 and aV integrin-expressing cells. Within the bone marrow, erythroblasts surround central macrophages forming erythroblastic islands. Evidence suggests that these islands are highly specialized subcompartments where cell adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. Since erythroblasts express a4b1 and ICAM-4 and macrophages exhibit aV, ICAM-4 is an attractive candidate for mediating cellular interactions within erythroblastic islands. To determine whether ICAM-4 binding properties are conserved across species, we first cloned and sequenced the murine homologue. The translated amino acid sequence showed 68 percent overall identity with human ICAM-4. Using recombinant murine ICAM-4 extracellular domains, we discovered that hematopoietic a4b1-expressing HEL cells and non-hematopoietic aV-expressing FLY cells adhered to mouse ICAM-4. Cell adhesion studies showed that FLY and HEL cells bound to mouse and human proteins with similar avidity. These data strongly suggest conservation of integrin-binding properties across species. Importantly, we characterized a novel second splice cDNA that would be predicted to encode an ICAM-4 isoform, lacking the membrane-spanning domain. Erythroblasts express both isoforms of ICAM-4. COS-7 cells transfected with GFP constructs of prototypic or novel ICAM-4 cDNA showed different cellular localization patterns. Moreover, analysis of tissue culture medium revealed that the novel ICAM-4 cDNA encodes a secreted protein. We postulate that secretion of this newly described isoform, ICAM-4S, may modulate binding of membrane-associated ICAM-4 and could thus play a critical regulatory role in erythroblast molecular attachments.

  9. Insulin receptor isoforms : an integrated view focused on gestational diabetes mellitus

    NARCIS (Netherlands)

    Westermeier, F.; Sáez, T.; Arroyo, P.; Toledo, F.; Gutierrez, J.; Sanhueza, C.; Pardo, F.; Leiva, A.; Sobrevia, L.

    2016-01-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms r

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms...

  12. Mitochondrial localization of the OAS1 p46 isoform associated with a common single nucleotide polymorphism

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Pahus, Jytte; Hansen, Mariann Fagernæs

    2014-01-01

    vacuoles/lysosomes. By using recombinantly expressed OAS1 mutant proteins, we found that the OAS1 SNP rs1131454 (former rs3741981) did not affect the enzymatic OAS1 activity. Conclusions: The SNP rs10774671 determines differential expression of the OAS1 isoforms. In Daudi and HT1080 cells the p46 isoform...

  13. Translational control of C/EBPalpha and C/EBPbeta isoform expression

    NARCIS (Netherlands)

    Calkhoven, C F; Müller, C; Leutz, A

    2000-01-01

    Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms re

  14. Differences in expression, actions and cocaine regulation of two isoforms for the brain transcriptional regulator NAC1.

    Science.gov (United States)

    Korutla, L; Wang, P J; Lewis, D M; Neustadter, J H; Stromberg, M F; Mackler, S A

    2002-01-01

    BTB/POZ proteins can influence the cell cycle and contribute to oncogenesis. Many family members are present in the mammalian CNS. Previous work demonstrated elevated NAC1 mRNA levels in the rat nucleus accumbens in response to cocaine. NAC1 acts like other BTB/POZ proteins that regulate transcription but is unusual because of the absence of identifiable DNA binding domains. cDNAs were isolated encoding two NAC1 isoforms differing by only 27 amino acids (the longer isoform contains 514 amino acids). The mRNAs for both isoforms were simultaneously expressed throughout the rat brain and peripheral tissues. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that the mRNA of the longer isoform was more abundant than the mRNA of the shorter isoform. Western blot analysis demonstrated a similar unequal distribution between the isoforms in the CNS. The longer isoform was the more abundant of the two NAC1 proteins and the ratio between them differed throughout the rat brain. The shorter isoform was not detected in most of the examined peripheral tissues, suggesting differences from the CNS in post-transcriptional processing. Both isoforms repressed transcription in H293T cells using a Gal4-luciferase reporter system. However, the shorter isoform did not repress transcription as effectively as the longer isoform. Transfection of different ratios for both isoforms, in order to replicate the relative amounts observed throughout the CNS, supported an interaction between the isoforms. The net effect on transcriptional repression was determined by the ratio of the two NAC1 isoforms. Each isoform exhibited the subnuclear localization that is characteristic of many BTB/POZ proteins. A rapid and transient increase in the level of the shorter isoform occurred in the nucleus accumbens 2 h following a single i.p. cocaine injection. We conclude that the two isoforms of NAC1 may differentially affect neuronal functions, including the regulation of

  15. 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.

  16. The OsCYP19-4 Gene Is Expressed as Multiple Alternatively Spliced Transcripts Encoding Isoforms with Distinct Cellular Localizations and PPIase Activities under Cold Stress

    Directory of Open Access Journals (Sweden)

    Areum Lee

    2016-07-01

    Full Text Available Alternative splicing (AS is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1 transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H and bimolecular fluoresence complementation (BiFC assays, although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.

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

    Science.gov (United States)

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

    2000-03-17

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

  18. Isolation and structural characterization of different isoforms of the hypusine-containing protein eIF-5A from HeLa cells.

    Science.gov (United States)

    Klier, H; Csonga, R; Joäo, H C; Eckerskorn, C; Auer, M; Lottspeich, F; Eder, J

    1995-11-14

    Posttranslational modification of a specific lysine residue in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability and proliferation. The product of this modification is hypusine, an amino acid unique to eIF-5A. We have purified and characterized one major and three minor isoforms of human eIF-5A from HeLa cells. The main form, which accounts for approximately 95% of the total eIF-5A, carries hypusine at position 50 and is amino-terminally acetylated as determined by amino acid composition analysis and electrospray ionization mass spectrometry. Analytical gel filtration indicates that this protein variant possesses a native apparent molecular weight that lies between that expected for a monomeric and dimeric form. Nevertheless, several experiments confirm this protein to be monomeric. It is further shown that eIF-5A have well-defined secondary structure. Both the far-UV circular dichroism spectrum as well as secondary structure predictions using different algorithms suggest this protein to have predominantly beta-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to the main form, all three minor isoforms of eIF-5A are characterized by acetylation of the epsilon-amino group of lysine at position 47. The minor isoforms are distinguishable by their state of modification of the lysine residue at position 50. Whereas the main form occurs in both the cytoplasmic and the nuclear fraction of HeLa cells, the minor isoforms were not detectable in the preparation of the nuclear fraction. Therefore, acetylation of lysine at position 47 might play a controlling role in the distribution of the minor isoforms to the nucleus.

  19. Insulin receptor isoforms A and B as well as insulin receptor substrates-1 and -2 are differentially expressed in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Martin Heni

    Full Text Available AIMS/HYPOTHESIS: In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation. METHODS: We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27(Kip1 was quantified by real-time RT-PCR and immunohistochemistry. RESULTS: Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05. IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05. These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27(Kip1 content (p<0.02. Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019. CONCLUSIONS/INTERPRETATION: We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.

  20. Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons

    Directory of Open Access Journals (Sweden)

    Smith Paula L

    2011-09-01

    Full Text Available Abstract Background Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. Results Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. Conclusions Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

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

    Directory of Open Access Journals (Sweden)

    Rafia A. Baba

    2012-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  3. A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

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    Frédérique Végran

    Full Text Available Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

  4. Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53(null cell lines.

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    Elisabeth Silden

    Full Text Available The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1, Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(PH quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

  5. Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

    Science.gov (United States)

    Park, Ji Yeon; Li, Wencheng; Zheng, Dinghai; Zhai, Peiyong; Zhao, Yun; Matsuda, Takahisa; Vatner, Stephen F.; Sadoshima, Junichi; Tian, Bin

    2011-01-01

    Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload. PMID:21799842

  6. Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

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    Roshan Mascarenhas

    Full Text Available mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs, and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs in lymphoblast cell lines (LCL and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T in ABCB1 (MDR1 on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

  7. Mutational epitope analysis and cross-reactivity of two isoforms of Api g 1, the major celery allergen.

    Science.gov (United States)

    Wangorsch, Andrea; Ballmer-Weber, Barbara K; Rösch, Paul; Holzhauser, Thomas; Vieths, Stefan

    2007-04-01

    For better understanding the cross-reactivity between the major birch pollen and celery allergens, Bet v 1 and Api g 1, respectively, putative epitope areas and structurally important positions for IgE-binding of the isoforms Api g 1.01 and Api g 1.02 were point mutated. The IgE binding capacities were measured in ELISA, the IgE cross-reactivity between the isoforms, mutants and Bet v 1 investigated by ELISA-inhibition experiments with serum pools from patients with confirmed celery allergy (DBPCFC). Api g 1.01 displayed a clearly higher frequency and capacity of IgE binding than Api g 1.02. In Api g 1.01, substitution of lysine against glutamic acid at amino acid position 44, a key residue of the Bet v 1 "P-loop", increased the IgE-binding properties. Structural instability due to proline insertion at position 111/112 resulted in loss of IgE binding of Api g 1.01, but not of Api g 1.02. Between Api g 1.01 and Api g 1.02 only partial cross-reactivity was seen. The data suggest that the IgE epitopes of the two isoforms are distinct and that in contrast to Api g 1.01, the "P-loop" region plays an important role for IgE binding of celery allergic subjects to Api g 1.02. Understanding and investigation of the molecular mechanisms in celery allergy is an important step to generate hypoallergenic proteins for safe and efficacious immunotherapy of food allergy.

  8. 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

    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...... production of these isoforms in the canine inflamed joint....

  9. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation.

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    Ansgar Zoch

    Full Text Available The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

  10. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

  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

    2016-01-01

    ABSTRACT 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. PMID:27044325

  12. 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.

  13. Estrogen and progesterone receptor isoforms expression in the stomach of Mongolian gerbils

    Institute of Scientific and Technical Information of China (English)

    Milena Saqui-Salces; Teresa Neri-Gómez; Armando Gamboa-Dominguez; Guillermo Ruiz-Palacios; Ignacio Camacho-Arroyo

    2008-01-01

    AIM:We studied the estrogen receptor (ER) and progesterone receptor (PR) isoforms expression in gastric antrum and corpus of female gerbils and their regulation by estradiol (E2) and progesterone (P4).METHODS: Ovariectomized adult female gerbils were subcutaneously treated with E2,and E2 + P4.Uteri and stomachs were removed,the latter were cut along the greater curvature,and antrum and corpus were excised.Proteins were immunoblotted using antibodies that recognize ER-alpha,ER-beta,and PR-A and PR-B receptor isoforms.Tissues from rats treated in the same way were used as controls.RESULTS: Specific bands were detected for ER-alpha (68 KDa),and PR isoforms (85 and 120 KDa for PR-A and PR-B isoforrns,respectively) in uteri,gastric antrum and corpus.We could not detect ER-beta isoform.PR isoforms were not regulated by E2 or P4 in uterus and gastric tissues of gerbils.ER-alpha isoform content was significantly down-regulated by E2 in the corpus,but not affected by hormones in uterus and gastric antrum.CONCLUSION: The presence of ER-alpha and PR isoforms in gerbils stomach suggests that E2 and P4 actions in this organ are in part mediated by their nuclear receptors.

  14. Revisiting the identification of canonical splice isoforms through integration of functional genomics and proteomics evidence.

    Science.gov (United States)

    Li, Hong-Dong; Menon, Rajasree; Omenn, Gilbert S; Guan, Yuanfang

    2014-12-01

    Canonical isoforms in different databases have been defined as the most prevalent, most conserved, most expressed, longest, or the one with the clearest description of domains or posttranslational modifications. In this article, we revisit these definitions of canonical isoforms based on functional genomics and proteomics evidence, focusing on mouse data. We report a novel functional relationship network-based approach for identifying the highest connected isoforms (HCIs). We show that 46% of these HCIs are not the longest transcripts. In addition, this approach revealed many genes that have more than one highly connected isoforms. Averaged across 175 RNA-seq datasets covering diverse tissues and conditions, 65% of the HCIs show higher expression levels than nonhighest connected isoforms at the transcript level. At the protein level, these HCIs highly overlap with the expressed splice variants, based on proteomic data from eight different normal tissues. These results suggest that a more confident definition of canonical isoforms can be made through integration of multiple lines of evidence, including HCIs defined by biological processes and pathways, expression prevalence at the transcript level, and relative or absolute abundance at the protein level. This integrative proteogenomics approach can successfully identify principal isoforms that are responsible for the canonical functions of genes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    Science.gov (United States)

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

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

    Institute of Scientific and Technical Information of China (English)

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

    2001-01-01

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

  17. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    Science.gov (United States)

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. 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

    2014-03-15

    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.

  19. 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.

  20. Non-muscle Myosin II Isoforms Co-assemble in Living Cells

    Science.gov (United States)

    Beach, Jordan R.; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A.

    2014-01-01

    SUMMARY Non-muscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB and IIC), each of which possesses distinct biophysical properties and supports unique, as well as redundant, cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear if NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or if filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged versions of NM IIA, IIB and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms co-assemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly-spread cells, arguing for the existence of sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NM II isoforms. PMID:24814144

  1. 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

    2013-10-01

    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 (1)H 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.

  2. Non-raft adenylyl cyclase 2 defines a cAMP signaling compartment that selectively regulates IL-6 expression in airway smooth muscle cells: differential regulation of gene expression by AC isoforms.

    Science.gov (United States)

    Bogard, Amy S; Birg, Anna V; Ostrom, Rennolds S

    2014-04-01

    Adenylyl cyclase (AC) isoforms differ in their tissue distribution, cellular localization, regulation, and protein interactions. Most cell types express multiple AC isoforms. We hypothesized that cAMP produced by different AC isoforms regulates unique cellular responses in human bronchial smooth muscle cells (BSMC). Overexpression of AC2, AC3, or AC6 had distinct effects on forskolin (Fsk)-induced expression of a number of known cAMP-responsive genes. These data show that different AC isoforms can differentially regulate gene expression. Most notable, overexpression and activation of AC2 enhanced interleukin 6 (IL-6) expression, but overexpression of AC3 or AC6 had no effect. IL-6 production by BSMC was induced by Fsk and select G protein-coupled receptor (GPCR) agonists, though IL-6 levels did not directly correlate with global cAMP levels. Treatment with PKA selective 6-Bnz-cAMP or Epac selective 8-CPT-2Me-cAMP cAMP analogs revealed a predominant role for PKA in cAMP-mediated induction of IL-6. IL-6 promoter mutations demonstrated that AP-1 and CRE transcription sites were required for Fsk to stimulate IL-6 expression. Our present study defines an AC2 cAMP signaling compartment that specifically regulates IL-6 expression in BSMC via Epac and PKA and demonstrates that other AC isoforms are excluded from this pool.

  3. An isoform of Taiman that contains a PRD-repeat motif is indispensable for transducing the vitellogenic juvenile hormone signal in Locusta migratoria.

    Science.gov (United States)

    Wang, Zhiming; Yang, Libin; Song, Jiasheng; Kang, Le; Zhou, Shutang

    2017-03-01

    Taiman (Tai) has been recently identified as the dimerizing partner of juvenile hormone (JH) receptor, Methoprene-tolerant (Met). However, the role of Tai isoforms in transducing vitellogenic signal of JH has not been determined. In this study, we show that the migratory locust Locusta migratoria has two Tai isoforms, which differ in an INDEL-1 domain with the PRD-repeat motif rich in histidine and proline at the C-terminus. Tai-A with the INDEL-1 is expressed at levels about 50-fold higher than Tai-B without the INDEL-1 in the fat body of vitellogenic adult females. Knockdown of Tai-A but not Tai-B results in a substantial reduction of vitellogenin expression in the fat body accompanied by the arrest of ovarian development and oocyte maturation, similar to that caused by depletion of both Tai isoforms. Either Tai-A or Tai-B combined with Met can induce target gene transcription in response to JH, but Tai-A appears to mediate a significantly higher transactivation. Our data suggest that the INDEL-1 domain plays a critical role in Tai function during reproduction as Tai-A appears be more active than Tai-B in transducing the vitellogenic JH signal in L. migratoria.

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

    Directory of Open Access Journals (Sweden)

    John C Castle

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

  5. Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

    Science.gov (United States)

    Yamanaka, Hidenori; Takeyoshi, Masahiro; Minobe, Yasushi; Yakabe, Yoshikuni; Takatsuki, Mineo; Sato, Hisaya

    2007-12-01

    Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

  6. Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds1

    Science.gov (United States)

    Abrecht, Helge; Wattiez, Ruddy; Ruysschaert, Jean-Marie; Homblé, Fabrice

    2000-01-01

    Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates. PMID:11080295

  7. 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.

  8. Expression of transforming growth factor-beta (TGF-beta) isoforms in osteosarcomas: TGF-beta3 is related to disease progression.

    Science.gov (United States)

    Kloen, P; Gebhardt, M C; Perez-Atayde, A; Rosenberg, A E; Springfield, D S; Gold, L I; Mankin, H J

    1997-12-15

    Transforming growth factor-beta (TGF-beta) is a multipotent growth factor affecting development, homeostasis, and tissue repair. In addition, increased expression of TGF-beta has been reported in different malignancies, suggesting a role for this growth factor in tumorigenesis. Using immunohistochemistry, the expression, prevalence, and distribution of TGF-beta isoforms were evaluated in 25 high grade human osteosarcomas. The Cox proportional hazards models and Kaplan-Meier curves were calculated correlating disease free survival with TGF-beta expression. Expression of one or more TGF-beta isoforms was found in all the osteosarcomas. Immunoreactivity for TGF-beta1 and TGF-beta3 generally was stronger than for TGF-beta2. The cytoplasm of the tumor cells showed stronger staining than their surrounding extracellular stroma. Most notably, osteoclasts showed strong to intense staining for all three isoforms. In 11 of 25 specimens angiogenic activity was noted with staining of multiple small vessels in the tumor stroma. Expression of TGF-beta3, but not of TGF-beta2 or TGF-beta1, related to disease progression, such that there was a statistically significant decrease in the disease free interval as the immunoreactivity for TGF-beta3 increased. All osteosarcomas expressed TGF-beta in the cytoplasm of the tumor cells as well as in their extracellular stroma. The presence of TGF-beta in the endothelial and perivascular layers of small vessels in the tumor stroma suggests angiogenic activity of this growth factor. The expression of TGF-beta3 was correlated strongly with disease progression (P = 0.027). These data suggest that increased expression of TGF-beta isoforms, especially TGF-beta3, may play a role in osteosarcoma progression.

  9. Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa.

    Science.gov (United States)

    Jimenez, Tamara; Sánchez, Gladis; Wertheimer, Eva; Blanco, Gustavo

    2010-05-01

    While the function of the ubiquitous Na,K-ATPase alpha1 subunit has been well documented, the role of the sperm-specific alpha4 isoform of this ion transporter is less known. We have explored the importance of alpha4 in rat sperm physiology by taking advantage of the high sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively block alpha4 activity, we found ouabain to reduce not only sperm total motility, but also multiple parameters of sperm movement, including progressive motility, straight line, curvilinear, and average path velocities, lateral head displacement, beat cross frequency, and linearity. According to a direct role of alpha4 in Na(+) transport, ouabain inhibition of alpha4 increased [Na(+)](i) in the male gametes. In addition, interference of alpha4 activity with ouabain produced cell membrane depolarization, diminished pH, and increased [Ca(2)(+)](i) in spermatozoa. Inhibition of alpha4 was sufficient to cause all these effects and additional blockage of alpha1, the other Na,K-ATPase alpha isoform expressed in sperm, and higher doses of ouabain did not result in further changes in the cell parameters studied. These results show that alpha4 is the Na,K-ATPase isoform primarily involved in controlling the transmembrane Na(+) gradient in sperm, and that alpha4 activity is necessary for maintaining membrane potential, [Ca(2)(+)](i), and [H(+)](i) in the cells. The high dependence of sperm motility on membrane excitability, [Ca(2)(+)](i), and acid-base balance suggests that their regulation is the mechanism by which alpha4 maintains motility of the male gametes.

  10. Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

    Science.gov (United States)

    Schjoldager, Katrine T; Joshi, Hiren J; Kong, Yun; Goth, Christoffer K; King, Sarah Louise; Wandall, Hans H; Bennett, Eric P; Vakhrushev, Sergey Y; Clausen, Henrik

    2015-12-01

    GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid metabolism. The results demonstrate that GalNAc-Ts have different non-redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc-T substrates. Our study provides a new view of the differential regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.

  11. Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR in neuromuscular junction and cardiac intercalated discs

    Directory of Open Access Journals (Sweden)

    Karpati George

    2004-11-01

    Full Text Available Abstract Background The Coxsackie and adenovirus receptor (CAR has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown. Results Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart. Conclusion The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.

  12. Acquiring Metastatic Competence by Oral Squamous Cell Carcinoma Cells Is Associated with Differential Expression of α-Tubulin Isoforms

    Directory of Open Access Journals (Sweden)

    Becky Lou

    2012-01-01

    Full Text Available We performed comparative global proteomics analyses of patient-matched primary (686Tu and metastatic (686Ln OSCC cells. The metastatic OSCC 686Ln cells showed greater in vitro migratory/invasive potential and distinct cell shape from their parental primary 686Tu cells. Ettan DIGE analysis revealed 1316 proteins spots in both cell lines with >85% to be quantitatively similar (<2 folds between the two cell lines. However, two protein spots among four serial spots were highly dominant in 686Ln cells. Mass spectrometry sequencing demonstrated all four spots to be α-tubulin isotypes. Further analysis showed no significant quantitative difference in the α-tubulin between the two cell lines either at mRNA or protein levels. Thus, two distinct isoforms of α-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells. Immunofluorescence demonstrated remarkable differences in the cytosolic α-tubulin distribution patterns between the two cells. In 686Tu cells, α-tubulin proteins formed a normal network composed of filaments. In contrast, α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with the majority of the protein diffusely distributed within the cytosol. Since α-tubulin is critical for cell shape and mobility, our finding suggests a role of α-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

  13. Loss of predominant Shank3 isoforms results in hippocampus-dependent impairments in behavior and synaptic transmission.

    Science.gov (United States)

    Kouser, Mehreen; Speed, Haley E; Dewey, Colleen M; Reimers, Jeremy M; Widman, Allie J; Gupta, Natasha; Liu, Shunan; Jaramillo, Thomas C; Bangash, Muhammad; Xiao, Bo; Worley, Paul F; Powell, Craig M

    2013-11-20

    The Shank3 gene encodes a scaffolding protein that anchors multiple elements of the postsynaptic density at the synapse. Previous attempts to delete the Shank3 gene have not resulted in a complete loss of the predominant naturally occurring Shank3 isoforms. We have now characterized a homozygous Shank3 mutation in mice that deletes exon 21, including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major naturally occurring Shank3 protein bands detected by C-terminal and N-terminal antibodies, allowing us to more definitively examine the role of Shank3 in synaptic function and behavior. This loss of Shank3 leads to an increased localization of mGluR5 to both synaptosome and postsynaptic density-enriched fractions in the hippocampus. These mice exhibit a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of the hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. These data suggest that Shank3 isoforms are required for normal synaptic transmission/plasticity in the hippocampus, as well as hippocampus-dependent spatial learning and memory.

  14. Decreased expression of hippocampal Na⁺/Ca²⁺ exchanger isoform-1 by pentylenetetrazole kindling in mice.

    Science.gov (United States)

    Kawanai, Takuya; Taruta, Atsuki; Inoue, Aya; Watanabe, Ryo; Ago, Yukio; Hashimoto, Hitoshi; Hasebe, Shigeru; Ooi, Yasuhiro; Takuma, Kazuhiro; Matsuda, Toshio

    2015-09-01

    Previous studies have shown that inhibitors of the Na(+)/Ca(2+) exchanger (NCX) attenuate seizure activity in drug-induced epilepsy models, but the role of NCX in epilepsy is not fully understood. The present study examined the effects of pentylenetetrazole (PTZ)-induced kindling on the mRNA expression of NCX isoforms (NCX1, NCX2 and NCX3) in mouse brain. Chronic administration of PTZ at 40mg/kg resulted in kindling seizure development. It caused decreases in the mRNA levels of NCX1 and NCX2, but not NCX3, in the hippocampus. Changes in NCX isoform expression levels were not observed in the prefrontal cortex or striatum. Acute PTZ at 40mg/kg, which caused little seizure activity, also decreased NCX2, but not NCX1 mRNA levels in the hippocampus. These results suggest that down-regulation of hippocampal NCX1 expression is associated with PTZ-induced kindling seizure development.

  15. Translational readthrough generates new astrocyte AQP4 isoforms that modulate supramolecular clustering, glial endfeet localization, and water transport.

    Science.gov (United States)

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Rosito, Stefania; Simone, Laura; Buccoliero, Cinzia; Trojano, Maria; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2017-05-01

    Regulation of water homeostasis is a central feature of central nervous system pathophysiology. In this context, several lines of evidence suggest a crucial role for the water channel aquaporin-4 (AQP4) and its plasma membrane supramolecular organization as the key element. Here, we demonstrate the expression in tissues of additional isoforms of AQP4 characterized by a C-terminal extension generated by programmed translational readthrough. These extended isoforms (AQP4ex) display a perivascular polarization and expression in dystrophin-dependent pools. AQP4ex reduces supramolecular clustering tendency and allows AQP4 interactions with syntrophin. Furthermore, site-directed mutagenesis of two serines in the extended C-terminus of AQP4ex showed potential regulation of water permeability by phosphorylation. Finally, AQP4ex expression can be positively modulated by gentamicin treatment, demonstrating the possibility of regulating the AQP4 translational readthrough frequency. This novel regulatory mechanism could have important pathophysiological implications for conditions in which alternations have been reported in AQP4 structure. © 2017 Wiley Periodicals, Inc.

  16. A cytosolic Ezh1 isoform modulates a PRC2–Ezh1 epigenetic adaptive response in postmitotic cells

    KAUST Repository

    Bodega, Beatrice

    2017-03-27

    The evolution of chromatin-based epigenetic cell memory may be driven not only by the necessity for cells to stably maintain transcription programs, but also by the need to recognize signals and allow plastic responses to environmental stimuli. The mechanistic role of the epigenome in adult postmitotic tissues, however, remains largely unknown. In vertebrates, two variants of the Polycomb repressive complex (PRC2-Ezh2 and PRC2-Ezh1) control gene silencing via methylation of histone H3 on Lys27 (H3K27me). Here we describe a reversible mechanism that involves a novel isoform of Ezh1 (Ezh1β). Ezh1β lacks the catalytic SET domain and acts in the cytoplasm of skeletal muscle cells to control nuclear PRC2-Ezh1 activity in response to atrophic oxidative stress, by regulating Eed assembly with Suz12 and Ezh1α (the canonical isoform) at their target genes. We report a novel PRC2-Ezh1 function that utilizes Ezh1β as an adaptive stress sensor in the cytoplasm, thus allowing postmitotic cells to maintain tissue integrity in response to environmental changes.

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

    Science.gov (United States)

    Moriya, Shogo; Takiguchi, Masaki; Seki, Naohiko

    2008-02-01

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

  18. Heterogeneous nuclear ribonucleoprotein K represses the production of pro-apoptotic Bcl-xS splice isoform.

    Science.gov (United States)

    Revil, Timothée; Pelletier, Jordan; Toutant, Johanne; Cloutier, Alexandre; Chabot, Benoit

    2009-08-01

    The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x(L) and the pro-apoptotic Bcl-x(S) isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5' splice site of Bcl-x(S). The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein. Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x(S), an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x(S) splice isoform.

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

    Science.gov (United States)

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

    2014-01-22

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

  20. Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

    Science.gov (United States)

    Montero-Pardo, Arnulfo; Diaz, Daniel; Olivares, Aleida; González-Padilla, Everardo; Murcia, Clara; Gómez-Chavarín, Margarita; Gutiérrez-Ospina, Gabriel; Perera-Marín, Gerardo

    2015-12-01

    Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

  1. Detection of isoform-specific fibroblast growth factor receptors by whole-mount in situ hybridization in early chick embryos.

    Science.gov (United States)

    Nishita, Junko; Ohta, Sho; Bleyl, Steven B; Schoenwolf, Gary C

    2011-06-01

    We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms.

  2. BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer

    Directory of Open Access Journals (Sweden)

    Brian J. Kluk, Yebo Fu, Trina A. Formolo, Lei Zhang, Anne K. Hindle, Yan-gao Man, Robert S. Siegel, Patricia E. Berg, Chuxia Deng, Timothy A. McCaffrey, Sidney W. Fu

    2010-01-01

    Full Text Available Introduction: Several lines of evidence point to an important role for BP1, an isoform of DLX4 homeobox gene, in breast carcinogenesis and progression. BRCA1 is a well-known player in the etiology of breast cancer. While familial breast cancer is often marked by BRCA1 mutation and subsequent loss of heterozygosity, sporadic breast cancers exhibit reduced expression of wild type BRCA1, and loss of BRCA1 expression may result in tumor development and progression.Methods: The Cister algorithm and Genomatix program were used to identify potential BP1 binding sites in BRCA1 gene. Real-time PCR, Western blot and immunohistochemistry analysis were performed to verify the expression of BRCA1 and BP1 in cell lines and breast cancer tissues. Double-stranded siRNA transfection was carried out for silencing BP1 expression. ChIP and EMSA were used to confirm that BP1 specifically binds to BRCA1.Results: A putative BP1 binding site was identified in the first intron of BRCA1, which was confirmed by chromatin immunoprecipiation and electrophoresis mobility shift assay. BP1 and BRCA1 expression were inversely correlated in breast cancer cell lines and tissues, suggesting that BP1 may suppress BRCA1 transcription through consensus sequence binding.Conclusions: BP1 homeoprotein represses BRCA1 expression through direct binding to its first intron, which is consistent with a previous study which identified a novel transcriptional repressor element located more than 500 base pairs into the first intron of BRCA1, suggesting that the first intron plays an important role in the negative regulation of BRCA1. Although further functional studies are necessary to confirm its repressor activity towards BRCA1, the elucidation of the role of BP1 in breast tumorigenesis holds great promise in establishing BP1 as a novel target for drug therapy.

  3. Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking.

    Science.gov (United States)

    Sadler, Jessica B A; Bryant, Nia J; Gould, Gwyn W

    2015-02-01

    The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

  4. Titin Isoform Size is Not Correlated with Thin Filament Length in Rat Skeletal Muscle

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    Marion Lewis Greaser

    2014-02-01

    Full Text Available The mechanisms controlling thin filament length in muscle remain controversial. It was recently reported that thin filament length was related to titin size, and that the latter might be involved in thin filament length determination. Titin plays several crucial roles in the sarcomere, but its function as it pertains to the thin filament has not been explored. We tested this relationship using several muscles from wild type rats and from a mutant rat model which results in increased titin size. Myofibrils were isolated from skeletal muscles (extensor digitorum longus, external oblique, gastrocnemius, longissimus dorsi, psoas major, and tibialis anterior using both adult wild type (WT and homozygous mutant (HM rats. Phalloidin and antibodies against tropomodulin-4 and nebulin’s N-terminus were used to determine thin filament length. The WT rats studied express skeletal muscle titin sizes ranging from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.7 MDa. No differences in phalloidin-based thin filament length, nebulin N terminus distances from the Z line, or tropomodulin distances from the Z line were observed across genotypes. The data indicates that, although titin performs many sarcomeric functions, its correlation with thin filament length and structure could not be demonstrated in the rat. Current models of thin filament assembly are inadequate to explain the phalloidin, nebulin N terminus, and tropomodulin staining patterns in the myofibril.

  5. A Single Aplysia Neurotrophin Mediates Synaptic Facilitation via Differentially Processed Isoforms

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    Stefan R. Kassabov

    2013-04-01

    Full Text Available Neurotrophins control the development and adult plasticity of the vertebrate nervous system. Failure to identify invertebrate neurotrophin orthologs, however, has precluded studies in invertebrate models, limiting our understanding of fundamental aspects of neurotrophin biology and function. We identified a neurotrophin (ApNT and Trk receptor (ApTrk in the mollusk Aplysia and found that they play a central role in learning-related synaptic plasticity. Blocking ApTrk signaling impairs long-term facilitation, whereas augmenting ApNT expression enhances it and induces the growth of new synaptic varicosities at the monosynaptic connection between sensory and motor neurons of the gill-withdrawal reflex. Unlike vertebrate neurotrophins, ApNT has multiple coding exons and exerts distinct synaptic effects through differentially processed and secreted splice isoforms. Our findings demonstrate the existence of bona fide neurotrophin signaling in invertebrates and reveal a posttranscriptional mechanism that regulates neurotrophin processing and the release of proneurotrophins and mature neurotrophins that differentially modulate synaptic plasticity.

  6. Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

    Science.gov (United States)

    Michaud, Morgane; Ubrig, Elodie; Filleur, Sophie; Erhardt, Mathieu; Ephritikhine, Geneviève; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

    2014-06-17

    Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

  7. GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions.

    Science.gov (United States)

    Moeton, Martina; Stassen, Oscar M J A; Sluijs, Jacqueline A; van der Meer, Vincent W N; Kluivers, Liselot J; van Hoorn, Hedde; Schmidt, Thomas; Reits, Eric A J; van Strien, Miriam E; Hol, Elly M

    2016-11-01

    Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP-GFAPδ was significantly slower than the exchange of GFP-GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP-GFAPα and GFP-GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition.

  8. Identification and expression analysis of alternatively spliced new transcript isoform of Bax gene in mouse.

    Science.gov (United States)

    Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Sarwar, Tarique; Rehman, Sayeed Ur; Tabish, Mohammad

    2017-07-20

    Bax, a pro-apoptotic member of Bcl-2 family regulates apoptosis through homodimerization/heterodimerization with Bcl-2. Bax-α is the only product of the Bax gene that has been extensively studied. Bax-α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene in mouse which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax-α1), having different N-terminus. The existence of the new transcript variant was confirmed by reverse transcriptase-PCR, semi-nested PCR using primers designed for the newly identified transcript variant. The identity of PCR product obtained after semi-nested PCR was confirmed by DNA sequencing. Relative expression of new transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of new transcript variant was further supported by the presence of clusters of overlapping ESTs from the database. Bax-α1 possibly displays heterogeneous properties as predicted by post-translational modification analysis tools. The differences in post-translational modifications might play important roles in divergent function of the new isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-03-01

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

  10. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

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    Fabian V Filipp

    2013-01-01

    Full Text Available 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 activation-induced tumorigenesis. These biochemical activities are controlled by a shift in the oligomeric state of PKM2 that includes acetylation, oxidation, phosphorylation, prolyl hydroxylation and sumoylation. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals. Metabolically inactive PKM2 dimer is imported into the nucleus and can function as protein kinase stimulating transcription. A systems biology approach to PKM2 at the genome, transcriptome, proteome, metabolome and fluxome level reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism. Cancer systems biology takes us beyond the Warburg effect, opening unprecedented therapeutic opportunities.

  11. ROCK-Isoform-Specific Polarization of Macrophages Associated with Age-Related Macular Degeneration

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    Souska Zandi

    2015-02-01

    Full Text Available Age is a major risk factor in age-related macular degeneration (AMD, but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

  12. Functional characterization of two defensin isoforms of the hard tick Ixodes ricinus

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    Růžek Daniel

    2011-04-01

    Full Text Available Abstract Background The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule. Results Two isoforms of the defensin gene (def1 and def2 were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established. Conclusions Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

  13. Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

    Science.gov (United States)

    Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

    2009-05-01

    Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation.

  14. Assignment and expression patterns of porcine muscle-specific isoform of phosphoglycerate mutase gene

    Institute of Scientific and Technical Information of China (English)

    Haifang Qiu; Shuhong Zhao; Xuewen Xu; Martine Yerle; Bang Liu

    2008-01-01

    It has been reported that the muscle-specific isoform (type M, PGAM2) of phosphoglycerate mutase (PGAM) is a housekeeping en-zyme; it catalyzes the conversion of 3-phospboglycerate into 2-phosphoglycerate in the glyeolysis process to release energy. It is encoded by the Pgam2 gene. In this study, the eDNA of the porcine Pgam2 was cloned. This gene contains an open reading frame of 765 bp en-coding a protein of 253 residues, and the predicted protein sequences share high similarity with other mammalians, 96% identity with humans, and 94% identity with mouse and rats. Pgam2 was mapped to SSC18q13-q21 by the RH panel. In this region, there are several QTLs, such as fat ratio, lean percentage, and diameter of muslce fiber, which affect meat production and quality. The reverse transcrip-tase-polymerase chain reaction revealed that the porcine Pgam2 gene was mainly expressed in the muscle tissue (skeletal muscle and cardiac muscle), and was expressed highly at skeletal muscle development stages (embryonic periods: 33, 65, and 90 days post-conception(dpe); postnatal pigs: 4 days and adult). This indicates that the Pgam2 gene plays an important role in muscle growth and development. In addition, it was demonstrated that PGAM2 locates both in cytoplasm and nuclei, and takes part in the glycometabolism process of cyto-plasm and nuclei.

  15. Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Steven L. K. Ching; Satinder K. Gidda; Amanda Rochon; Owen R. van Cauwenberghe; Barry J. Shelp; Robert T. Mullen

    2012-01-01

    Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye,an intermediate of the γ-aminobutyrate (GABA) pathway.Two isoforms of GLYR exist in plants,GLYR1 and GLYR2,and while GLYR2 is known to be localized in plastids,GLYR1 has been reported to be localized in either peroxisomes or the cytosol.Here,we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L.Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants,in combination with fluorescence microscopy.The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species,tissue and/or cell type,or exposure of plants to environmental stresses that would increase flux through the GABA pathway.Moreover,the C-terminal tripeptide sequence of GLYR1,-SRE,despite its resemblance to a type 1 peroxisomal targeting signal,is not sufficient for targeting to peroxisomes.Collectively,these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells,but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

  16. [Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Arabtseva, M A; Lavrinenko, I A; Parfenova, I V; Grechkina, M V; Abud, F S

    2011-01-01

    Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

  17. Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration

    Science.gov (United States)

    Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S.

    2016-11-01

    Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

  18. Neuronal and intestinal protein kinase d isoforms mediate Na+ (salt taste)-induced learning.

    Science.gov (United States)

    Fu, Ya; Ren, Min; Feng, Hui; Chen, Lu; Altun, Zeynep F; Rubin, Charles S

    2009-08-11

    Ubiquitously expressed protein kinase D (PKD) isoforms are poised to disseminate signals carried by diacylglycerol (DAG). However, the in vivo regulation and functions of PKDs are poorly understood. We show that the Caenorhabditis elegans gene, dkf-2, encodes not just DKF-2A, but also a second previously unknown isoform, DKF-2B. Whereas DKF-2A is present mainly in intestine, we show that DKF-2B is found in neurons. Characterization of dkf-2 null mutants and transgenic animals expressing DKF-2B, DKF-2A, or both isoforms revealed that PKDs couple DAG signals to regulation of sodium ion (Na+)-induced learning. EGL-8 (a phospholipase Cbeta4 homolog) and TPA-1 (a protein kinase Cdelta homolog) are upstream regulators of DKF-2 isoforms in vivo. Thus, pathways containing EGL-8-TPA-1-DKF-2 enable learning and behavioral plasticity by receiving, transmitting, and cooperatively integrating environmental signals targeted to both neurons and intestine.

  19. Glial fibrillary acidic protein isoform expression in plaque related astrogliosis in Alzheimer's disease

    NARCIS (Netherlands)

    Kamphuis, W.; Middeldorp, Jinte; Kooijman, Lieneke; Sluijs, Jacqueline A; Kooi, Evert-Jan; Moeton, Martina; Freriks, Michel; Mizee, Mark R; Hol, Elly M

    2014-01-01

    In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of ast

  20. Receptor-isoform-selective insulin analogues give tissue-preferential effects

    DEFF Research Database (Denmark)

    Vienberg, Sara Gry; Bouman, Stephan D; Sørensen, Heidi

    2011-01-01

    The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can...... be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI...... (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI...

  1. 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.

  2. Evolution of a TRIM5-CypA splice isoform in old world monkeys.

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    Ruchi M Newman

    2008-02-01

    Full Text Available The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA. We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101 of rhesus macaques (M. mulatta. The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a

  3. Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression.

    Science.gov (United States)

    Wagener, F A; da Silva, J L; Farley, T; de Witte, T; Kappas, A; Abraham, N G

    1999-10-01

    Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme

  4. Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2005-06-01

    Full Text Available Abstract Background Interferon (IFN-α is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs. This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS stimulation in vitro. Results Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-β, IFN-α2b/α2, IFN-αI, IFN-α2, IFN-αC, IFN-αJ1, IFN-αH2, and INF-α4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-γ. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs. Conclusion Seven IFN-α isoforms and IFN-β participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-α in

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

    Science.gov (United States)

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

    2017-01-01

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

  6. P120-catenin isoforms 1 and 3 regulate proliferation and cell cycle of lung cancer cells via β-catenin and Kaiso respectively.

    Directory of Open Access Journals (Sweden)

    Guiyang Jiang

    Full Text Available BACKGROUND: The different mechanisms involved in p120-catenin (p120ctn isoforms' 1/3 regulation of cell cycle progression are still not elucidated to date. METHODS AND FINDINGS: We found that both cyclin D1 and cyclin E could be effectively restored by restitution of p120ctn-1A or p120ctn-3A in p120ctn depleted lung cancer cells. When the expression of cyclin D1 was blocked by co-transfection with siRNA-cyclin D1 in p120ctn depleted cells restoring p120ctn-1A or 3A, the expression of cyclin E was slightly decreased, not increased, implying that p120ctn isoforms 1 and 3 cannot up-regulate cyclin E directly but may do so through up-regulation of cyclin D1. Interestingly, overexpression of p120ctn-1A increased β-catenin and cyclin D1 expression, while co-transfection with siRNA targeting β-catenin abolishes the effect of p120ctn-1A on up-regulation of cyclin D1, suggesting a role of β-catenin in mediating p120ctn-1A's regulatory function on cyclin D1 expression. On the other hand, overexpression of p120ctn isoform 3A reduced nuclear Kaiso localization, thus decreasing the binding of Kaiso to KBS on the cyclin D1 promoter and thereby enhancing the expression of cyclin D1 gene by relieving the repressor effect of Kaiso. Because overexpressing NLS-p120ctn-3A (p120ctn-3A nuclear target localization plasmids or inhibiting nuclear export of p120ctn-3 by Leptomycin B (LMB caused translocation of Kaiso to the nucleus, it is plausible that the nuclear export of Kaiso is p120ctn-3-dependent. CONCLUSIONS: Our results suggest that p120ctn isoforms 1 and 3 up-regulate cyclin D1, and thereby cyclin E, resulting in the promotion of cell proliferation and cell cycle progression in lung cancer cells probably via different protein mediators, namely, β-catenin for isoform 1 and Kaiso, a negative transcriptional factor of cyclin D1, for isoform 3.

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

    OpenAIRE

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

    2010-01-01

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

  8. 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.

  9. Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells.

    Science.gov (United States)

    Cappelletti, Vera; Miodini, Patrizia; Di Fronzo, Giovanni; Daidone, Maria Grazia

    2006-05-01

    High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

  10. 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.

  11. 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....

  12. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  14. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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    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.

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

    Science.gov (United States)

    2009-02-01

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

  16. The impact of tropomyosins on actin filament assembly is isoform specific.

    Science.gov (United States)

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  17. Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae.

    Science.gov (United States)

    Gorman, Maureen J; Sullivan, L