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Sample records for abnormal isoform prpsc

  1. Tetracycline affects abnormal properties of synthetic PrP peptides and PrP(Sc) in vitro.

    PubMed

    Tagliavini, F; Forloni, G; Colombo, L; Rossi, G; Girola, L; Canciani, B; Angeretti, N; Giampaolo, L; Peressini, E; Awan, T; De Gioia, L; Ragg, E; Bugiani, O; Salmona, M

    2000-07-28

    Prion diseases are characterized by the accumulation of altered forms of the prion protein (termed PrP(Sc)) in the brain. Unlike the normal protein, PrP(Sc) isoforms have a high content of beta-sheet secondary structure, are protease-resistant, and form insoluble aggregates and amyloid fibrils. Evidence indicates that they are responsible for neuropathological changes (i.e. nerve cell degeneration and glial cell activation) and transmissibility of the disease process. Here, we show that the antibiotic tetracycline: (i) binds to amyloid fibrils generated by synthetic peptides corresponding to residues 106-126 and 82-146 of human PrP; (ii) hinders assembly of these peptides into amyloid fibrils; (iii) reverts the protease resistance of PrP peptide aggregates and PrP(Sc) extracted from brain tissue of patients with Creutzfeldt-Jakob disease; (iv) prevents neuronal death and astrocyte proliferation induced by PrP peptides in vitro. NMR spectroscopy revealed several through-space interactions between aromatic protons of tetracycline and side-chain protons of Ala(117-119), Val(121-122) and Leu(125) of PrP 106-126. These properties make tetracycline a prototype of compounds with the potential of inactivating the pathogenic forms of PrP. PMID:10903871

  2. Detection of PrPSc in Formalin Fixed Paraffin Embedded Tissue by Western Blot Differentiates Classical Scrapie, Nor98 Scrapie, and BSE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible spongiform encephalopathies including bovine spongiform encephalopathy and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions (PrP**Sc). Identification of PrP**Sc in the CNS is typicall...

  3. Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

    PubMed

    Tanaka, Misaki; Fujiwara, Ai; Suzuki, Akio; Yamasaki, Takeshi; Hasebe, Rie; Masujin, Kentaro; Horiuchi, Motohiro

    2016-08-01

    We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation. PMID:27267758

  4. Accumulation of PrP-Sc in hemal nodes of naturally and experimentally scrapie-infected sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical scrapie is a naturally occurring fatal disease of sheep and goats which is caused by prions, a novel class of infectious agent. Infection is accompanied by accumulation of abnormal isoforms of the prion protein (PrP-Sc) in certain neural and lymphoid tissues. Hemal nodes, which are unique ...

  5. PK-sensitive PrPSc is infectious and shares basic structural features with PK-resistant PrPSc

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One of the main characteristics of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrPSc following Western blot or ELISA. More recently, researchers determ...

  6. Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity.

    PubMed

    Lewis, Patrick A; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R; Collinge, John; Jackson, Graham S

    2006-04-15

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity. PMID:16441239

  7. Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

    PubMed Central

    Lewis, Patrick A.; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R.; Collinge, John; Jackson, Graham S.

    2006-01-01

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrPC. Removal of the GPI anchor from PrPSc requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrPSc in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrPSc, (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrPSc or on prion infectivity. PMID:16441239

  8. Infectivity-associated PrPSc and disease duration-associated PrPSc of mouse BSE prions

    PubMed Central

    Miyazawa, Kohtaro; Okada, Hiroyuki; Masujin, Kentaro; Iwamaru, Yoshifumi; Yokoyama, Takashi

    2015-01-01

    ABSTRACT Disease-related prion protein (PrPSc), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrPSc in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrPSc in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrPSc was precipitated into the pellet, and the supernatant contained only a slight amount of PrPSc. Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrPSc may be responsible for prion infectivity and that large, aggregated PrPSc may contribute to determining prion disease duration. PMID:26555211

  9. Small molecules and antibodies: a means of distinguishing between PrPC and PrPSc

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PrPSc and PrPC are isoforms, since they possess identical covalent structures and identical post-translational modifications. The same amino acid may react differently with the same chemical reagent in an isoform-dependent manner. The site of covalent modification can be identified by mass spectrom...

  10. Distinguishing between PrPC and PrPSc using small molecule reagents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. The structural difference between PrPSc and PrPC is entirely conformational: they are isoforms. Both isoforms possess identical covalent structures and identical post-translational modifications. This means that the same amino acid can react differently with the same chemic...

  11. Distinguishing between PrPC and PrPSc using small molecule reagents(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. The structural difference between PrPSc and PrPC is entirely conformational: they are isoforms. Both isoforms possess identical covalent structures and identical post-translational modifications. This means that the same amino acid can react differently with the same chemica...

  12. L-Arginine ethylester enhances in vitro amplification of PrP(Sc) in macaques with atypical L-type bovine spongiform encephalopathy and enables presymptomatic detection of PrP(Sc) in the bodily fluids.

    PubMed

    Murayama, Y; Ono, F; Shimozaki, N; Shibata, H

    2016-02-12

    Protease-resistant, misfolded isoforms (PrP(Sc)) of a normal cellular prion protein (PrP(C)) in the bodily fluids, including blood, urine, and saliva, are expected to be useful diagnostic markers of prion diseases, and nonhuman primate models are suited for performing valid diagnostic tests for human Creutzfeldt-Jakob disease (CJD). We developed an effective amplification method for PrP(Sc) derived from macaques infected with the atypical L-type bovine spongiform encephalopathy (L-BSE) prion by using mouse brain homogenate as a substrate in the presence of polyanions and L-arginine ethylester. This method was highly sensitive and detected PrP(Sc) in infected brain homogenate diluted up to 10(10) by sequential amplification. This method in combination with PrP(Sc) precipitation by sodium phosphotungstic acid is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF), saliva, urine, and plasma of macaques that have been intracerebrally inoculated with the L-BSE prion. Furthermore, PrP(Sc) was detectable in the saliva or urine samples as well as CSF samples obtained at the preclinical phases of the disease. Thus, our novel method may be useful for furthering the understanding of bodily fluid leakage of PrP(Sc) in nonhuman primate models. PMID:26802462

  13. Detection of PrP(Sc) in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease that affects cattle and can be transmitted to human beings as new variant Creutzfeldt-Jakob disease (vCJD). A protease-resistant, disease-associated isoform of the prion protein (PrP**Sc) accumulates in the central ner...

  14. Phosphorothioate Oligonucleotides Reduce PrPSc Levels and Prion Infectivity in Cultured Cells

    PubMed Central

    Karpuj, Marcela V; Giles, Kurt; Gelibter-Niv, Sagit; Scott, Michael R; Lingappa, Vishwanath R; Szoka, Francis C; Peretz, David; Denetclaw, Wilfred; Prusiner, Stanley B

    2007-01-01

    Prions are composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process. Here we report that short phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels was independent of the nucleotide sequence. The effective concentration (EC50) of PS-DNA required to achieve half-maximal diminution of PrPSc was ~70 nM, whereas the EC50 of PS-DNA for PrPC was more than 50-fold greater. This finding indicated that diminished levels of PrPSc after exposure to PS-DNA are unlikely to be due to decreased PrPC levels. Bioassays in transgenic mice demonstrated a substantial diminution in the prion infectivity after ScN2a cells were exposed to PS-DNAs. Whether PS-DNA will be useful in the treatment of prion disease in people or livestock remains to be established. PMID:17592554

  15. Role of cell death in the propagation of PrP(Sc) in immune cells.

    PubMed

    Takahashi, Kenichi; Inoshima, Yasuo; Ishiguro, Naotaka

    2015-03-01

    A number of studies have suggested that macrophages, dendritic cells, and follicular dendritic cells play an important role in the propagation of PrP(Sc). Both accumulation and proteolysis of PrP(Sc) have been demonstrated in peripheral macrophages. Macrophages may act as reservoirs for PrP(Sc) particles if the cells die during transient PrP(Sc) propagation. However, whether cell death plays a role in PrP(Sc) propagation in macrophages remains unclear. In this study, we investigated the possibility of propagation and transmission of PrP(Sc) between dead immune cells and living neural cells. We found that under specific conditions, transient PrP(Sc) propagation occurs in dead cells, indicating that interaction between PrP(C) and PrP(Sc) on plasma membrane lipid rafts might be important for PrP(Sc) propagation. Co-culturing of killed donor PrP(Sc)-infected macrophages with recipient N2a-3 neuroblastoma cells accelerated PrP(Sc) transmission. Our results suggest that cell death may play an important role in PrP(Sc) propagation, whereas transient PrP(Sc) propagation in macrophages has little effect on PrP(Sc) transmission. PMID:25559669

  16. N(ε)-Carboxymethyl Modification of Lysine Residues in Pathogenic Prion Isoforms.

    PubMed

    Choi, Yeong-Gon; Shin, Hae-Young; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

    2016-07-01

    The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP(Sc), at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP(Sc)) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N(ε)-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP(Sc). We show that CML is linked to at least one Lys residue at the N-terminus of PrP(Sc) in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP(Sc). The nonenzymatic glycation of the Lys residue(s) of PrP(Sc) with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP(C) but is seen in the pathologic PrP(Sc) isoform. Furthermore, the modification of PrP(Sc) with CML may be closely involved in prion propagation and deposition in pathological brain areas. PMID:25983034

  17. Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms.

    PubMed Central

    Barbas, J A; Galceran, J; Torroja, L; Prado, A; Ferrús, A

    1993-01-01

    The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor. Images PMID:7680094

  18. Complement factors alter the amount of PrP(Sc) in primary-cultured mouse cortical neurons associated with increased membrane permeability.

    PubMed

    Hasebe, Rie; Tanaka, Misaki; Suzuki, Akio; Yamasaki, Takeshi; Horiuchi, Motohiro

    2016-09-01

    We examined the effects of complement factors on primary-cultured neurons infected with prions. The amount of protease K (PK)-resistant abnormal form of prion protein (PrP(Sc)) reached a maximum level at 12 and 16 days post exposure (dpe) in 22L- and Chandler-infected neurons, respectively. In Chandler-infected neurons, the reaction of complement factors C1q, C3 and C9 significantly increased membrane permeability. This was followed by a decrease of PK-resistant PrP(Sc) at 16 and 20dpe. In contrast, in 22L-infected neurons, the effects of complement factors were observed at 12 and 16dpe, but not at 20dpe. Membrane permeability also increased in 22L-infected neurons by reaction of complement factor C3, but interestingly, the amount of PK-resistant PrP(Sc) initially decreased, and then increased. These results suggest that the reactivity of complement factors in prion-infected neurons depends on the amount of PrP(Sc) and the prion strain. PMID:27236741

  19. Comparison of trichloroacetic acid with other protein-precipitating agents in enriching abnormal prion protein for Western blot analysis.

    PubMed

    LeBrun, Matthew; Huang, Hongsheng; He, Runtao; Booth, Stephanie; Balachandran, Aru; Li, Xuguang

    2008-06-01

    Detection of the abnormal or the pathogenic form of prion protein (PrP(Sc)) by Western blot (WB) is challenging, especially, for samples derived from cell cultures that contain low levels of PrP(Sc). A variety of PrP(Sc) concentration methods have been reported with various PrP(Sc) recovery efficiencies. Ultracentrifugation is one of the methods used frequently to enrich the pathogenic form of PrP(Sc) prior to WB analyses. The resulting PrP(Sc) pellet is extremely insoluble and often requires sonication to be dissolved, potentially generating aerosols. We modified the common protein-precipitating protocol using trichloroacetic acid to concentrate PrP(Sc) by slow-speed centrifugation, followed by solubilization of the pellets with 6 mol/L urea prior to sodium dodecyl sulphate -- polyacrylamide gel electrophoresis and WB analyses. Comparative studies suggest this simple trichloroacetic acid protocol was more effective in enriching PrP(Sc) presented in cell cultures and brain homogenates than other reported protein-precipitating methods. Furthermore, incorporation of the urea treatment step to dissolve the precipitated PrP(Sc) pellets helped to reduce the infectivity of PrP(Sc). PMID:18535632

  20. COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform

    PubMed Central

    Kaneko, Kiyotoshi; Vey, Martin; Scott, Michael; Pilkuhn, Susanne; Cohen, Fred E.; Prusiner, Stanley B.

    1997-01-01

    Efficient formation of scrapie isoform of prion protein (PrPSc) requires targeting PrPSc by glycophosphatidyl inositol (GPI) anchors to caveolae-like domains (CLDs). Redirecting the cellular isoform of prion protein (PrPC) to clathrin-coated pits by creating chimeric PrP molecules with four different COOH-terminal transmembrane domains prevented the formation of PrPSc. To determine if these COOH-terminal transmembrane segments prevented PrPC from refolding into PrPSc by altering the structure of the polypeptide, we fused the 28-aa COOH termini from the Qa protein. Two COOH-terminal Qa segments differing by a single residue direct the transmembrane protein to clathrin-coated pits or the GPI form to CLDs; PrPSc was formed from GPI-anchored PrPC but not from transmembrane PrPC. Our findings argue that PrPSc formation is restricted to a specific subcellular compartment and as such, it is likely to involve auxiliary macromolecules found within CLDs. PMID:9122195

  1. Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks.

    PubMed

    Caplazi, Patrick; O'Rourke, Katherine; Wolf, Cynthia; Shaw, Daniel; Baszler, Timothy V

    2004-11-01

    Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171

  2. Targeted mutations in the Na,K-ATPase α 2 isoform confer ouabain resistance and result in abnormal behavior in mice.

    PubMed

    Schaefer, Tori L; Lingrel, Jerry B; Moseley, Amy E; Vorhees, Charles V; Williams, Michael T

    2011-06-01

    Sodium and potassium-activated adenosine triphosphatases (Na,K-ATPase) are ubiquitous, participate in osmotic balance and membrane potential, and are composed of α, β, and γ subunits. The α subunit is required for the catalytic and transport properties of the enzyme and contains binding sites for cations, ATP, and digitalis-like compounds including ouabain. There are four known α isoforms; three that are expressed in the CNS in a regional and cell-specific manner. The α2 isoform is most commonly found in astrocytes, pyramidal cells of the hippocampus in adults, and developmentally in several other neuronal types. Ouabain-like compounds are thought to be produced endogenously in mammals, bind the Na,K-ATPase, and function as a stress-related hormone, however, the impact of the Na,K-ATPase ouabain binding site on neurobehavioral function is largely unknown. To determine if the ouabain binding site of the α2 isoform plays a physiological role in CNS function, we examined knock-in mice in which the normally ouabain-sensitive α2 isoform was made resistant (α2(R/R) ) while still retaining basal Na,K-ATPase enzymatic function. Egocentric learning (Cincinnati water maze) was impaired in adult α2(R/R) mice compared to wild type (WT) mice. They also exhibited decreased locomotor activity in a novel environment and increased responsiveness to a challenge with an indirect sympathomimetic agonist (methamphetamine) relative to WT mice. The α2(R/R) mice also demonstrated a blunted acoustic startle reflex and a failure to habituate to repeated acoustic stimuli. The α2(R/R) mice showed no evidence of altered anxiety (elevated zero maze) nor were they impaired in spatial learning or memory in the Morris water maze and neither group could learn in a large Morris maze. These results suggest that the ouabain binding site is involved in specific types of learning and the modulation of dopamine-mediated locomotor behavior. PMID:20936682

  3. Targeted Mutations in the Na,K-ATPase Alpha 2 Isoform Confer Ouabain Resistance and Result in Abnormal Behavior in Mice

    PubMed Central

    Schaefer, Tori L.; Lingrel, Jerry B; Moseley, Amy E.; Vorhees, Charles V.; Williams, Michael T.

    2011-01-01

    Sodium and potassium-activated adenosine triphosphatases (Na,K-ATPase) are ubiquitous, participate in osmotic balance and membrane potential, and are composed of α, β, and γ subunits. The α subunit is required for the catalytic and transport properties of the enzyme and contains binding sites for cations, ATP, and digitalis-like compounds including ouabain. There are four known α isoforms; three that are expressed in the CNS in a regional and cell-specific manner. The α2 isoform is most commonly found in astrocytes, pyramidal cells of the hippocampus in adults, and developmentally in several other neuronal types. Ouabain-like compounds are thought to be produced endogenously in mammals, bind the Na,K-ATPase, and function as a stress-related hormone, however, the impact of the Na,K-ATPase ouabain binding site on neurobehavioral function is largely unknown. To determine if the ouabain binding site of the α2 isoform plays a physiological role in CNS function, we examined knock-in mice in which the normally ouabain-sensitive α2 isoform was made resistant (α2R/R) while still retaining basal Na,K-ATPase enzymatic function. Egocentric learning (Cincinnati water maze) was impaired in adult α2R/R mice compared to wild type (WT) mice. They also exhibited decreased locomotor activity in a novel environment and increased responsiveness to a challenge with an indirect sympathomimetic agonist (methamphetamine) relative to WT mice. The α2R/R mice also demonstrated a blunted acoustic startle reflex and a failure to habituate to repeated acoustic stimuli. The α2R/R mice showed no evidence of altered anxiety (elevated zero maze) nor were they impaired in spatial learning or memory in the Morris water maze and neither group could learn in a large Morris maze. These results suggest that the ouabain binding site is involved in specific types of learning and the modulation of dopamine-mediated locomotor behavior. PMID:20936682

  4. Distribution of Peripheral PrPSc in Sheep with Naturally Acquired Scrapie

    PubMed Central

    Garza, María Carmen; Monzón, Marta; Marín, Belén; Badiola, Juan José; Monleón, Eva

    2014-01-01

    Accumulation of prion protein (PrPSc) in the central nervous system is the hallmark of transmissible spongiform encephalopathies. However, in some of these diseases such as scrapie or chronic wasting disease, the PrPSc can also accumulate in other tissues, particularly in the lymphoreticular system. In recent years, PrPSc in organs other than nervous and lymphoid have been described, suggesting that distribution of this protein in affected individuals may be much larger than previously thought. In the present study, 11 non-nervous/non-lymphoid organs from 16 naturally scrapie infected sheep in advanced stages of the disease were examined for the presence of PrPSc. Fourteen infected sheep were of the ARQ/ARQ PRNP genotype and 2 of the VRQ/VRQ, where the letters A, R, Q, and V represent the codes for amino-acids alanine, arginine, glutamine and valine, respectively. Adrenal gland, pancreas, heart, skin, urinary bladder and mammary gland were positive for PrPSc by immunohistochemistry and IDEXX HerdChek scrapie/BSE Antigen EIA Test in at least one animal. Lung, liver, kidney and skeletal muscle exhibited PrPSc deposits by immunohistochemistry only. To our knowledge, this is the first report regarding the presence of PrPSc in the heart, pancreas and urinary bladder in naturally acquired scrapie infections. In some other organs examined, in which PrPSc had been previously detected, PrPSc immunolabeling was observed to be associated with new structures within those organs. The results of the present study illustrate a wide dissemination of PrPSc in both ARQ/ARQ and VRQ/VRQ infected sheep, even when the involvement of the lymphoreticular system is scarce or absent, thus highlighting the role of the peripheral nervous system in the spread of PrPSc. PMID:24828439

  5. INSIGHTS ON SCRAPIE PRION PROTEIN (PrPSc) STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elucidation of the structure of PrPSc, essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research, and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on...

  6. Characterization of PrP(Sc) transmission from immune cells to neuronal cells.

    PubMed

    Tanaka, Yufuko; Sadaike, Tetsuji; Inoshima, Yasuo; Ishiguro, Naotaka

    2012-10-01

    We investigated PrP(Sc) transmission in neuronal cells, spleen cells and several immune cells using an in vitro cell-to-cell transmission system. The transmission of PrP(Sc) in the supernatant of PrP(Sc)-infected neuronal cells was also investigated. We found that PrP(Sc) transmission was more efficient in the cell-to-cell transmission system than in the supernatant-mediated system. PrP(Sc) was more efficiently transmitted from adherent spleen cells to neuronal cells than from floating spleen cells. The adherent spleen cells were composed of macrophages (80%), dendritic cells (8%) and follicular dendritic cells (3%), indicating that macrophages play an important role in PrP(Sc) transmission from immune cells to neuronal cells. Although PrP(Sc) in the immune cells used as donor cells was gradually degraded, the PrP(Sc) transmitted to neuronal cells was observed by Western blot analysis. Investigation of the mechanism of PrP(Sc) transmission between cells represents an important step towards understanding the pathogenesis of prion diseases. PMID:23246505

  7. The effect of PrPSc accumulation on inflammatory gene expression within sheep peripheral lymphoid tissue

    PubMed Central

    Gossner, Anton G.; Hopkins, John

    2015-01-01

    Accumulation of the misfolded prion protein, PrPSc in the central nervous system (CNS) is strongly linked to progressive neurodegenerative disease. For many transmissible spongiform encephalopathies (TSEs), peripheral lymphoid tissue is an important site of PrPSc amplification but without gross immunological consequence. Susceptible VRQ homozygous New Zealand Cheviot sheep were infected with SSBP/1 scrapie by inoculation in the drainage area of the prescapular lymph nodes. The earliest time that PrPSc was consistently detected by immunohistology in these nodes was D50 post infection. This transcriptomic study of lymph node taken before (D10) and after (D50) the detection of PrPSc, aimed to identify the genes and physiological pathways affected by disease progression within the nodes as assessed by PrPSc detection. Affymetrix Ovine Gene arrays identified 75 and 80 genes as differentially-expressed at D10 and D50, respectively, in comparison with control sheep inoculated with uninfected brain homogenate. Approximately 70% of these were repressed at each time point. RT-qPCR analysis of seven genes showed statistically significant correlation with the array data, although the results for IL1RN and TGIF were different between the two technologies. The ingenuity pathway analysis (IPA) and general low level of repression of gene expression in lymphoid tissue, including many inflammatory genes, contrasts with the pro-inflammatory and pro-apoptotic events that occur within the CNS at equivalent stages of disease progression as assessed by PrPSc accumulation. PMID:26507419

  8. Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

    PubMed Central

    Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Takatsuki, Hanae; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrPSc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrPSc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrPSc degradation were evaluated. The accumulation of PrPSc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrPSc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrPSc from either the 22L or the Chandler strain, indicating that the degradation of PrPSc in host cells might be strain-dependent. PMID:26368533

  9. Stability of PrP**Sc from cattle inoculated with different BSE strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases are a class of transmissible neurodegenerative diseases, caused by an infectious protein, that afflict humans and other mammals. The infectious agent is a host encoded protein known as PrP that has adopted a misfolded conformation termed PrP**Sc. The diseases have been shown to manife...

  10. Probing the structure of GPI-less PrPSc by limited proteolysis(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Limited proteolysis is a very useful tool to pinpoint flexible regions within scrapie prion protein (PrPSc), but due to carbohydrate and glysosylphosphatidylinositol (GPI) moieties, and limitations of the analytical techniques, until now it was impossible to characterize accurately these regions. To...

  11. Stability properties of PrPSc from cattle with experimental transmissible spongiform encephalopathies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible Spongiform Encephalopathies (TSEs), including scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE), are fatal diseases of the nervous system associated with accumulation of misfolded prion protein (PrPSc). Different strains of BSE exist...

  12. Isolation and characterization of a proteinase K sensitive PrPSc fraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies have shown that a sizeable fraction of PrPSc present in prion-infected tissues is,contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in att...

  13. Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

    PubMed Central

    Rouvinski, Alexander; Karniely, Sharon; Kounin, Maria; Moussa, Sanaa; Goldberg, Miri D.; Warburg, Gabriela; Lyakhovetsky, Roman; Papy-Garcia, Dulce; Kutzsche, Janine; Korth, Carsten; Carlson, George A.; Godsave, Susan F.; Peters, Peter J.; Luhr, Katarina; Kristensson, Krister

    2014-01-01

    Mammalian prions refold host glycosylphosphatidylinositol-anchored PrPC into β-sheet–rich PrPSc. PrPSc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrPSc rather than on its truncated PrP27-30 product. We show that N-terminal PrPSc epitopes are exposed in their physiological context and visualize, for the first time, PrPSc in living cells. PrPSc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrPSc amyloids. PMID:24493590

  14. Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

    PubMed Central

    Kaneko, K; Peretz, D; Pan, K M; Blochberger, T C; Wille, H; Gabizon, R; Griffith, O H; Cohen, F E; Baldwin, M A; Prusiner, S B

    1995-01-01

    Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479957

  15. Co-existence of distinct prion types enables conformational evolution of human PrPSc by competitive selection.

    PubMed

    Haldiman, Tracy; Kim, Chae; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Qing, Liuting; Cohen, Mark L; Langeveld, Jan; Telling, Glenn C; Kong, Qingzhong; Safar, Jiri G

    2013-10-11

    The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrP(Sc)). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrP(Sc) particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrP(Sc) particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrP(C) substrate, the dominant PrP(Sc) conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrP(Sc) is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrP(Sc) conformers. PMID:23974118

  16. PrP(Sc) detection and infectivity in semen from scrapie-infected sheep.

    PubMed

    Rubenstein, Richard; Bulgin, Marie S; Chang, Binggong; Sorensen-Melson, Sharon; Petersen, Robert B; LaFauci, Giuseppe

    2012-06-01

    A scrapie-positive ewe was found in a flock that had been scrapie-free for 13 years, but housed adjacent to scrapie-positive animals, separated by a wire fence. Live animal testing of the entire flock of 24 animals revealed seven more subclinical scrapie-positive ewes. We hypothesized that they may have contracted the disease from scrapie-positive rams used for breeding 4 months prior, possibly through the semen. The genotypes of the ewe flock were highly scrapie-susceptible and the rams were infected with the 'Caine' scrapie strain having a short incubation time of 4.3-14.6 months in sheep with 136/171 VQ/VQ and AQ/VQ genotypes. PrP(Sc) accumulates in a variety of tissues in addition to the central nervous system. Although transmission of prion diseases, or transmissible spongiform encephalopathies, has been achieved via peripheral organ or tissue homogenates as well as by blood transfusion, neither infectivity nor PrP(Sc) have been found in semen from scrapie-infected animals. Using serial protein misfolding cyclic amplification followed by a surround optical fibre immunoassay, we demonstrate that semen from rams infected with a short-incubation-time scrapie strain contains prion disease-associated-seeding activity that generated PrP(Sc) in sPMCA (serial protein misfolding cyclic amplification). Injection of the ovinized transgenic mouse line TgSShpPrP with semen from scrapie-infected sheep resulted in PrP(Sc)-seeding activity in clinical and, probably as a result of the low titre, non-clinical mouse brain. These results suggest that the transmissible agent, or at least the seeding activity, for sheep scrapie is present in semen. This may be a strain-specific phenomenon. PMID:22323531

  17. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John

    2014-01-01

    Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent. PMID:25005024

  18. Transmission of the agent of sheep scrapie to deer results in PrPSc with two distinct molecular profiles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this work was to determine susceptibility of white-tailed deer (WTD) to the agent of sheep scrapie and to compare the resultant PrPSc to that of the original inoculum and chronic wasting disease (CWD). We inoculated WTD by a natural route of exposure (concurrent oral and intranasal (I...

  19. Western-blot detection of PrP**sc in archived paraffin-embedded brainstem from scrapie-affected sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies or prion diseases. Immunoassays that identify disease-associated prion protein (PrP**Sc) are integral to the diagnosis o...

  20. Relationships between PrPSc stability and incubation time for United States scrapie strains in a natural host system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP**C) into a beta-rich conformer (PrP**Sc) that accumulates into higher-order structures in the brain and other ti...

  1. Tropomyosin isoforms and reagents

    PubMed Central

    Schevzov, Galina; Whittaker, Shane P; Fath, Thomas; Lin, Jim JC

    2011-01-01

    Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments.1 The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike. PMID:22069507

  2. Early Generation of New PrPSc on Blood Vessels after Brain Microinjection of Scrapie in Mice

    PubMed Central

    Striebel, James; Rangel, Alejandra; Phillips, Katie; Hughson, Andrew; Caughey, Byron; Race, Brent

    2015-01-01

    ABSTRACT Aggregation of misfolded host proteins in the central nervous system is believed to be important in the pathogenic process in several neurodegenerative diseases of humans, including prion diseases, Alzheimer’s disease, and Parkinson’s disease. In these diseases, protein misfolding and aggregation appear to expand through a process of seeded polymerization. Prion diseases occur in both humans and animals and are experimentally transmissible orally or by injection, thus providing a controllable model of other neurodegenerative protein misfolding diseases. In rodents and ruminants, prion disease has a slow course, lasting months to years. Although prion infectivity has been detected in brain tissue at 3 to 4 weeks postinfection (p.i.), the details of early prion replication in the brain are not well understood. Here we studied the localization and quantitation of PrPSc generation in vivo starting at 30 min postmicroinjection of scrapie into the brain. In C57BL mice at 3 days p.i., generation of new PrPSc was detected by immunohistochemistry and immunoblot assays, and at 7 days p.i., new generation was confirmed by real-time quaking-induced conversion assay. The main site of new PrPSc generation was near the outer basement membrane of small and medium blood vessels. The finding and localization of replication at this site so early after injection have not been reported previously. This predominantly perivascular location suggested that structural components of the blood vessel basement membrane or perivascular astrocytes might act as cofactors in the initial generation of PrPSc. The location of PrPSc replication at the basement membrane also implies a role for the brain interstitial fluid drainage in the early infection process. PMID:26396245

  3. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  4. Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

    PubMed Central

    James, Thomas L.; Liu, He; Ulyanov, Nikolai B.; Farr-Jones, Shauna; Zhang, Hong; Donne, David G.; Kaneko, Kiyotoshi; Groth, Darlene; Mehlhorn, Ingrid; Prusiner, Stanley B.; Cohen, Fred E.

    1997-01-01

    The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition. PMID:9294167

  5. Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship.

    PubMed

    Konold, Timm; Phelan, Laura J; Cawthraw, Saira; Simmons, Marion M; Chaplin, Melanie J; González, Lorenzo

    2016-01-01

    Scrapie is transmissible spongiform encephalopathy (TSE), which causes neurological signs in sheep, but confirmatory diagnosis is usually made postmortem on examination of the brain for TSE-associated markers like vacuolar changes and disease-associated prion protein (PrP(Sc)). The objective of this study was to evaluate whether testing of brainstem auditory evoked potentials (BAEPs) at two different sound levels could aid in the clinical diagnosis of TSEs in sheep naturally or experimentally infected with different TSE strains [classical and atypical scrapie and bovine spongiform encephalopathy (BSE)] and whether any BAEP abnormalities were associated with TSE-associated markers in the auditory pathways. BAEPs were recorded from 141 clinically healthy sheep of different breeds and ages that tested negative for TSEs on postmortem tests to establish a reference range and to allow comparison with 30 sheep clinically affected or exposed to classical scrapie (CS) without disease confirmation (test group 1) and 182 clinically affected sheep with disease confirmation (test group 2). Abnormal BAEPs were found in 7 sheep (23%) of group 1 and 42 sheep (23%) of group 2. The proportion of sheep with abnormalities did not appear to be influenced by TSE strain or PrP(Sc) gene polymorphisms. When the magnitude of TSE-associated markers in the auditory pathways was compared between a subset of 12 sheep with and 12 sheep without BAEP abnormalities in group 2, no significant differences in the total PrP(Sc) or vacuolation scores in the auditory pathways could be found. However, the data suggested that there was a difference in the PrP(Sc) scores depending on the TSE strain because PrP(Sc) scores were significantly higher in sheep with BAEP abnormalities infected with classical and L-type BSE, but not with CS. The results indicated that BAEPs may be abnormal in sheep infected with TSEs but the test is not specific for TSEs and that neither vacuolation nor PrP(Sc) accumulation

  6. Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship

    PubMed Central

    Konold, Timm; Phelan, Laura J.; Cawthraw, Saira; Simmons, Marion M.; Chaplin, Melanie J.; González, Lorenzo

    2016-01-01

    Scrapie is transmissible spongiform encephalopathy (TSE), which causes neurological signs in sheep, but confirmatory diagnosis is usually made postmortem on examination of the brain for TSE-associated markers like vacuolar changes and disease-associated prion protein (PrPSc). The objective of this study was to evaluate whether testing of brainstem auditory evoked potentials (BAEPs) at two different sound levels could aid in the clinical diagnosis of TSEs in sheep naturally or experimentally infected with different TSE strains [classical and atypical scrapie and bovine spongiform encephalopathy (BSE)] and whether any BAEP abnormalities were associated with TSE-associated markers in the auditory pathways. BAEPs were recorded from 141 clinically healthy sheep of different breeds and ages that tested negative for TSEs on postmortem tests to establish a reference range and to allow comparison with 30 sheep clinically affected or exposed to classical scrapie (CS) without disease confirmation (test group 1) and 182 clinically affected sheep with disease confirmation (test group 2). Abnormal BAEPs were found in 7 sheep (23%) of group 1 and 42 sheep (23%) of group 2. The proportion of sheep with abnormalities did not appear to be influenced by TSE strain or PrPSc gene polymorphisms. When the magnitude of TSE-associated markers in the auditory pathways was compared between a subset of 12 sheep with and 12 sheep without BAEP abnormalities in group 2, no significant differences in the total PrPSc or vacuolation scores in the auditory pathways could be found. However, the data suggested that there was a difference in the PrPSc scores depending on the TSE strain because PrPSc scores were significantly higher in sheep with BAEP abnormalities infected with classical and L-type BSE, but not with CS. The results indicated that BAEPs may be abnormal in sheep infected with TSEs but the test is not specific for TSEs and that neither vacuolation nor PrPSc accumulation appears to be

  7. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  8. DNA signals at isoform promoters.

    PubMed

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  9. CD21-positive follicular dendritic cells: A possible source of PrPSc in lymph node macrophages of scrapie-infected sheep.

    PubMed

    Herrmann, Lynn M; Cheevers, William P; Davis, William C; Knowles, Donald P; O'Rourke, Katherine I

    2003-04-01

    Natural sheep scrapie is a prion disease characterized by the accumulation of PrP(Sc) in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrP(Sc). In this study, lymph nodes were analyzed for the presence of PrP(Sc) and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrP(Sc) by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrP(Sc) to N-terminally truncated PrP(Sc). PMID:12651600

  10. Protease-sensitive prion species in neoplastic spleens of prion-infected mice with uncoupling of PrP(Sc) and prion infectivity.

    PubMed

    Krasemann, Susanne; Neumann, Melanie; Szalay, Beata; Stocking, Carol; Glatzel, Markus

    2013-02-01

    Prion diseases are fatal neurodegenerative disorders. An important step in disease pathophysiology is the conversion of cellular prion protein (PrP(C)) to disease-associated misfolded conformers (PrP(Sc)). These misfolded PrP variants are a common component of prion infectivity and are detectable in diseased brain and lymphoreticular organs such as spleen. In the latter, PrP(Sc) is thought to replicate mainly in follicular dendritic cells within spleen follicles. Although the presence of PrP(Sc) is a hallmark for prion disease and serves as a main diagnostic criterion, in certain instances the amount of PrP(Sc) does not correlate well with neurotoxicity or prion infectivity. Therefore, it has been proposed that prions might be a mixture of different conformers and aggregates with differing properties. This study investigated the impact of disruption of spleen architecture by neoplasia on the abundance of different PrP species in spleens of prion-infected mice. Although follicular integrity was completely disturbed, titres of prion infectivity in neoplastic spleens were not significantly altered, yet no protease-resistant PrP(Sc) was detectable. Instead, unique protease-sensitive prion species could be detected in neoplastic spleens. These results indicate the dissociation of PrP(Sc) and prion infectivity and showed the presence of non-PrP(Sc) PrP species in spleen with divergent biochemical properties that become apparent after tissue architecture disruption. PMID:23136363

  11. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  12. Congenital Abnormalities

    MedlinePlus

    ... serious health problems (e.g. Down syndrome ). Single-Gene Abnormalities Sometimes the chromosomes are normal in number, ... blood flow to the fetus impair fetal growth. Alcohol consumption and certain drugs during pregnancy significantly increase ...

  13. Craniofacial Abnormalities

    MedlinePlus

    ... of the skull and face. Craniofacial abnormalities are birth defects of the face or head. Some, like cleft ... palate, are among the most common of all birth defects. Others are very rare. Most of them affect ...

  14. Walking abnormalities

    MedlinePlus

    ... include: Arthritis of the leg or foot joints Conversion disorder (a psychological disorder) Foot problems (such as a ... injuries. For an abnormal gait that occurs with conversion disorder, counseling and support from family members are strongly ...

  15. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  16. Nail abnormalities

    MedlinePlus

    Nail abnormalities are problems with the color, shape, texture, or thickness of the fingernails or toenails. ... Fungus or yeast cause changes in the color, texture, and shape of the nails. Bacterial infection may ...

  17. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

    PubMed Central

    Zoch, Ansgar; Mayerl, Steffen; Schulz, Alexander; Greither, Thomas; Frappart, Lucien; Rübsam, Juliane; Heuer, Heike; Giovannini, Marco; Morrison, Helen

    2015-01-01

    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. PMID:26258444

  18. Identification and characterization of a novel retinal isoform of dystrophin

    SciTech Connect

    D`Souza, V.N.; Sigesmund, D.A.; Man, N.

    1994-09-01

    We have shown that dystrophin is required for normal function of the retina as measured by electroretinography (ERG). In these studies a genotype/phenotype correlation was found in which DMD/BMD patients with deletions in the central to distal region of the gene had abnormal ERGs, while patients with deletions in the 5{prime} end of the gene had a mild or normal retinal phenotype. A similar correlation was also observed in the mouse in which the mdx mouse having a mutation in exon 23 had a normal retinal phenotype, whereas the mdx{sup Cv3} mouse (mutation in intron 65) had an abnormal phenotype. Molecular analysis of both human and mouse retina indicated that at least two isoforms of dystrophin are expressed in the retina and localize to the outer plexiform layer, the synaptic junction between the photoreceptors, the bipolar cells, and the horizontal cells. Using a panel of monoclonal dystrophin antisera to analyze mdx mouse retina which does not contain full length dystrophin antisera, we showed that a shorter dystrophin isoform (approximately 260 kDa) was present and contained part of the rod, the cysteine-rich and C-terminal domains. The 5{prime} end of the transcript giving rise to this isoform was characterized and cloned using 5{prime}RACE. Sequence analysis indicated that this transcript contained a novel exon 1 consisting of 240 nucleotides and coded for a unique N-terminus of 13 amino acids. This isoform is distinct from the DP116 dystrophin isoform identified in peripheral nerve. From the functional analysis of DMD patients and dystrophic mice we conclude that this 260 kDa dystrophin isoform is required for normal retinal electrophysiology.

  19. Relationship of PrPSc molecular properties with incubation time in a natural prion disease host: a characterization of three isolates of U.S. sheep scrapie

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determination of aspects of tertiary and quaternary structure of PrPSc associated with differences in disease presentation in the host is a key area of interest in the prion field. Previously, we determined that a U.S. scrapie isolate (136-VDEP) with a short incubation time upon passage in sheep als...

  20. Use of an ELISA-based stability assay to examine host genotype, PrP**Sc stability, and incubation time relationships in U.S. livestock prion strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transmissible spongiform encephalopathies (TSEs) are caused by the misfolding of the cellular prion protein (PrP**C) into a disease-associated version (PrP**Sc) that accumulates in certain tissues, leading to pathological changes in the brain and eventual death. Different strains of TSEs have been d...

  1. Differential Roles of PML Isoforms.

    PubMed

    Nisole, Sébastien; Maroui, Mohamed Ali; Mascle, Xavier H; Aubry, Muriel; Chelbi-Alix, Mounira K

    2013-01-01

    The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed. PMID:23734343

  2. Modulation of neuronal differentiation by CD40 isoforms

    SciTech Connect

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

    2008-05-02

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

  3. Detection of PrPSc in lung and mammary gland is favored by the presence of Visna/maedi virus lesions in naturally coinfected sheep

    PubMed Central

    Salazar, Eider; Monleón, Eva; Bolea, Rosa; Acín, Cristina; Pérez, Marta; Álvarez, Neila; Leginagoikoa, Iratxe; Juste, Ramón; Minguijón, Esmeralda; Reina, Ramsés; Glaria, Idoia; Berriatua, Eduardo; de Andrés, Damián; Badiola, Juan José; Amorena, Beatriz; Luján, Lluís

    2010-01-01

    There are few reports on the pathogenesis of scrapie (Sc) and Visna/maedi virus (VMV) coinfections. The aim of this work was to study in vivo as well as post mortem both diseases in 91 sheep. Diagnosis of Sc and VMV infections allowed the distribution of animals into five groups according to the presence (+) or absence (−) of infection by Sc and VMV: Sc−/VMV−, Sc−/VMV+, Sc+/VMV− and Sc+/VMV+. The latter was divided into two subgroups, with and without VMV-induced lymphoid follicle hyperplasia (LFH), respectively. In both the lung and mammary gland, PrPSc deposits were found in the germinal center of hyperplasic lymphoid follicles in the subgroup of Sc+/VMV+ having VMV-induced LFH. This detection was always associated with (and likely preceded by) PrPSc observation in the corresponding lymph nodes. No PrPSc was found in other VMV-associated lesions. Animals suffering from scrapie had a statistically significantly lower mean age than the scrapie free animals at the time of death, with no apparent VMV influence. ARQ/ARQ genotype was the most abundant among the 91 ewes and the most frequent in scrapie-affected sheep. VMV infection does not seem to influence the scrapie risk group distribution among animals from the five groups established in this work. Altogether, these data indicate that certain VMV-induced lesions can favor PrPSc deposits in Sc non-target organs such as the lung and the mammary gland, making this coinfection an interesting field that warrants further research for a better comprehension of the pathogenesis of both diseases. PMID:20423698

  4. Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains.

    PubMed

    Carroll, James A; Striebel, James F; Rangel, Alejandra; Woods, Tyson; Phillips, Katie; Peterson, Karin E; Race, Brent; Chesebro, Bruce

    2016-04-01

    Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice. PMID:27046083

  5. Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains

    PubMed Central

    Carroll, James A.; Striebel, James F.; Rangel, Alejandra; Woods, Tyson; Phillips, Katie; Peterson, Karin E.; Race, Brent; Chesebro, Bruce

    2016-01-01

    Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice. PMID:27046083

  6. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    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.

  7. Glycosylation differences between the normal and pathogenic prion protein isoforms

    PubMed Central

    Rudd, Pauline M.; Endo, Tama; Colominas, Cristina; Groth, Darlene; Wheeler, Susan F.; Harvey, David J.; Wormald, Mark R.; Serban, Hana; Prusiner, Stanley B.; Kobata, Akira; Dwek, Raymond A.

    1999-01-01

    Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrPC) and pathogenic (PrPSc) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrPC into PrPSc is not confined to a subset of PrPs that contain specific sugars. Compared with PrPC, PrPSc contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrPC in cells where PrPSc is formed and argues that, in at least some cells forming PrPSc, the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions. PMID:10557270

  8. Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions

    PubMed Central

    Murayama, Yuichi; Yoshioka, Miyako; Okada, Hiroyuki; Takata, Eri; Masujin, Kentaro; Iwamaru, Yoshifumi; Shimozaki, Noriko; Yamamura, Tomoaki; Yokoyama, Takashi; Mohri, Shirou; Tsutsumi, Yuji

    2015-01-01

    The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrPSc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrPSc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrPSc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrPSc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was

  9. Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

    PubMed Central

    Pinard, Melissa A.

    2015-01-01

    The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3−. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors. PMID:25811028

  10. Effectiveness of GH isoform differential immunoassay for detecting rhGH doping on application of various growth factors.

    PubMed

    Okano, Masato; Nishitani, Yasunori; Sato, Mitsuhiko; Kageyama, Shinji

    2012-09-01

    The analytical method for detecting growth hormone (GH) doping, the so-called GH isoform differential immunoassay, is currently approved by the World Anti-Doping Agency (WADA). Anti-doping laboratories often face challenges by athletes' lawyers and need to have various types of scientific evidence against the claim that the adverse analytical finding (AAF) result was caused by excess ectopic or abnormal excretion. In this work, a population study of Japanese athletes (255 male and 256 female) and administration studies of recombinant human GH (rhGH) in Japanese females were conducted to confirm the applicability of GH isoform differential immunoassay. The present paper describes the effectiveness of the GH isoform differential immunoassay under abnormal excretion of endogenous GH as determined by administration studies of GH releasing hormone (GHRH(1-44)) and insulin-like growth factor-1 (IGF-1). No false positive findings were found in Japanese athletes. The GH isoform differential immunoassays could detect application of rhGH for approximately 12-24 h. The administration of GHRH(1-44) and IGF-1 as well as ghrelin receptor agonists did not affect the isoform ratio (no false positives). We conclude that the GH isoform differential immunoassay is a highly specific method for detecting rhGH doping. Subject-based profiling (i.e. athlete biological passport) very likely will represent a highly sensitive approach for detecting rhGH doping. PMID:22733714

  11. Comparison of the anti-prion mechanism of four different anti-prion compounds, anti-PrP monoclonal antibody 44B1, pentosan polysulfate, chlorpromazine, and U18666A, in prion-infected mouse neuroblastoma cells.

    PubMed

    Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2014-01-01

    Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP(Sc) formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents. PMID:25181483

  12. Abnormal Head Position

    MedlinePlus

    ... cause. Can a longstanding head turn lead to any permanent problems? Yes, a significant abnormal head posture could cause permanent ... occipitocervical synostosis and unilateral hearing loss. Are there any ... postures? Yes. Abnormal head postures can usually be improved depending ...

  13. Urine - abnormal color

    MedlinePlus

    ... straw-yellow. Abnormally colored urine may be cloudy, dark, or blood-colored. Causes Abnormal urine color may ... red blood cells, or mucus in the urine. Dark brown but clear urine is a sign of ...

  14. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  15. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  16. Oral inoculation of neonatal Suffolk sheep with the agent of classical scrapie results in PrPSc accumulation in sheep with the PRNP ARQ/ARQ but not the ARQ/ARR genotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Scrapie is a transmissible spongiform encephalopathy that can be transmitted amongst susceptible sheep. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent. Findings This study reports the failure to detect PrPSc in nervous or lymphoid tis...

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

    PubMed

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

    2016-03-01

    Abnormally active glycogen synthase kinase-3 (GSK3) contributes to pathological processes in multiple psychiatric and neurological disorders. Modeled in mice, this includes increasing susceptibility to dysregulation of mood-relevant behaviors, impairing performance in several cognitive tasks and impairing adult hippocampal neural precursor cell (NPC) proliferation. These deficits are all evident in GSK3α/β knockin mice, in which serine-to-alanine mutations block the inhibitory serine phosphorylation regulation of both GSK3 isoforms, leaving GSK3 hyperactive. It was unknown if both GSK3 isoforms perform redundant actions in these processes, or if hyperactivity of one GSK3 isoform has a predominant effect. To test this, we examined GSK3α or GSK3β knockin mice in which only one isoform was mutated to a hyperactive form. Only GSK3β, not GSK3α, knockin mice displayed heightened vulnerability to the learned helplessness model of depression-like behavior. Three cognitive measures impaired in GSK3α/β knockin mice showed differential regulation by GSK3 isoforms. Novel object recognition was impaired in GSK3β, not in GSK3α, knockin mice, whereas temporal order memory was not impaired in GSK3α or GSK3β knockin mice, and co-ordinate spatial processing was impaired in both GSK3α and GSK3β knockin mice. Adult hippocampal NPC proliferation was severely impaired in GSK3β knockin mice, but not impaired in GSK3α knockin mice. Increased activity of GSK3β, in the absence of overexpression or disease pathology, is sufficient to impair mood regulation, novel object recognition and hippocampal NPC proliferation, whereas hyperactive GSK3α individually does not impair these processes. These results show that hyperactivity of the two GSK3 isoforms execute non-redundant effects on these processes. PMID:26749572

  18. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    PubMed Central

    Volpi, Nila; Pecorelli, Alessandra; Lorenzoni, Paola; Di Lazzaro, Francesco; Belmonte, Giuseppe; Aglianò, Margherita; Giannini, Fabio; Grasso, Giovanni

    2013-01-01

    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. PMID:23840094

  19. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... The appearance of normal teeth varies, especially the molars. ... conditions. Specific diseases can affect tooth shape, tooth ...

  20. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... from many different conditions. Specific diseases can affect tooth shape, tooth color, time of appearance, or absence ...

  1. The anti-angiogenic isoforms of VEGF in health and disease.

    PubMed

    Qiu, Yan; Hoareau-Aveilla, Coralie; Oltean, Sebastian; Harper, Steven J; Bates, David O

    2009-12-01

    Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF(165)b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGF(xxx)b isoforms to the pro-angiogenic VEGF(xxx) isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGF(xxx)b isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology. PMID:19909248

  2. The anti-angiogenic isoforms of VEGF in health and disease

    PubMed Central

    Qiu, Yan; Hoareau-Aveilla, Coralie; Oltean, Sebastian; Harper, Steven J.; Bates, David O.

    2010-01-01

    Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology. PMID:19909248

  3. Structurally abnormal human autosomes

    SciTech Connect

    1993-12-31

    Chapter 25, discusses structurally abnormal human autosomes. This discussion includes: structurally abnormal chromosomes, chromosomal polymorphisms, pericentric inversions, paracentric inversions, deletions or partial monosomies, cri du chat (cat cry) syndrome, ring chromosomes, insertions, duplication or pure partial trisomy and mosaicism. 71 refs., 8 figs.

  4. Abnormal prion protein in the retina of Rocky Mountain elk (Cervus Elaphus Nelsoni)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging mule deer (Odocoileus hemionus hemionus), white-tailed deer (Odocoileus virginianus) and Rocky Mountain elk (Cervus elaphus nelsoni). An abnormal isoform of a prion pro...

  5. Role of nuclear progesterone receptor isoforms in uterine pathophysiology

    PubMed Central

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

    2015-01-01

    cellular signaling pathways required for growth. In contrast, progesterone via PR activation appears to increase leiomyoma growth. The exact role of PRs in cervical cancer is unclear. PRs regulate implantation and therefore aberrant PR function may be implicated in recurrent pregnancy loss (RPL). PRs likely regulate key immunogenic factors involved in RPL. However, the exact role of PRs in the pathophysiology of RPL and the use of progesterone for therapeutic benefit remains uncertain. CONCLUSIONS PRs are key mediators of progesterone action in uterine tissues and are essential for normal uterine function. Aberrant PR function (due to abnormal expression and/or function) is a major cause of uterine pathophysiology. Further investigation of the underlying mechanisms of PR isoform action in the uterus is required, as this knowledge will afford the opportunity to create progestin/PR-based therapeutics to treat various uterine pathologies. PMID:25406186

  6. TNNT1, TNNT2, and TNNT3: Isoform genes, regulation, and structure-function relationships.

    PubMed

    Wei, Bin; Jin, J-P

    2016-05-10

    Troponin T (TnT) is a central player in the calcium regulation of actin thin filament function and is essential for the contraction of striated muscles. Three homologous genes have evolved in vertebrates to encode three muscle type-specific TnT isoforms: TNNT1 for slow skeletal muscle TnT, TNNT2 for cardiac muscle TnT, and TNNT3 for fast skeletal muscle TnT. Alternative splicing and posttranslational modifications confer additional structural and functional variations of TnT during development and muscle adaptation to various physiological and pathological conditions. This review focuses on the TnT isoform genes and their molecular evolution, alternative splicing, developmental regulation, structure-function relationships of TnT proteins, posttranslational modifications, and myopathic mutations and abnormal splicing. The goal is to provide a concise summary of the current knowledge and some perspectives for future research and translational applications. PMID:26774798

  7. Apolipoprotein E isoform-dependent microglia migration

    PubMed Central

    Cudaback, Eiron; Li, Xianwu; Montine, Kathleen S.; Montine, Thomas J.; Keene, C. Dirk

    2011-01-01

    Complement component C5a and ATP are potent effectors of microglial movement and are increased in diverse neurodegenerative diseases and at sites of injury. Apolipoprotein E (apoE) influences microglial function, and different human apoE isoforms confer variable risk for development of neurodegenerative disorders, especially Alzheimer's disease. The purpose of this investigation was to test the hypothesis that mouse apoE and human apoE isoforms influence microglial migration. Using primary wild-type and apoE-deficient microglia, we show that C5a- and ATP-stimulated chemotaxis are largely apoE-dependent processes with different molecular bases. Although the C5a-dependent chemotaxis of wild-type microglia was completely blocked by receptor-associated protein (RAP), suggesting apoE receptor involvement, ATP-stimulated migration was unaffected by RAP but was associated with differential ERK phosphorylation. Studies using primary microglia derived from targeted replacement mice “humanized” for the coding exons (protein isoform) of human ε2 (apoE2), ε3 (apoE3), or ε4 (apoE4) allele of APOE revealed that primary mouse microglia expressing apoE4 or apoE2 exhibited significantly reduced C5a- and ATP-stimulated migration compared with microglia expressing human apoE3. This study, for the first time, demonstrates apoE dependence and apoE isoform-specific modulation of microglial migration in response to distinct chemotactic stimuli commonly associated with neurodegenerative disease.—Cudaback, E., Li, X., Montine, K. S., Montine, T. J., Keene, C. D. Apolipoprotein E isoform-dependent microglia migration. PMID:21385991

  8. "Jeopardy" in Abnormal Psychology.

    ERIC Educational Resources Information Center

    Keutzer, Carolin S.

    1993-01-01

    Describes the use of the board game, Jeopardy, in a college level abnormal psychology course. Finds increased student interaction and improved application of information. Reports generally favorable student evaluation of the technique. (CFR)

  9. Abnormal Uterine Bleeding

    MedlinePlus

    ... Abnormal uterine bleeding is any bleeding from the uterus (through your vagina) other than your normal monthly ... or fibroids (small and large growths) in the uterus can also cause bleeding. Rarely, a thyroid problem, ...

  10. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... as cancer of the uterus, cervix, or vagina • Polycystic ovary syndrome How is abnormal bleeding diagnosed? Your health care ... before the fetus can survive outside the uterus. Polycystic Ovary Syndrome: A condition characterized by two of the following ...

  11. Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers

    PubMed Central

    Staszewska, Ilona; Fischer, Irmgard; Wiche, Gerhard

    2015-01-01

    Plectin is a highly versatile cytoskeletal protein that acts as a mechanical linker between intermediate filament (IF) networks and various cellular structures. The protein is crucial for myofiber integrity. Its deficiency leads to severe pathological changes in skeletal muscle fibers of patients suffering from epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Skeletal muscle fibers express four major isoforms of plectin which are distinguished solely by alternative, relatively short, first exon-encoded N-terminal sequences. Each one of these isoforms is localized to a different subcellular compartment and plays a specific role in maintaining integrity and proper function(s) of myofibers. The unique role of individual isoforms is supported by distinct phenotypes of isoform-specific knockout mice and recently discovered mutations in first coding exons of plectin that lead to distinct, tissue-specific, pathological abnormalities in humans. In this study, we demonstrate that the lack of plectin isoform 1 (P1) in myofibers of mice leads to alterations of nuclear morphology, similar to those observed in various forms of MD. We show that P1-mediated targeting of desmin IFs to myonuclei is essential for maintenance of their typically spheroidal architecture as well as their proper positioning and movement along the myofiber. Furthermore, we show that P1 deficiency affects chromatin modifications and the expression of genes involved in various cellular functions, including signaling pathways mediating mechanotransduction. Mechanistically, P1 is shown to specifically interact with the myonuclear membrane-associated (BAR domain-containing) protein endophilin B. Our results open a new perspective on cytoskeleton-nuclear crosstalk via specific cytolinker proteins. PMID:26487297

  12. Chromosomal Abnormalities and Schizophrenia

    PubMed Central

    BASSETT, ANNE S.; CHOW, EVA W.C.; WEKSBERG, ROSANNA

    2011-01-01

    Schizophrenia is a common and serious psychiatric illness with strong evidence for genetic causation, but no specific loci yet identified. Chromosomal abnormalities associated with schizophrenia may help to understand the genetic complexity of the illness. This paper reviews the evidence for associations between chromosomal abnormalities and schizophrenia and related disorders. The results indicate that 22q11.2 microdeletions detected by fluorescence in-situ hybridization (FISH) are significantly associated with schizophrenia. Sex chromosome abnormalities seem to be increased in schizophrenia but insufficient data are available to indicate whether schizophrenia or related disorders are increased in patients with sex chromosome aneuploidies. Other reports of chromosomal abnormalities associated with schizophrenia have the potential to be important adjuncts to linkage studies in gene localization. Advances in molecular cytogenetic techniques (i.e., FISH) have produced significant increases in rates of identified abnormalities in schizophrenia, particularly in patients with very early age at onset, learning difficulties or mental retardation, or dysmorphic features. The results emphasize the importance of considering behavioral phenotypes, including adult onset psychiatric illnesses, in genetic syndromes and the need for clinicians to actively consider identifying chromosomal abnormalities and genetic syndromes in selected psychiatric patients. PMID:10813803

  13. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  14. A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical scrapie is a form of transmissible spongiform encephalopathies (TSE) affecting domestic goats and sheep and disease is characterized by the accumulation of abnormal conformational isoform (PrP-Sc) of normal cellular prion protein (PrP-C) in the central nervous system and, in most cases, ly...

  15. Sonication Induced Intermediate in Prion Protein Conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vivo conversion of prion protein (PrPC) to its abnormal pathogenic isoform (PrPSc) is associated with conformational transition of alpha-helices and unstructured regions to beta-sheets. Protein misfolding cyclic amplification (PMCA) is thought to mimics this conversion in vitro. PMCA involves son...

  16. Classical natural ovine scrapie prions are detected in practical volumes of blood by lamb and transgenic mouse bioassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro ligand-based immunoassay studies revealed abnormal isoforms of prion protein (PrP-Sc) are primarily associated with B lymphocytes of scrapie-infected sheep. Our recent study also demonstrated efficient transmission of scrapie to lambs following a transfusion of B lymphocytes isolated from 5...

  17. Cell surface expression of PrP-c and the presence of scrapie prions in the blood of goats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical scrapie is a naturally occurring fatal brain disease of goats and sheep which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

  18. Classical scrapie prions in ovine blood are associated with B lymphocytes and platelets-rich plasma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical scrapie is a naturally occurring fatal brain disease of sheep and goats which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

  19. Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

  20. Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

    PubMed Central

    Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

    2015-01-01

    The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

  1. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms

    PubMed Central

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. PMID:26170631

  2. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

    PubMed

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. PMID:26170631

  3. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

    Ivanov, Sergey V.; Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I.

    2009-05-08

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

  4. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    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.

  5. Decoding RAS isoform and codon-specific signalling

    PubMed Central

    Newlaczyl, Anna U.; Hood, Fiona E.; Coulson, Judy M.; Prior, Ian A.

    2014-01-01

    RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms. PMID:25109951

  6. Separation of plasmid DNA isoforms using centrifugal ultrafiltration.

    PubMed

    Borujeni, Ehsan Espah; Zydney, Andrew L

    2012-07-01

    Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms. PMID:22780319

  7. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    PubMed Central

    Dlamini, Zodwa; Tshidino, Shonisani C.; Hull, Rodney

    2015-01-01

    Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets. PMID:26580598

  8. Morphological abnormalities in elasmobranchs.

    PubMed

    Moore, A B M

    2015-08-01

    A total of 10 abnormal free-swimming (i.e., post-birth) elasmobranchs are reported from The (Persian-Arabian) Gulf, encompassing five species and including deformed heads, snouts, caudal fins and claspers. The complete absence of pelvic fins in a milk shark Rhizoprionodon acutus may be the first record in any elasmobranch. Possible causes, including the extreme environmental conditions and the high level of anthropogenic pollution particular to The Gulf, are briefly discussed. PMID:25903257

  9. Chromosome abnormalities in glioma

    SciTech Connect

    Li, Y.S.; Ramsay, D.A.; Fan, Y.S.

    1994-09-01

    Cytogenetic studies were performed in 25 patients with gliomas. An interesting finding was a seemingly identical abnormality, an extra band on the tip of the short arm of chromosome 1, add(1)(p36), in two cases. The abnormality was present in all cells from a patient with a glioblastoma and in 27% of the tumor cells from a patient with a recurrent irradiated anaplastic astrocytoma; in the latter case, 7 unrelated abnormal clones were identified except 4 of those clones shared a common change, -Y. Three similar cases have been described previously. In a patient with pleomorphic astrocytoma, the band 1q42 in both homologues of chromosome 1 was involved in two different rearrangements. A review of the literature revealed that deletion of the long arm of chromosome 1 including 1q42 often occurs in glioma. This may indicate a possible tumor suppressor gene in this region. Cytogenetic follow-up studies were carried out in two patients and emergence of unrelated clones were noted in both. A total of 124 clonal breakpoints were identified in the 25 patients. The breakpoints which occurred three times or more were: 1p36, 1p22, 1q21, 1q25, 3q21, 7q32, 8q22, 9q22, 16q22, and 22q13.

  10. [Congenital foot abnormalities].

    PubMed

    Delpont, M; Lafosse, T; Bachy, M; Mary, P; Alves, A; Vialle, R

    2015-03-01

    The foot may be the site of birth defects. These abnormalities are sometimes suspected prenatally. Final diagnosis depends on clinical examination at birth. These deformations can be simple malpositions: metatarsus adductus, talipes calcaneovalgus and pes supinatus. The prognosis is excellent spontaneously or with a simple orthopedic treatment. Surgery remains outstanding. The use of a pediatric orthopedist will be considered if malposition does not relax after several weeks. Malformations (clubfoot, vertical talus and skew foot) require specialized care early. Clubfoot is characterized by an equine and varus hindfoot, an adducted and supine forefoot, not reducible. Vertical talus combines equine hindfoot and dorsiflexion of the forefoot, which is performed in the midfoot instead of the ankle. Skew foot is suspected when a metatarsus adductus is resistant to conservative treatment. Early treatment is primarily orthopedic at birth. Surgical treatment begins to be considered after walking age. Keep in mind that an abnormality of the foot may be associated with other conditions: malposition with congenital hip, malformations with syndromes, neurological and genetic abnormalities. PMID:25524290

  11. Basal activity of GIRK5 isoforms.

    PubMed

    Salvador, Carolina; Mora, Silvia I; Ordaz, Benito; Antaramian, Anaid; Vaca, Luis; Escobar, Laura I

    2003-02-14

    G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results. PMID:12535718

  12. Abnormal pressures as hydrodynamic phenomena

    USGS Publications Warehouse

    Neuzil, C.E.

    1995-01-01

    So-called abnormal pressures, subsurface fluid pressures significantly higher or lower than hydrostatic, have excited speculation about their origin since subsurface exploration first encountered them. Two distinct conceptual models for abnormal pressures have gained currency among earth scientists. The static model sees abnormal pressures generally as relict features preserved by a virtual absence of fluid flow over geologic time. The hydrodynamic model instead envisions abnormal pressures as phenomena in which flow usually plays an important role. This paper develops the theoretical framework for abnormal pressures as hydrodynamic phenomena, shows that it explains the manifold occurrences of abnormal pressures, and examines the implications of this approach. -from Author

  13. Feeling Abnormal: Simulation of Deviancy in Abnormal and Exceptionality Courses.

    ERIC Educational Resources Information Center

    Fernald, Charles D.

    1980-01-01

    Describes activity in which student in abnormal psychology and psychology of exceptional children classes personally experience being judged abnormal. The experience allows the students to remember relevant research, become sensitized to the feelings of individuals classified as deviant, and use caution in classifying individuals as abnormal.…

  14. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  15. Exercises to Improve Gait Abnormalities

    MedlinePlus

    ... Home About iChip Articles Directories Videos Resources Contact Exercises to Improve Gait Abnormalities Home » Article Categories » Exercise and Fitness Font Size: A A A A Exercises to Improve Gait Abnormalities Next Page The manner ...

  16. Uptake and dynamics of infectious prion protein in the intestine.

    PubMed

    Ano, Yasuhisa; Sakudo, Akikazu; Nakayama, Hiroyuki; Onodera, Takashi

    2009-01-01

    Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a protease-resistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyer's patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection. PMID:19275737

  17. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

    PubMed

    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

    2015-09-21

    Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). 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. Significantly, both isoforms form fully functional photopigments. 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

  18. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  19. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    PubMed Central

    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

  20. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  1. IL-33 isoforms: their future as vaccine adjuvants?

    PubMed Central

    Villarreal, Daniel O; Weiner, David B

    2015-01-01

    The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy. PMID:25656504

  2. Spirometric abnormalities among welders

    SciTech Connect

    Rastogi, S.K.; Gupta, B.N.; Husain, T.; Mathur, N.; Srivastava, S. )

    1991-10-01

    A group of manual welders age group 13-60 years having a mean exposure period of 12.4 {plus minus} 1.12 years were subjected to spirometry to evaluate the prevalence of spirometric abnormalities. The welders showed a significantly higher prevalence of respiratory impairment than that observed among the unexposed controls as a result of exposure to welding gases which comprised fine particles of lead, zinc, chromium, and manganese. This occurred despite the lower concentration of the pollutants at the work place. In the expose group, the smoking welders showed a prevalence of respiratory impairment significantly higher than that observed in the nonsmoking welders. The results of the pulmonary function tests showed a predominantly restrictive type of pulmonary impairment followed by a mixed ventilatory defect among the welders. The effect of age on pulmonary impairment was not discernible. Welders exposed for over 10 years showed a prevalence of respiratory abnormalities significantly higher than those exposed for less than 10 years. Smoking also had a contributory role.

  3. [Unfolding chaperone as a prion protein relating molecule].

    PubMed

    Hachiya, Naomi S; Sakasegawa, Yuji; Kaneko, Kiyotoshi

    2003-11-01

    Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent. PMID:15152473

  4. Eye movement abnormalities.

    PubMed

    Moncayo, Jorge; Bogousslavsky, Julien

    2012-01-01

    Generation and control of eye movements requires the participation of the cortex, basal ganglia, cerebellum and brainstem. The signals of this complex neural network finally converge on the ocular motoneurons of the brainstem. Infarct or hemorrhage at any level of the oculomotor system (though more frequent in the brain-stem) may give rise to a broad spectrum of eye movement abnormalities (EMAs). Consequently, neurologists and particularly stroke neurologists are routinely confronted with EMAs, some of which may be overlooked in the acute stroke setting and others that, when recognized, may have a high localizing value. The most complex EMAs are due to midbrain stroke. Horizontal gaze disorders, some of them manifesting unusual patterns, may occur in pontine stroke. Distinct varieties of nystagmus occur in cerebellar and medullary stroke. This review summarizes the most representative EMAs from the supratentorial level to the brainstem. PMID:22377853

  5. Development and assessment of sensitive immuno-PCR assays for the quantification of cerebrospinal fluid three- and four-repeat tau isoforms in tauopathies.

    PubMed

    Luk, Connie; Compta, Yaroslau; Magdalinou, Nadia; Martí, Maria José; Hondhamuni, Geshanthi; Zetterberg, Henrik; Blennow, Kaj; Constantinescu, Radu; Pijnenburg, Yolande; Mollenhauer, Brit; Trenkwalder, Claudia; Van Swieten, John; Chiu, Wan Zheng; Borroni, Barbara; Cámara, Ana; Cheshire, Perdita; Williams, David R; Lees, Andrew J; de Silva, Rohan

    2012-11-01

    Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration-tau (FTDP-17/FTLD-tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP-17 cause elevation of tau isoforms with four microtubule-binding repeat domains (4R-tau) compared to those with three repeats (3R-tau). On the basis of two well-characterised monoclonal antibodies against 3R- and 4R-tau, we developed novel, sensitive immuno-PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R-tau in CSF of PSP and AD patients compared with controls, and lower 4R-tau levels in AD compared with PDD. These decreases could be related to the disease-specific conformational masking of the RD4-binding epitope because of abnormal folding and/or aggregation of the 4R-tau isoforms in tauopathies or increased sequestration of the 4R-tau isoforms in brain tau pathology. PMID:22862741

  6. Essential role of the nuclear isoform of RBFOX1, a candidate gene for autism spectrum disorders, in the brain development

    PubMed Central

    Hamada, Nanako; Ito, Hidenori; Nishijo, Takuma; Iwamoto, Ikuko; Morishita, Rika; Tabata, Hidenori; Momiyama, Toshihiko; Nagata, Koh-Ichi

    2016-01-01

    Gene abnormalities in RBFOX1, encoding an mRNA-splicing factor, have been shown to cause autism spectrum disorder and other neurodevelopmental disorders. Since pathophysiological significance of the dominant nuclear isoform in neurons, RBFOX1-isoform1 (iso1), remains to be elucidated, we performed comprehensive analyses of Rbfox1-iso1 during mouse corticogenesis. Knockdown of Rbfox1-iso1 by in utero electroporation caused abnormal neuronal positioning during corticogenesis, which was attributed to impaired migration. The defects were found to occur during radial migration and terminal translocation, perhaps due to impaired nucleokinesis. Axon extension and dendritic arborization were also suppressed in vivo in Rbfox1-iso1-deficient cortical neurons. In addition, electrophysiology experiments revealed significant defects in the membrane and synaptic properties of the deficient neurons. Aberrant morphology was further confirmed by in vitro analyses; Rbfox1-iso1-konckdown in hippocampal neurons resulted in the reduction of primary axon length, total length of dendrites, spine density and mature spine number. Taken together, this study shows that Rbfox1-iso1 plays an important role in neuronal migration and synapse network formation during corticogenesis. Defects in these critical processes may induce structural and functional defects in cortical neurons, and consequently contribute to the pathophysiology of neurodevelopmental disorders with RBFOX1 abnormalities. PMID:27481563

  7. Essential role of the nuclear isoform of RBFOX1, a candidate gene for autism spectrum disorders, in the brain development.

    PubMed

    Hamada, Nanako; Ito, Hidenori; Nishijo, Takuma; Iwamoto, Ikuko; Morishita, Rika; Tabata, Hidenori; Momiyama, Toshihiko; Nagata, Koh-Ichi

    2016-01-01

    Gene abnormalities in RBFOX1, encoding an mRNA-splicing factor, have been shown to cause autism spectrum disorder and other neurodevelopmental disorders. Since pathophysiological significance of the dominant nuclear isoform in neurons, RBFOX1-isoform1 (iso1), remains to be elucidated, we performed comprehensive analyses of Rbfox1-iso1 during mouse corticogenesis. Knockdown of Rbfox1-iso1 by in utero electroporation caused abnormal neuronal positioning during corticogenesis, which was attributed to impaired migration. The defects were found to occur during radial migration and terminal translocation, perhaps due to impaired nucleokinesis. Axon extension and dendritic arborization were also suppressed in vivo in Rbfox1-iso1-deficient cortical neurons. In addition, electrophysiology experiments revealed significant defects in the membrane and synaptic properties of the deficient neurons. Aberrant morphology was further confirmed by in vitro analyses; Rbfox1-iso1-konckdown in hippocampal neurons resulted in the reduction of primary axon length, total length of dendrites, spine density and mature spine number. Taken together, this study shows that Rbfox1-iso1 plays an important role in neuronal migration and synapse network formation during corticogenesis. Defects in these critical processes may induce structural and functional defects in cortical neurons, and consequently contribute to the pathophysiology of neurodevelopmental disorders with RBFOX1 abnormalities. PMID:27481563

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

    PubMed Central

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

    2015-01-01

    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. PMID:26573433

  9. Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis.

    PubMed

    Baltes-Breitwisch, Michelle M; Artac, Robin A; Bott, Rebecca C; McFee, Renee M; Kerl, Jill G; Clopton, Debra T; Cupp, Andrea S

    2010-08-01

    Vascular endothelial growth factor A (VEGFA) plays a role in both angiogenesis and seminiferous cord formation, and alternative splicing of the Vegfa gene produces both proangiogenic isoforms and antiangiogenic isoforms (B-isoforms). The objectives of this study were to evaluate the expression of pro- and antiangiogenic isoforms during testis development and to determine the role of VEGFA isoforms in testis morphogenesis. Quantitative RT-PCR determined that Vegfa_165b mRNA was most abundant between embryonic days 13.5 and 16 (E13.5 and 16; P<0.05). Compared with ovarian mRNA levels, Vegfa_120 was more abundant at E13-14 (P<0.05), Vegfa_164 was less abundant at E13 (P<0.05), and Vegfa_165b tended to be less abundant at E13 (P<0.09) in testes. Immunohistochemical staining localized antiangiogenic isoforms to subsets of germ cells at E14-16, and western blot analysis revealed similar protein levels for VEGFA_165B, VEGFA_189B, and VEGFA_206B at this time point. Treatment of E13 organ culture testes with VEGFA_120, VEGFA_164, and an antibody to antiangiogenic isoforms (anti-VEGFAxxxB) resulted in less organized and defined seminiferous cords compared with paired controls. In addition, 50 ng/ml VEGFA_120 and VEGFA_164 treatments increased vascular density in cultured testes by 60 and 48% respectively, and treatment with VEGFAxxxB antibody increased vascular density by 76% in testes (0.5 ng/ml) and 81% in ovaries (5 ng/ml) compared with controls (P<0.05). In conclusion, both pro- and antiangiogenic VEGFA isoforms are involved in the development of vasculature and seminiferous cords in rat testes, and differential expression of these isoforms may be important for normal gonadal development. PMID:20457593

  10. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  11. Ictal Cardiac Ryhthym Abnormalities

    PubMed Central

    Ali, Rushna

    2016-01-01

    Cardiac rhythm abnormalities in the context of epilepsy are a well-known phenomenon. However, they are under-recognized and often missed. The pathophysiology of these events is unclear. Bradycardia and asystole are preceded by seizure onset suggesting ictal propagation into the cortex impacting cardiac autonomic function, and the insula and amygdala being possible culprits. Sudden unexpected death in epilepsy (SUDEP) refers to the unanticipated death of a patient with epilepsy not related to status epilepticus, trauma, drowning, or suicide. Frequent refractory generalized tonic-clonic seizures, anti-epileptic polytherapy, and prolonged duration of epilepsy are some of the commonly identified risk factors for SUDEP. However, the most consistent risk factor out of these is an increased frequency of generalized tonic–clonic seizures (GTC). Prevention of SUDEP is extremely important in patients with chronic, generalized epilepsy. Since increased frequency of GTCS is the most consistently reported risk factor for SUDEP, effective seizure control is the most important preventive strategy. PMID:27347227

  12. Abnormal uterine bleeding.

    PubMed

    Whitaker, Lucy; Critchley, Hilary O D

    2016-07-01

    Abnormal uterine bleeding (AUB) is a common and debilitating condition with high direct and indirect costs. AUB frequently co-exists with fibroids, but the relationship between the two remains incompletely understood and in many women the identification of fibroids may be incidental to a menstrual bleeding complaint. A structured approach for establishing the cause using the Fédération International de Gynécologie et d'Obstétrique (FIGO) PALM-COEIN (Polyp, Adenomyosis, Leiomyoma, Malignancy (and hyperplasia), Coagulopathy, Ovulatory disorders, Endometrial, Iatrogenic and Not otherwise classified) classification system will facilitate accurate diagnosis and inform treatment options. Office hysteroscopy and increasing sophisticated imaging will assist provision of robust evidence for the underlying cause. Increased availability of medical options has expanded the choice for women and many will no longer need to recourse to potentially complicated surgery. Treatment must remain individualised and encompass the impact of pressure symptoms, desire for retention of fertility and contraceptive needs, as well as address the management of AUB in order to achieve improved quality of life. PMID:26803558

  13. IDENTIFICATION AND REMOVAL OF PROTEINS THAT CO-PURIFY WITH INFECTIOUS PRION PROTEIN IMPROVES THE ANALYSIS OF ITS SECONDARY STRUCTURE

    PubMed Central

    Moore, Roger A.; Timmes, Andrew; Wilmarth, Phillip A.; Safronetz, David; Priola, Suzette A.

    2013-01-01

    Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrPSc) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies suggesting that other proteins might be confounding the analysis of PrPSc secondary structure. We have used FTIR and tandem mass spectrometry to analyze enriched PrPSc from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrPSc. Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn, and β-sheet structures attributed to PrPSc. The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652cm−1 in the FTIR spectrum associated with PrPSc. However, even the removal of greater than 99% of the ferritin from PrPSc did not completely abolish absorbance at 1652cm−1. Our results show that contaminating proteins alter the FTIR spectrum attributed to PrPSc and suggest that the α-helical, loop/turn, and β-sheet secondary structure that remains following their removal are derived from PrPSc itself. PMID:21805638

  14. p53 Isoforms: Key Regulators of the Cell Fate Decision.

    PubMed

    Joruiz, Sebastien M; Bourdon, Jean-Christophe

    2016-01-01

    It is poorly understood how a single protein, p53, can be responsive to so many stress signals and orchestrates very diverse cell responses to maintain/restore cell/tissue functions. The uncovering that TP53 gene physiologically expresses, in a tissue-dependent manner, several p53 splice variants (isoforms) provides an explanation to its pleiotropic biological activities. Here, we summarize a decade of research on p53 isoforms. The clinical studies and the diverse cellular and animal models of p53 isoforms (zebrafish, Drosophila, and mouse) lead us to realize that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the coexpressed p53 isoforms and that unbalancing expression of different p53 isoforms leads to cancer, premature aging, (neuro)degenerative diseases, inflammation, embryo malformations, or defects in tissue regeneration. Cracking the p53 isoforms' code is, thus, a necessary step to improve cancer treatment. It also opens new exciting perspectives in tissue regeneration. PMID:26801896

  15. Frac-seq reveals isoform-specific recruitment to polyribosomes

    PubMed Central

    Sterne-Weiler, Timothy; Martinez-Nunez, Rocio Teresa; Howard, Jonathan M.; Cvitovik, Ivan; Katzman, Sol; Tariq, Muhammad A.; Pourmand, Nader; Sanford, Jeremy R.

    2013-01-01

    Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms. PMID:23783272

  16. A Network of Splice Isoforms for the Mouse.

    PubMed

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  17. Isoform dependent regulation of human HCN channels by cholesterol

    PubMed Central

    Fürst, Oliver; D’Avanzo, Nazzareno

    2015-01-01

    Cholesterol has been shown to regulate numerous ion channels. HCN channels represent the molecular correlate of If or Ih in sinoatrial node (SAN) and neuronal cells. Previous studies have implicated a role for cholesterol in the regulation of rabbit HCN4 channels with effects on pacing in the rabbit SAN. Using electrophysiological and biochemical approaches, we examined the effect of cholesterol modulation on human HCN1, HCN2 and HCN4 isoforms. Patch-clamp experiments uncovered isoform specific differences in the effect of cholesterol on gating kinetics upon depletion by MβCD or mevastatin or enrichment using MβCD/cholesterol. Most dramatically cholesterol had isoform specific effects on mode-shifting, which has been suggested to play a key role in stabilizing firing rate and preventing arrhythmic firing in SAN cells and neurons. Mode-shifting in HCN1 channels was insensitive to cholesterol manipulation, while HCN2 and HCN4 were strongly affected. Trafficking of each isoform to the plasma membrane was also affected by cholesterol modulation differentially between isoforms, however, each isoform remained localized in lipid raft domains after cholesterol depletion. These effects may contribute to the side effects of cholesterol reducing therapies including disrupted heart rhythm and neuropathic pain, as well as the susceptibility of sinus dysfunction in patients with elevated cholesterol. PMID:26404789

  18. A Network of Splice Isoforms for the Mouse

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S.; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  19. Electrocardiograph abnormalities revealed during laparoscopy

    PubMed Central

    Nijjer, Sukhjinder; Dubrey, Simon William

    2010-01-01

    This brief case presents a well patient in whom an electrocardiograph abnormality consistent with an accessory pathway was found during a routine procedure. We present the electrocardiographs, explain the underlying condition, and consider why the abnormality was revealed in this manner. PMID:22419949

  20. Abnormal pressure in hydrocarbon environments

    USGS Publications Warehouse

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  1. Haem degradation in abnormal haemoglobins.

    PubMed Central

    Brown, S B; Docherty, J C

    1978-01-01

    The coupled oxidation of certain abnormal haemoglobins leads to different bile-pigment isomer distributions from that of normal haemoglobin. The isomer pattern may be correlated with the structure of the abnormal haemoglobin in the neighbourhood of the haem pocket. This is support for haem degradation by an intramolecular reaction. PMID:708385

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

    PubMed Central

    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 streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa. PMID:9518500

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

    PubMed Central

    Bultmann-Mellin, Insa; Conradi, Anne; Maul, Alexandra C.; Dinger, Katharina; Wempe, Frank; Wohl, Alexander P.; Imhof, Thomas; Wunderlich, F. Thomas; Bunck, Alexander C.; Nakamura, Tomoyuki; Koli, Katri; Bloch, Wilhelm; Ghanem, Alexander; Heinz, Andrea; von Melchner, Harald; Sengle, Gerhard; Sterner-Kock, Anja

    2015-01-01

    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. PMID:25713297

  4. Probing PrPSc structure using chemical cross-linking and mass spectrometry: evidence of the proximity of Gly90 amino termini in the PrP 27-30 aggregate.

    PubMed

    Onisko, Bruce; Fernández, Esteban Guitián; Freire, María Louro; Schwarz, Anja; Baier, Michael; Camiña, Félix; García, Javier Rodríguez; Rodríguez-Segade Villamarín, Santiago; Requena, Jesús R

    2005-08-01

    Elucidation of the structure of PrP(Sc) continues to be one of the most important and difficult challenges in prion research. This task, essential for gaining an understanding of the basis of prion infectivity, has been hampered by the insoluble, aggregated nature of this molecule. We used a combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), in an attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamsters infected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residues that are close enough to each other to react with a bifunctional reagent of a given chain length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), obtaining dimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followed by mass spectrometric analysis showed that BS(3) reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP 27-30 dimers, but not in intramolecularly cross-linked monomers or control samples. This observation indicates the spatial proximity of Gly90 amino termini in PrP 27-30 fibrils. The Gly90-Gly90 cross-link is consistent with a recent model of PrP 27-30, based on electron crystallographic data, featuring a fiber composed of stacked trimers of PrP monomers; specifically, it is compatible with cross-linking of monomers stacked vertically along the fiber axis but not those adjacent to each other horizontally in the trimeric building block. Our results constitute the first measured distance constraint in PrP(Sc). PMID:16042387

  5. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

  6. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    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. PMID:23923011

  7. Haematological abnormalities in mitochondrial disorders

    PubMed Central

    Finsterer, Josef; Frank, Marlies

    2015-01-01

    INTRODUCTION This study aimed to assess the kind of haematological abnormalities that are present in patients with mitochondrial disorders (MIDs) and the frequency of their occurrence. METHODS The blood cell counts of a cohort of patients with syndromic and non-syndromic MIDs were retrospectively reviewed. MIDs were classified as ‘definite’, ‘probable’ or ‘possible’ according to clinical presentation, instrumental findings, immunohistological findings on muscle biopsy, biochemical abnormalities of the respiratory chain and/or the results of genetic studies. Patients who had medical conditions other than MID that account for the haematological abnormalities were excluded. RESULTS A total of 46 patients (‘definite’ = 5; ‘probable’ = 9; ‘possible’ = 32) had haematological abnormalities attributable to MIDs. The most frequent haematological abnormality in patients with MIDs was anaemia. 27 patients had anaemia as their sole haematological problem. Anaemia was associated with thrombopenia (n = 4), thrombocytosis (n = 2), leucopenia (n = 2), and eosinophilia (n = 1). Anaemia was hypochromic and normocytic in 27 patients, hypochromic and microcytic in six patients, hyperchromic and macrocytic in two patients, and normochromic and microcytic in one patient. Among the 46 patients with a mitochondrial haematological abnormality, 78.3% had anaemia, 13.0% had thrombopenia, 8.7% had leucopenia and 8.7% had eosinophilia, alone or in combination with other haematological abnormalities. CONCLUSION MID should be considered if a patient’s abnormal blood cell counts (particularly those associated with anaemia, thrombopenia, leucopenia or eosinophilia) cannot be explained by established causes. Abnormal blood cell counts may be the sole manifestation of MID or a collateral feature of a multisystem problem. PMID:26243978

  8. Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes

    PubMed Central

    Brim, Svetlana; Groschup, Martin H.; Kuczius, Thorsten

    2016-01-01

    Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC–Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays. PMID:27093554

  9. Heterogeneity of presynaptic proteins: do not forget isoforms

    PubMed Central

    Bragina, Luca; Fattorini, Giorgia; Giovedì, Silvia; Bosco, Federica; Benfenati, Fabio; Conti, Fiorenzo

    2013-01-01

    Analysis of presynaptic protein expression in glutamatergic and GABAergic central synapses performed in several laboratories and with different techniques is unveiling a complex scenario, largely because each presynaptic protein exists in several isoforms. The interpretation of these findings is generally based on the notion that each synapse and each synaptic vesicle contains one of the isoforms of each family of presynaptic proteins. We verified whether this interpretation is tenable by performing triple labeling and immunoisolation studies with the aim of detecting two isoforms of a given presynaptic protein in glutamatergic or GABAergic axon terminals and/or synaptic vesicles (SVs). Here, we show that: (1) the possibility that not all families of presynaptic proteins are expressed in all terminals must be taken into serious account; (2) the expression of a given protein isoform in a terminal does not exclude the expression of other isoforms of the same protein in the same terminal and in the same vesicle. These conclusions open new and interesting problems; their experimental analysis might improve our understanding of the physiology and pathophysiology of central synapses. PMID:23382710

  10. SURVIV for survival analysis of mRNA isoform variation.

    PubMed

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  11. p53 isoform profiling in glioblastoma and injured brain

    PubMed Central

    Takahashi, Rie; Giannini, Caterina; Sarkaria, Jann N.; Schroeder, Mark; Rogers, Joseph; Mastroeni, Diego; Scrable, Heidi

    2014-01-01

    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 10–70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, which have been shown to 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 RT-PCR. At the protein level, 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 to 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. PMID:22824800

  12. SURVIV for survival analysis of mRNA isoform variation

    PubMed Central

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  13. Differential regulation of renal phospholipase C isoforms by catecholamines.

    PubMed

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  14. Cryo-immunogold EM for prions: towards identification of a conversion site

    PubMed Central

    Godsave, S. F.; Wille, H.; Kujala, P.; Latawiec, D.; DeArmond, S.J.; Serban, A.; Prusiner, S. B.; Peters, P. J.

    2009-01-01

    Summary Prion diseases are caused by accumulation of an abnormally folded isoform (PrPSc) of the cellular prion protein (PrPC). The subcellular distribution of PrPSc and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the RML strain of prions. Two antibodies were used: R2, which recognizes both PrPC and PrPSc; and F4–31, which only detects PrPC in undenatured sections. At a late subclinical stage of prion infection, both PrPC and PrPSc were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24 fold) was found on the early / recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrPSc may be oligomeric, protease-sensitive PrPSc (sPrPSc). PMID:19020041

  15. Using small molecule reagents to selectively modify epitopes based on their conformation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PrPSc is an infectious protein. The only experimentally verified difference between PrPSc and their normal cellular isoform (PrPC) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrPSc isoform. The N-hydroxys...

  16. Laminin isoforms in endothelial and perivascular basement membranes

    PubMed Central

    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. PMID:23263631

  17. A penalized likelihood approach for robust estimation of isoform expression

    PubMed Central

    2016-01-01

    Ultra high-throughput sequencing of transcriptomes (RNA-Seq) has enabled the accurate estimation of gene expression at individual isoform level. However, systematic biases introduced during the sequencing and mapping processes as well as incompleteness of the transcript annotation databases may cause the estimates of isoform abundances to be unreliable, and in some cases, highly inaccurate. This paper introduces a penalized likelihood approach to detect and correct for such biases in a robust manner. Our model extends those previously proposed by introducing bias parameters for reads. An L1 penalty is used for the selection of non-zero bias parameters. We introduce an efficient algorithm for model fitting and analyze the statistical properties of the proposed model. Our experimental studies on both simulated and real datasets suggest that the model has the potential to improve isoform-specific gene expression estimates and identify incompletely annotated gene models.

  18. Identification and characterization of novel NuMA isoforms

    SciTech Connect

    Wu, Jin; Xu, Zhe; He, Dacheng; Lu, Guanting

    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.

  19. Oxygenation properties and isoform diversity of snake hemoglobins.

    PubMed

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

    2015-11-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. PMID:26354849

  20. Apolipoprotein E isoform-specific effects on lipoprotein receptor processing

    PubMed Central

    Bachmeier, Corbin; Shackleton, Ben; Ojo, Joseph; Paris, Daniel; Mullan, Michael; Crawford, Fiona

    2014-01-01

    Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood-brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained elevated. Likewise, intracranial administration of Aβ to apoE targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4>apoE3>apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain. PMID:25015123

  1. A68 proteins in Alzheimer's disease are composed of several tau isoforms in a phosphorylated state which affects their electrophoretic mobilities.

    PubMed Central

    Brion, J P; Hanger, D P; Couck, A M; Anderton, B H

    1991-01-01

    The tau-immunoreactive A68 polypeptides found in brains from patients with Alzheimer's disease have been studied by Western blotting using (1) antibodies to synthetic peptides corresponding to sequences that span the complete human tau molecule, and (2) antibodies specific for inserts 1 and 2 found towards the N-terminus of some tau isoforms. The three major A68 polypeptides were labelled by all of the antibodies to sequences common to all tau isoforms, but the faster-migrating A68 polypeptides was not labelled by either of the two antibodies specific for inserts 1 and 2. Treatment with alkaline phosphatase of non-solubilized A68 did not change its electrophoretic mobility on SDS/PAGE under the conditions described here. However, A68 that was solubilized before treating it with alkaline phosphatase was found to move faster on SDS/PAGE than untreated A68, to a position similar to that of normal tau. We also confirmed that A68 preparations contain numerous paired helical filaments (PHF). These PHF were labelled by all anti-tau antibodies, including insert-specific antibodies. Our results further support the notion that PHF contain abnormally phosphorylated tau in an aggregated state, and indicate that these abnormally phosphorylated tau forms are composed of several tau isoforms and that the full length of the tau molecule is present in these polypeptides. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1953678

  2. Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

    PubMed Central

    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. PMID:24244129

  3. Molecular abnormalities in Ewing's sarcoma.

    PubMed

    Burchill, Susan Ann

    2008-10-01

    Ewing's sarcoma is one of the few solid tumors for which the underlying molecular genetic abnormality has been described: rearrangement of the EWS gene on chromosome 22q12 with an ETS gene family member. These translocations define the Ewing's sarcoma family of tumors (ESFT) and provide a valuable tool for their accurate and unequivocal diagnosis. They also represent ideal targets for the development of tumor-specific therapeutics. Although secondary abnormalities occur in over 80% of primary ESFT the clinical utility of these is currently unclear. However, abnormalities in genes that regulate the G(1)/S checkpoint are frequently described and may be important in predicting outcome and response. Increased understanding of the molecular events that arise in ESFT and their role in the development and maintenance of the malignant phenotype will inform the improved stratification of patients for therapy and identify targets and pathways for the design of more effective cancer therapeutics. PMID:18925858

  4. Complex patterns of abnormal heartbeats

    NASA Technical Reports Server (NTRS)

    Schulte-Frohlinde, Verena; Ashkenazy, Yosef; Goldberger, Ary L.; Ivanov, Plamen Ch; Costa, Madalena; Morley-Davies, Adrian; Stanley, H. Eugene; Glass, Leon

    2002-01-01

    Individuals having frequent abnormal heartbeats interspersed with normal heartbeats may be at an increased risk of sudden cardiac death. However, mechanistic understanding of such cardiac arrhythmias is limited. We present a visual and qualitative method to display statistical properties of abnormal heartbeats. We introduce dynamical "heartprints" which reveal characteristic patterns in long clinical records encompassing approximately 10(5) heartbeats and may provide information about underlying mechanisms. We test if these dynamics can be reproduced by model simulations in which abnormal heartbeats are generated (i) randomly, (ii) at a fixed time interval following a preceding normal heartbeat, or (iii) by an independent oscillator that may or may not interact with the normal heartbeat. We compare the results of these three models and test their limitations to comprehensively simulate the statistical features of selected clinical records. This work introduces methods that can be used to test mathematical models of arrhythmogenesis and to develop a new understanding of underlying electrophysiologic mechanisms of cardiac arrhythmia.

  5. [Emotion Disorders and Abnormal Perspiration].

    PubMed

    Umeda, Satoshi

    2016-08-01

    This article reviewed the relationship between emotional disorders and abnormal perspiration. First, I focused on local brain areas related to emotional processing, and summarized the functions of the emotional network involving those local areas. Functional disorders followed by the damage in the amygdala, orbitofrontal cortex, and insular cortex were reviewed, including related abnormal perspiration. I then addressed the mechanisms of how autonomic disorders influence emotional processing. Finally, possible future directions for integrated understanding of the connection between neural activities and bodily reactions were discussed. PMID:27503817

  6. Ultrasonographic assessment of abnormal pregnancy.

    PubMed

    England, G C

    1998-07-01

    Ultrasonographic imaging is widely used in small animal practice for the diagnosis of pregnancy and the determination of fetal number. Ultrasonography can also be used to monitor abnormal pregnancies, for example, conceptuses that are poorly developed for their gestational age (and therefore are likely to fail), and pregnancies in which there is embryonic resorption or fetal abortion. An ultrasound examination may reveal fetal abnormalities and therefore alter the management of the pregnant bitch or queen prior to parturition. There are, however, a number of ultrasonographic features of normal pregnancies that may mimic disease, and these must be recognized. PMID:9698618

  7. Role of p53 isoforms and aggregations in cancer.

    PubMed

    Kim, SeJin; An, Seong Soo A

    2016-06-01

    p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  8. Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

    PubMed

    Lopez-Mejia, Isabel C; de Toledo, Marion; Chavey, Carine; Lapasset, Laure; Cavelier, Patricia; Lopez-Herrera, Celia; Chebli, Karim; Fort, Philippe; Beranger, Guillaume; Fajas, Lluis; Amri, Ez Z; Casas, Francois; Tazi, Jamal

    2014-05-01

    Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals. PMID:24639560

  9. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    PubMed Central

    Zhang, Jifeng; Tan, Minghui; Yin, Yichen; Ren, Bingyu; Jiang, Nannan; Guo, Guoqing; Chen, Yuan

    2015-01-01

    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. PMID:26682072

  10. Characterization of multiple nestin isoforms in the goldfish brain.

    PubMed

    Venables, Maddie J; Navarro-Martín, Laia; Basak, Ajoy; Baum, Bernard R; Zhang, Dapeng; Trudeau, Vance L

    2016-09-01

    Nestin is an intermediate filament protein involved in neurogenesis in fish, mice, and humans. In this study we used rapid amplification of cDNA ends PCR to isolate goldfish nestin (nes). PCR analysis and sequencing revealed three different nes transcripts of 4003, 2446, and 2126 nucleotides, which are predicted to generate proteins of 860, 274, and 344 amino acids in length. Sequence analysis suggests that these nes transcripts are likely a result of alternative splicing. We next applied a multiple-antigenic peptide strategy to generate a goldfish-specific nestin antibody. Western blotting with this antibody together with mass spectrometry verified the presence of major nestin protein isoforms with differing molecular weights (~70, 40 and 30kDa). We further examined expression patterns of these nestin protein isoforms in different parts of the goldfish brain and pituitary and found the telencephalon to express all three isoforms at a distinct level and abundance. We report that multiple nestin isoforms are present indicating another level of complexity for the regulation of intermediate filaments in comparison to mammals. Studying the differential roles and regulation of these nestins could lead to a better understanding of cellular remodeling during neurogenesis and the unparalleled regenerative abilities after damage in the teleost CNS. PMID:27254106

  11. Alternative splicing results in RET isoforms with distinct trafficking properties

    PubMed Central

    Richardson, Douglas S.; Rodrigues, David M.; Hyndman, Brandy D.; Crupi, Mathieu J. F.; Nicolescu, Adrian C.; Mulligan, Lois M.

    2012-01-01

    RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling. PMID:22875993

  12. Differential isoform expression and selective muscle involvement in muscular dystrophies.

    PubMed

    Huovinen, Sanna; Penttilä, Sini; Somervuo, Panu; Keto, Joni; Auvinen, Petri; Vihola, Anna; Huovinen, Sami; Pelin, Katarina; Raheem, Olayinka; Salenius, Juha; Suominen, Tiina; Hackman, Peter; Udd, Bjarne

    2015-10-01

    Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies. PMID:26269091

  13. Role of p53 isoforms and aggregations in cancer

    PubMed Central

    Kim, SeJin; An, Seong Soo A.

    2016-01-01

    Abstract p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers. Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways. Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects. As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  14. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    PubMed

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities. PMID:27091317

  15. Regulatory Divergence of Transcript Isoforms in a Mammalian Model System

    PubMed Central

    Thybert, David; Stefflova, Klara; Watt, Stephen; Flicek, Paul; Brazma, Alvis; Marioni, John C.; Odom, Duncan T.

    2015-01-01

    Phenotypic differences between species are driven by changes in gene expression and, by extension, by modifications in the regulation of the transcriptome. Investigation of mammalian transcriptome divergence has been restricted to analysis of bulk gene expression levels and gene-internal splicing. Using allele-specific expression analysis in inter-strain hybrids of Mus musculus, we determined the contribution of multiple cellular regulatory systems to transcriptome divergence, including: alternative promoter usage, transcription start site selection, cassette exon usage, alternative last exon usage, and alternative polyadenylation site choice. Between mouse strains, a fifth of genes have variations in isoform usage that contribute to transcriptomic changes, half of which alter encoded amino acid sequence. Virtually all divergence in isoform usage altered the post-transcriptional regulatory instructions in gene UTRs. Furthermore, most genes with isoform differences between strains contain changes originating from multiple regulatory systems. This result indicates widespread cross-talk and coordination exists among different regulatory systems. Overall, isoform usage diverges in parallel with and independently to gene expression evolution, and the cis and trans regulatory contribution to each differs significantly. PMID:26339903

  16. Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (U...

  17. APPRIS: annotation of principal and alternative splice isoforms.

    PubMed

    Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L

    2013-01-01

    Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform. PMID:23161672

  18. Trangenic misexpression of the differentiation-specific desmocollin isoform 1 in basal keratinocytes.

    PubMed

    Henkler, F; Strom, M; Mathers, K; Cordingley, H; Sullivan, K; King, I

    2001-01-01

    Keratinocytes undergoing terminal differentiation are characterized by well-defined changes in protein expression, which contribute towards the transformation of cytoarchitecture and epithelial morphology. Characteristic patterns of desmosomal cadherins are tightly regulated and distinct isoforms are expressed during development and differentiation of epithelial tissues. Desmocollin-1 is strictly confined to suprabasal layers of epidermis, but it is absent in mitotically active, basal keratinocytes. This raises the question of whether basal desmocollin-1 could alter desmosomal functions and compromise keratinocyte proliferation, stratification, or early differentiation in skin. In this study, we misexpressed human desmocollin-1 in mouse epidermis, under control of the keratin-14 promoter. Transgenic animals were generated, which showed a specific expression of transgenic human desmocollin-1 in epidermal basal cells. High level transgenic expression, which was equal to or greater than endogenous protein levels, was observed in mice with multiple copy integration of the transgene. A punctate distribution of desmocollin-1 was demonstrated at the cell membrane by indirect immunofluorescence. Transgenic human desmocollin-1 colocalized with endogenous desmosomal marker proteins, indicating efficient incorporation into desmosomes. Transgenic mice did not display any obvious abnormalities, either in the histology of skin and hair follicles, or in the ultrastructure of desmosomes. These observations suggest that desmocollin-1 can function as a desmosomal cadherin both in basal and suprabasal cells. We propose that the differentiation-specific desmocollin isoforms desmocollin-1 and desmocollin-3 are functionally equivalent in basal epidermal cells and suggest that their changing expression patterns are markers, but not regulators, of the initial steps in keratinocyte differentiation. PMID:11168810

  19. Cell, Isoform, and Environment Factors Shape Gradients and Modulate Chemotaxis

    PubMed Central

    Chang, S. Laura; Cavnar, Stephen P.; Takayama, Shuichi; Luker, Gary D.; Linderman, Jennifer J.

    2015-01-01

    Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment. PMID:25909600

  20. Species barrier in prion diseases: a kinetic interpretation based on the conformational adaptation of the prion protein.

    PubMed Central

    Kellershohn, N; Laurent, M

    1998-01-01

    Prion diseases are thought to result from the conformational change of the normal cellular prion protein to a pathogenic protease-resistant isoform. However, brain extracts not containing the protease-resistant isoform of the prion protein can be infectious following interspecies transmission. The 'protein-only' hypothesis of pathogenesis is extended to provide possible explanations which could be interpreted in terms of a different infectious agent. It is proposed that normal cellular protein (PrPC) may be transformed into a form (PrP*) that is conformationally distinct from the host-specific abnormal isoform (PrPSc). In infection from a heterologous donor, the dimeric forms of heterologous PrPSc, which may catalyse the formation of host PrP* from PrPC, host PrP* and host PrPSc are all considered to be capable of catalysing, to some extent, the conversion of PrPC into PrPSc. However, depending on the species involved, PrP* may, or may not, be pathogenic, and may, or may not, be sensitive to proteolysis. It is shown, by numerical integration of the differential rate equations derived from this model, that a strain may be stabilized after two or three passages through a different species and that transmission might occur in the absence of detectable protease-resistant prion protein. The natural transmission of scrapie to cattle is discussed in relation to the model. PMID:9729459

  1. Extracellular Matrix Abnormalities in Schizophrenia

    PubMed Central

    Berretta, Sabina

    2011-01-01

    Emerging evidence points to the involvement of the brain extracellular matrix (ECM) in the pathophysiology of schizophrenia (SZ). Abnormalities affecting several ECM components, including Reelin and chondroitin sulfate proteoglycans (CSPGs), have been described in subjects with this disease. Solid evidence supports the involvement of Reelin, an ECM glycoprotein involved in corticogenesis, synaptic functions and glutamate NMDA receptor regulation, expressed prevalently in distinct populations of GABAergic neurons, which secrete it into the ECM. Marked changes of Reelin expression in SZ have typically been reported in association with GABA-related abnormalities in subjects with SZ and bipolar disorder. Recent findings from our group point to substantial abnormalities affecting CSPGs, a main ECM component, in the amygdala and entorhinal cortex of subjects with schizophrenia, but not bipolar disorder. Striking increases of glial cells expressing CSPGs were accompanied by reductions of perineuronal nets, CSPG- and Reelin-enriched ECM aggregates enveloping distinct neuronal populations. CSPGs developmental and adult functions, including neuronal migration, axon guidance, synaptic and neurotransmission regulation are highly relevant to the pathophysiology of SZ. Together with reports of anomalies affecting several other ECM components, these findings point to the ECM as a key component of the pathology of SZ. We propose that ECM abnormalities may contribute to several aspects of the pathophysiology of this disease, including disrupted connectivity and neuronal migration, synaptic anomalies and altered GABAergic, glutamatergic and dopaminergic neurotransmission. PMID:21856318

  2. SUGARBEET ROOT SUCROSE SYNTHASE ISOFORMS DIFFER IN DEVELOPMENTAL EXPRESSION, SUBUNIT COMPOSITION AND RESPONSE TO PH.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two sucrose synthase isoforms have been identified by activity stained isoelectric focused polyacrylamide electrophoresis in developing sugarbeet (Beta vulgaris L.) root. Sucrose synthase isoform I (SuSyI) was present from the early stages of development to maturity. Sucrose synthase isoform II (S...

  3. The Three Maize Sucrose synthase Isoforms Differ in Distribution, Localization, and Phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although sucrose synthase (SUS) is widely appreciated for its role in plant metabolism and growth, very little is known about the contribution of each of the SUS isoforms to these processes. Using isoform-specific antibodies, we evaluated the three known isoforms individually at the protein level. ...

  4. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

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

    SciTech Connect

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

    2013-08-01

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

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

    PubMed

    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. PMID:22623992

  8. Haplotype and isoform specific expression estimation using multi-mapping RNA-seq reads

    PubMed Central

    2011-01-01

    We present a novel pipeline and methodology for simultaneously estimating isoform expression and allelic imbalance in diploid organisms using RNA-seq data. We achieve this by modeling the expression of haplotype-specific isoforms. If unknown, the two parental isoform sequences can be individually reconstructed. A new statistical method, MMSEQ, deconvolves the mapping of reads to multiple transcripts (isoforms or haplotype-specific isoforms). Our software can take into account non-uniform read generation and works with paired-end reads. PMID:21310039

  9. [PHF10 isoforms are phosphorylated in the PBAF mammalian chromatin remodeling complex].

    PubMed

    Brechalov, A V; Valieva, M E; Georgieva, S G; Soshnikova, N V

    2016-01-01

    Chromatin remodeling complex PBAF(SWI/SNF) alters the structure of chromatin and controls gene expression. PHF10 is a specific subunit of PBAF complex and is expressed as four isoforms in mammalian cells. We demonstrated that all isoforms are expressed in various human cell types of different histological origins. All four isoforms are extensively phosphorylated and their phosphorylation level is depended on the cell type. Phosphorylation of PHF10 isoforms occurs while they are incorporated as a subunit of the PBAF complex, and therefore phosphorylation of PHF10 isoforms may play an essential role in regulation of PBAF complex's function and mechanism of action. PMID:27239853

  10. GLIAL ABNORMALITIES IN MOOD DISORDERS

    PubMed Central

    Öngür, Dost; Bechtholt, Anita J.; Carlezon, William A.; Cohen, Bruce M.

    2015-01-01

    Multiple lines of evidence indicate that mood disorders are associated with abnormalities in the brain's cellular composition, especially in glial cells. Considered inert support cells in the past, glial cells are now known to be important for brain function. Treatments for mood disorders enhance glial cell proliferation, and experimental stimulation of cell growth has antidepressant effects in animal models of mood disorders. These findings suggest that the proliferation and survival of glial cells may be important in the pathogenesis of mood disorders and may be possible targets for the development of new treatments. In this chapter, we will review the evidence for glial abnormalities in mood disorders. We will discuss glial cell biology and evidence from postmortem studies of mood disorders. This is not carry out a comprehensive review; rather we selectively discuss existing evidence in building an argument for the role of glial cells in mood disorders. PMID:25377605

  11. PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes

    PubMed Central

    Sasamoto, Yuzuru; Hayashi, Ryuhei; Park, Sung-Joon; Saito-Adachi, Mihoko; Suzuki, Yutaka; Kawasaki, Satoshi; Quantock, Andrew J.; Nakai, Kenta; Tsujikawa, Motokazu; Nishida, Kohji

    2016-01-01

    PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype. PMID:26899008

  12. Functional impact of splice isoform diversity in individual cells

    PubMed Central

    Yap, Karen; Makeyev, Eugene V.

    2016-01-01

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

  13. Direct Activation of Epac by Sulfonylurea is Isoform Selective

    PubMed Central

    Herbst, Katie J.; Coltharp, Carla; Amzel, L. Mario; Zhang, Jin

    2011-01-01

    Summary Commonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators. PMID:21338921

  14. Comparison of liver oncogenic potential among human RAS isoforms

    PubMed Central

    Chung, Sook In; Moon, Hyuk; Ju, Hye-Lim; Kim, Dae Yeong; Cho, Kyung Joo; Ribback, Silvia; Dombrowski, Frank; Calvisi, Diego F.; Ro, Simon Weonsang

    2016-01-01

    Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene. PMID:26799184

  15. Structural differences between C-terminal regions of tropomyosin isoforms

    PubMed Central

    Śliwińska, Małgorzata

    2013-01-01

    Tropomyosins are actin-binding regulatory proteins which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins in the absence of actin. Conformations of the junction regions bound to actin are unknown. In this work, orientation of the overlap on actin alone and on actin–myosin complex was evaluated by measuring FRET distances between a donor (AEDANS) attached to tropomyosin and an acceptor (DABMI) bound to actin’s Cys374. Donor was attached to the Cys residue introduced by site-directed mutagenesis near the C-terminal half of the overlap. The recombinant alpha-tropomyosin isoforms used in this study – skeletal muscle skTM, non-muscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the N- and C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 Å and 48.1 Å. Rigor binding of myosin S1 increased the apparent FRET distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions. PMID:24167776

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

    PubMed

    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. PMID:27315173

  17. Specific roles of GABAB(1) receptor isoforms in cognition

    PubMed Central

    Jacobson, Laura H.; Kelly, Peter H.; Bettler, Bernhard; Kaupmann, Klemens; Cryan, John F.

    2010-01-01

    The GABAB receptor is a heterodimer of GABAB(1) and GABAB(2) subunits. There are two isoforms of the GABAB(1) subunit: GABAB(1a) and GABAB(1b). Recent studies with mutant mice suggest a differential role for the two GABAB(1) isoforms in behavioural processes. As pharmacological and genetic studies have implicated GABAB receptors in cognition we investigated the behaviour of GABAB(1a) −/− and GABAB(1b) −/− mice in different types of cognitive paradigms. GABAB(1a) −/− and GABAB(1b) −/− mice were both impaired relative to wildtype controls in a continuous spontaneous alternation behaviour test of working spatial memory. In contrast to the reported phenotype of GABAB(1) −/− mice, however, neither GABAB(1a) −/− nor GABAB(1b) −/− mice were deficient in a passive avoidance task. On the other hand, GABAB(1a) −/− mice were impaired in familiar and novel object recognition. We conclude that GABAB(1) isoforms contribute differentially to GABAB receptor-mediated cognitive processes. PMID:17498817

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

    PubMed Central

    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 plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. PMID:27254302

  19. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. Conclusions These findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans. PMID:24996838

  20. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    PubMed Central

    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. PMID:27315173

  1. GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

    PubMed Central

    Greenberg, Anthony J.; Schedl, Paul

    2001-01-01

    The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo. PMID:11713290

  2. Expression profile of parkin isoforms in human gliomas.

    PubMed

    Maugeri, Grazia; D'Amico, Agata Grazia; Magro, Gaetano; Salvatorelli, Lucia; Barbagallo, Giuseppe M V; Saccone, Salvatore; Drago, Filippo; Cavallaro, Sebastiano; D'Agata, Velia

    2015-10-01

    Mutations of parkin gene are not restricted to familial forms of Parkinsonism but they also occur in a wide variety of malignancies including gliomas. Parkin over-expression reduces glioma cells proliferation and analysis of its expression is predictive for the survival outcome of patients with glioma. To date have been identified 21 parkin alternative splice variants. However, most of the studies have focused their attention exclusively on full-length protein. In the present study, the expression profile of parkin isoforms in different grades of astrocytomas was analyzed for the first time, in order to evaluate their involvement in this malignancy. Furthermore, to investigate their role in cellular processes, their expression in three glioblastoma cell lines was analyzed following treatment with the proteasome inhibitor MG132, or induction of mitophagy with CCCP, or after serum deprivation. Results suggested that H20, H1 and H5 isoforms are always expressed in tumors both in vivo and in vitro models. Therefore, these isoforms might be used as specific biomarkers to develop a prognostic tool for brain tumors. PMID:26238155

  3. An isoform of Nedd4-2 is critically involved in the renal adaptation to high salt intake in mice

    PubMed Central

    Minegishi, Shintaro; Ishigami, Tomoaki; Kino, Tabito; Chen, Lin; Nakashima-Sasaki, Rie; Araki, Naomi; Yatsu, Keisuke; Fujita, Megumi; Umemura, Satoshi

    2016-01-01

    Epithelial sodium channels (ENaCs) play critical roles in the maintenance of fluid and electrolyte homeostasis, and their genetic abnormalities cause one type of hereditary salt-sensitive hypertension, Liddle syndrome. As we reported previously, both human and rodent Nedd4L/Nedd4-2 showed molecular diversity, with and without a C2 domain in their N-terminal. Nedd4L/Nedd4-2 isoforms with a C2 domain are hypothesized to be related closely to ubiquitination of ENaCs. We generated Nedd4-2 C2 domain knockout mice. We demonstrate here that loss of Nedd4-2 C2 isoform causes salt-sensitive hypertension under conditions of a high dietary salt intake in vivo. The knockout mice had reduced urinary sodium excretion, osmotic pressure and increased water intake and urine volume with marked dilatation of cortical tubules while receiving a high salt diet. To the contrary, there was no difference in metabolic data between wild-type and knockout mice receiving a normal control diet. In the absence of Nedd4-2 C2 domain, a high salt intake accelerated ENaC expression. Coimmunoprecipitation studies revealed suppressed ubiquitination for ENaC with a high salt intake. Taken together, our findings demonstrate that during a high oral salt intake the Nedd4-2 C2 protein plays a pivotal role in maintaining adaptive salt handling in the kidney. PMID:27256588

  4. An isoform of Nedd4-2 is critically involved in the renal adaptation to high salt intake in mice.

    PubMed

    Minegishi, Shintaro; Ishigami, Tomoaki; Kino, Tabito; Chen, Lin; Nakashima-Sasaki, Rie; Araki, Naomi; Yatsu, Keisuke; Fujita, Megumi; Umemura, Satoshi

    2016-01-01

    Epithelial sodium channels (ENaCs) play critical roles in the maintenance of fluid and electrolyte homeostasis, and their genetic abnormalities cause one type of hereditary salt-sensitive hypertension, Liddle syndrome. As we reported previously, both human and rodent Nedd4L/Nedd4-2 showed molecular diversity, with and without a C2 domain in their N-terminal. Nedd4L/Nedd4-2 isoforms with a C2 domain are hypothesized to be related closely to ubiquitination of ENaCs. We generated Nedd4-2 C2 domain knockout mice. We demonstrate here that loss of Nedd4-2 C2 isoform causes salt-sensitive hypertension under conditions of a high dietary salt intake in vivo. The knockout mice had reduced urinary sodium excretion, osmotic pressure and increased water intake and urine volume with marked dilatation of cortical tubules while receiving a high salt diet. To the contrary, there was no difference in metabolic data between wild-type and knockout mice receiving a normal control diet. In the absence of Nedd4-2 C2 domain, a high salt intake accelerated ENaC expression. Coimmunoprecipitation studies revealed suppressed ubiquitination for ENaC with a high salt intake. Taken together, our findings demonstrate that during a high oral salt intake the Nedd4-2 C2 protein plays a pivotal role in maintaining adaptive salt handling in the kidney. PMID:27256588

  5. Loss of Predominant Shank3 Isoforms Results in Hippocampus-Dependent Impairments in Behavior and Synaptic Transmission

    PubMed Central

    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.

    2013-01-01

    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. PMID:24259569

  6. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain.

    PubMed

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs. PMID:26241350

  7. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain

    PubMed Central

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABAAR). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1–6, β1–3 or γ2 GABAAR subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABAAR subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABAARs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABAAR subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N=16) and comparison (N=14) subjects and found evidence of abnormal localization of the β1 and β2 GABAAR subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β252 kDa), 50 kDa (β250 kDa) and 48 kDa (β248 kDa). In the ER, we found increased total β2 GABAAR subunit (β2ALL) expression driven by increased β250 kDa, a decreased ratio of β248 kDa:β2ALL and an increased ratio of β250 kDa:β248 kDa. Decreased ratios of β1:β2ALL and β1:β250 kDa in both the ER and SYN fractions and an increased ratio of β252 kDa:β248 kDa at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABAARs. PMID:26241350

  8. Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging cervids. An abnormal isoform of a prion protein (PrP-CWD) has been associated with CWD in Rocky Mountain elk (Cervus elaphus nelsoni) and this prion protein can be detected with i...

  9. Generation and characterization of mice transgenic for human IL-18-binding protein isoform a.

    PubMed

    Fantuzzi, Giamila; Banda, Nirmal K; Guthridge, Carla; Vondracek, Andrea; Kim, Soo-Hyun; Siegmund, Britta; Azam, Tania; Sennello, Joseph A; Dinarello, Charles A; Arend, William P

    2003-11-01

    Interleukin (IL)-18 binding protein (IL-18BP) is a natural inhibitor of the pleiotropic cytokine IL-18. To study the role of IL-18BP in modulating inflammatory responses in vivo, mice transgenic for human IL-18BP isoform a (IL-18BP-Tg) were generated. The transgene was expressed at high levels in each organ examined. High levels of bioactive human IL-18BPa were detectable in the circulation of IL-18BP-Tg mice, which were viable, fertile, and had no tissue or organ abnormality. The high levels of IL-18BP in the transgenic mice were able to completely neutralize the interferon-gamma (IFN-gamma)-inducing activity of exogenously administered IL-18. Following administration of endotoxin, with or without prior sensitization with heat-inactivated Propionibacterium acnes, IL-18BP-Tg mice produced significantly lower serum levels of IFN-gamma and macrophage-inflammatory protein-2 compared with nontransgenic littermates. Significantly reduced production of IFN-gamma in response to endotoxin was also observed in cultures of IL-18BP-Tg splenocytes. Finally, IL-18BP-Tg mice were completely protected in a model of hepatotoxicity induced by administration of concanavalin A. These results indicate that high endogenous levels of IL-18BP in trangenic mice effectively neutralize IL-18 and are protective in response to different inflammatory stimuli. PMID:12960225

  10. Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

    PubMed

    Amador, Fátima Camões; Henriques, Ana Gabriela; da Cruz E Silva, Odete A B; da Cruz E Silva, Edgar F

    2004-01-01

    Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future. PMID:15113600

  11. Protein 4.1R–deficient mice are viable but have erythroid membrane skeleton abnormalities

    PubMed Central

    Shi, Zheng-Tao; Afzal, Veena; Coller, Barry; Patel, Dipti; Chasis, Joel A.; Parra, Marilyn; Lee, Gloria; Paszty, Chris; Stevens, Mary; Walensky, Loren; Peters, Luanne L.; Mohandas, Narla; Rubin, Edward; Conboy, John G.

    1999-01-01

    A diverse family of protein 4.1R isoforms is encoded by a complex gene on human chromosome 1. Although the prototypical 80-kDa 4.1R in mature erythrocytes is a key component of the erythroid membrane skeleton that regulates erythrocyte morphology and mechanical stability, little is known about 4.1R function in nucleated cells. Using gene knockout technology, we have generated mice with complete deficiency of all 4.1R protein isoforms. These 4.1R-null mice were viable, with moderate hemolytic anemia but no gross abnormalities. Erythrocytes from these mice exhibited abnormal morphology, lowered membrane stability, and reduced expression of other skeletal proteins including spectrin and ankyrin, suggesting that loss of 4.1R compromises membrane skeleton assembly in erythroid progenitors. Platelet morphology and function were essentially normal, indicating that 4.1R deficiency may have less impact on other hematopoietic lineages. Nonerythroid 4.1R expression patterns, viewed using histochemical staining for lacZ reporter activity incorporated into the targeted gene, revealed focal expression in specific neurons in the brain and in select cells of other major organs, challenging the view that 4.1R expression is widespread among nonerythroid cells. The 4.1R knockout mice represent a valuable animal model for exploring 4.1R function in nonerythroid cells and for determining pathophysiological sequelae to 4.1R deficiency. PMID:9927493

  12. Making chromosome abnormalities treatable conditions.

    PubMed

    Cody, Jannine DeMars; Hale, Daniel Esten

    2015-09-01

    Individuals affected by the classic chromosome deletion syndromes which were first identified at the beginning of the genetic age, are now positioned to benefit from genomic advances. This issue highlights five of these conditions (4p-, 5p-, 11q-, 18p-, and 18q-). It focuses on the increased in understanding of the molecular underpinnings and envisions how these can be transformed into effective treatments. While it is scientifically exciting to see the phenotypic manifestations of hemizygosity being increasingly understood at the molecular and cellular level, it is even more amazing to consider that we are now on the road to making chromosome abnormalities treatable conditions. PMID:26351122

  13. Foot abnormalities of wild birds

    USGS Publications Warehouse

    Herman, C.M.; Locke, L.N.; Clark, G.M.

    1962-01-01

    The various foot abnormalities that occur in birds, including pox, scaly-leg, bumble-foot, ergotism and freezing are reviewed. In addition, our findings at the Patuxent Wildlife Research Center include pox from dove, mockingbird, cowbird, grackle and several species of sparrows. Scaly-leg has been particularly prevalent on icterids. Bumble foot has been observed in a whistling swan and in a group of captive woodcock. Ergotism is reported from a series of captive Canada geese from North Dakota. Several drug treatments recommended by others are presented.

  14. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes

    PubMed Central

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  15. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

    PubMed

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  16. Abnormality on Liver Function Test

    PubMed Central

    2013-01-01

    Children with abnormal liver function can often be seen in outpatient clinics or inpatients wards. Most of them have respiratory disease, or gastroenteritis by virus infection, accompanying fever. Occasionally, hepatitis by the viruses causing systemic infection may occur, and screening tests are required. In patients with jaundice, the tests for differential diagnosis and appropriate treatment are important. In the case of a child with hepatitis B virus infection vertically from a hepatitis B surface antigen positive mother, the importance of the recognition of immune clearance can't be overstressed, for the decision of time to begin treatment. Early diagnosis changes the fate of a child with Wilson disease. So, screening test for the disease should not be omitted. Non-alcoholic fatty liver disease, which is mainly discovered in obese children, is a new strong candidate triggering abnormal liver function. Muscular dystrophy is a representative disease mimicking liver dysfunction. Although muscular dystrophy is a progressive disorder, and early diagnosis can't change the fate of patients, it will be better to avoid parent's blame for delayed diagnosis. PMID:24511518

  17. Medical management of abnormal pregnancy.

    PubMed

    Ratnam, S S; Prasad, R N

    1990-06-01

    Medical termination of abnormal pregnancy requires specific techniques since some conditions make therapy more effective, e.g., missed abortion intrauterine death and molar pregnancy, and others less so, e.g. anencephalic pregnancy. In all cases it is best to terminate the pregnancy as soon as possible to reduce anguish and risks of complications such as consumptive coagulopathy. Oxytocin is not consistently effective, but intraamniotic rivanol has oxytocic properties, and prostaglandins (PGs) are effective by several routes. Surgical methods are more popular in Japan and the US. A diagnostic flow chart is included and described. For missed abortion and fetal death vacuum aspiration or dilatation and evacuation are appropriate for early pregnancy, or PGs are used for later pregnancy, unless there are medical contraindications. Anencephalic pregnancy, usually diagnoses in 2nd or 3rd trimester, is resistant to medical therapy and must often be terminated by cesarean section. Molar pregnancy can be managed with vacuum aspiration at any length of gestation, but must be completed by curettage. Intraamniotic PGs are not advised for mole or fetal death. PG analogs can be administered intramuscularly, or vaginally in gel form. Other types of abnormal pregnancy that can be managed with PGs are spina bifida, hydrocephalus, hydrops fetalis, Dandy-Walker syndrome and Down's syndrome. Tubal pregnancy can be evacuated with intratubally administered PGs under laparoscopic control, thereby preserving tubal integrity. PMID:2225605

  18. Evolutionary, environmental and tissue controls on the occurrence of multiple isoforms of acyl carrier protein

    SciTech Connect

    Battey, J.F.; Ohlrogge, J.B. )

    1989-04-01

    Previous research has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP). We have examined the development of this trait in evolutionarily diverse species. Isoforms were resolved by Western blotting and native PAGE of {sup 3}H-palmitate labelled ACP's. Multiple isoforms of ACP were observed in primitive vascular plants including gymnosperms, ferns and Psilotum and the nonvascular liverworts and mosses. Therefore, the development of ACP isoforms occurred early in evolution. However, unicellular algae and bacteria such as Chlamydomonas, Dunaliella, Synechocystis and Agmnellum have only a single electrophoretic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants. We have also examined light and tissue control over the expression of ACP isoforms. The expression of multiple forms of ACP in leaf of Spinacia and Avena is altered very little by light. Rather, the different patterns of ACP isoforms are primarily dependant on tissue source.

  19. Abnormal Synaptic Vesicle Biogenesis in Drosophila Synaptogyrin Mutants

    PubMed Central

    Stevens, Robin J.; Akbergenova, Yulia; Jorquera, Ramon A.; Littleton, J. Troy

    2012-01-01

    Sustained neuronal communication relies on the coordinated activity of multiple proteins that regulate synaptic vesicle biogenesis and cycling within the presynaptic terminal. Synaptogyrin and synaptophysin are conserved MARVEL domain-containing transmembrane proteins that are among the most abundant synaptic vesicle constituents, although their role in the synaptic vesicle cycle has remained elusive. To further investigate the function of these proteins, we generated and characterized a synaptogyrin (gyr) null mutant in Drosophila, whose genome encodes a single synaptogyrin isoform and lacks a synaptophysin homolog. We demonstrate that Drosophila synaptogyrin plays a modulatory role in synaptic vesicle biogenesis at larval neuromuscular junctions. Drosophila lacking synaptogyrin are viable and fertile and have no overt deficits in motor function. However, ultrastructural analysis of gyr larvae revealed increased synaptic vesicle diameter and enhanced variability in the size of synaptic vesicles. In addition, the resolution of endocytic cisternae into synaptic vesicles in response to strong stimulation is defective in gyr mutants. Electrophysiological analysis demonstrated an increase in quantal size and a concomitant decrease in quantal content, suggesting functional consequences for transmission caused by the loss of synaptogyrin. Furthermore, high-frequency stimulation resulted in increased facilitation and a delay in recovery from synaptic depression, indicating that synaptic vesicle exo-endocytosis is abnormally regulated during intense stimulation conditions. These results suggest that synaptogyrin modulates the synaptic vesicle exo-endocytic cycle and is required for the proper biogenesis of synaptic vesicles at nerve terminals. PMID:23238721

  20. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew

    2015-01-01

    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting non-processive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs. PMID:25712463

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

    PubMed

    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. PMID:26026127

  2. Different motifs regulate trafficking of SorCS1 isoforms.

    PubMed

    Nielsen, Morten S; Keat, Sady J; Hamati, Jida W; Madsen, Peder; Gutzmann, Jakob J; Engelsberg, Arne; Pedersen, Karen M; Gustafsen, Camilla; Nykjaer, Anders; Gliemann, Jørgen; Hermans-Borgmeyer, Irm; Kuhl, Dietmar; Petersen, Claus M; Hermey, Guido

    2008-06-01

    The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the alphaC/sigma2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi-endosomal transport to a significant degree. PMID:18315530

  3. Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

    PubMed

    Cardona, Carolina; Sánchez-Mejías, Elisabeth; Dávila, José C; Martín-Rufián, Mercedes; Campos-Sandoval, José A; Vitorica, Javier; Alonso, Francisco J; Matés, José M; Segura, Juan A; Norenberg, Michael D; Rama Rao, Kakulavarapu V; Jayakumar, Arumugan R; Gutiérrez, Antonia; Márquez, Javier

    2015-03-01

    The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes. PMID:25297978

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

    SciTech Connect

    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. - 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.

  5. Abnormalities of the Erythrocyte Membrane

    PubMed Central

    Gallagher, Patrick G.

    2014-01-01

    Synopsis Primary abnormalities of the erythrocyte membrane, including the hereditary spherocytosis and hereditary elliptocytosis syndromes, are an important group of inherited hemolytic anemias. Classified by distinctive morphology on peripheral blood smear, these disorders are characterized by clinical, laboratory, and genetic heterogeneity. Among this group, hereditary spherocytosis patients are more likely to experience symptomatic anemia. Treatment of hereditary spherocytosis with splenectomy is curative in most patients. Once considered routine, growing recognition of the longterm risks of splenectomy, including cardiovascular disease, thrombotic disorders, and pulmonary hypertension, as well as the emergence of penicillin-resistant pneumococci, a concern for infection in overwhelming postsplenectomy infection, have led to re-evaluation of the role of splenectomy. Current management guidelines acknowledge these important considerations when entertaining splenectomy and recommend detailed discussion between health care providers, patient, and family. The hereditary elliptocytosis syndromes are the most common primary disorders of erythrocyte membrane proteins. However, most elliptocytosis patients are asymptomatic and do not require therapy. PMID:24237975

  6. Adults with Chromosome 18 Abnormalities.

    PubMed

    Soileau, Bridgette; Hasi, Minire; Sebold, Courtney; Hill, Annice; O'Donnell, Louise; Hale, Daniel E; Cody, Jannine D

    2015-08-01

    The identification of an underlying chromosome abnormality frequently marks the endpoint of a diagnostic odyssey. However, families are frequently left with more questions than answers as they consider their child's future. In the case of rare chromosome conditions, a lack of longitudinal data often makes it difficult to provide anticipatory guidance to these families. The objective of this study is to describe the lifespan, educational attainment, living situation, and behavioral phenotype of adults with chromosome 18 abnormalities. The Chromosome 18 Clinical Research Center has enrolled 483 individuals with one of the following conditions: 18q-, 18p-, Tetrasomy 18p, and Ring 18. As a part of the ongoing longitudinal study, we collect data on living arrangements, educational level attained, and employment status as well as data on executive functioning and behavioral skills on an annual basis. Within our cohort, 28 of the 483 participants have died, the majority of whom have deletions encompassing the TCF4 gene or who have unbalanced rearrangement involving other chromosomes. Data regarding the cause of and age at death are presented. We also report on the living situation, educational attainment, and behavioral phenotype of the 151 participants over the age of 18. In general, educational level is higher for people with all these conditions than implied by the early literature, including some that received post-high school education. In addition, some individuals are able to live independently, though at this point they represent a minority of patients. Data on executive function and behavioral phenotype are also presented. Taken together, these data provide insight into the long-term outcome for individuals with a chromosome 18 condition. This information is critical in counseling families on the range of potential outcomes for their child. PMID:25403900

  7. The multigene families of actinoporins (part I): Isoforms and genetic structure.

    PubMed

    Valle, A; Alvarado-Mesén, J; Lanio, M E; Álvarez, C; Barbosa, J A R G; Pazos, I F

    2015-09-01

    Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function. PMID:26187849

  8. Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System

    PubMed Central

    Neville, Megan C.; Nojima, Tetsuya; Ashley, Elizabeth; Parker, Darren J.; Walker, John; Southall, Tony; Van de Sande, Bram; Marques, Ana C.; Fischer, Bettina; Brand, Andrea H.; Russell, Steven; Ritchie, Michael G.; Aerts, Stein; Goodwin, Stephen F.

    2014-01-01

    Summary Background In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system. Results To characterize the roles of sex-specific Fru isoforms in specifying male behavior, we generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior. Conclusions These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system. PMID:24440396

  9. Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

    PubMed

    Ryan, M C; Lee, K; Miyashita, Y; Carter, W G

    1999-06-14

    Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium. PMID:10366601

  10. Breathing abnormalities in sleep in achondroplasia.

    PubMed Central

    Waters, K A; Everett, F; Sillence, D; Fagan, E; Sullivan, C E

    1993-01-01

    Overnight sleep studies were performed in 20 subjects with achondroplasia to document further the respiratory abnormalities present in this group. Somatosensory evoked potentials (SEPs) were recorded in 19 of the subjects to screen for the presence of brainstem abnormalities, which are one of the potential aetiological mechanisms. Fifteen children aged 1 to 14 years, and five young adults, aged 20 to 31 years were included. All had upper airway obstruction and 15 (75%) had a pathological apnoea index (greater than five per hour). Other sleep associated respiratory abnormalities, including partial obstruction, central apnoea, and abnormal electromyographic activity of accessory muscles of respiration, also showed a high prevalence. SEPs were abnormal in eight (42%), but there was no correlation between abnormal SEPs and apnoea during sleep, either qualitatively or quantitatively. A high prevalence of both sleep related respiratory abnormalities and abnormal SEPs in young subjects with achondroplasia was demonstrated. However, the sleep related respiratory abnormalities do not always result in significant blood gas disturbances or correlate with abnormal SEPs in this group. PMID:8215519

  11. Role of Rho kinase isoforms in murine allergic airway responses.

    PubMed

    Zhu, M; Liu, P-Y; Kasahara, D I; Williams, A S; Verbout, N G; Halayko, A J; Fedulov, A; Shoji, T; Williams, E S; Noma, K; Shore, S A; Liao, J K

    2011-10-01

    Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses. PMID:21565918

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

    PubMed

    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. PMID:27167299

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

    PubMed

    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

    2014-10-21

    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

  14. A New View of Ras Isoforms in Cancers.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum

    2016-01-01

    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  15. Role of cysteines in mammalian VDAC isoforms' function.

    PubMed

    De Pinto, Vito; Reina, Simona; Gupta, Ankit; Messina, Angela; Mahalakshmi, Radhakrishnan

    2016-08-01

    In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26947058

  16. Functional Networks of Highest-Connected Splice Isoforms: From The Chromosome 17 Human Proteome Project.

    PubMed

    Li, Hong-Dong; Menon, Rajasree; Govindarajoo, Brandon; Panwar, Bharat; Zhang, Yang; Omenn, Gilbert S; Guan, Yuanfang

    2015-09-01

    Alternative splicing allows a single gene to produce multiple transcript-level splice isoforms from which the translated proteins may show differences in their expression and function. Identifying the major functional or canonical isoform is important for understanding gene and protein functions. Identification and characterization of splice isoforms is a stated goal of the HUPO Human Proteome Project and of neXtProt. Multiple efforts have catalogued splice isoforms as "dominant", "principal", or "major" isoforms based on expression or evolutionary traits. In contrast, we recently proposed highest connected isoforms (HCIs) as a new class of canonical isoforms that have the strongest interactions in a functional network and revealed their significantly higher (differential) transcript-level expression compared to nonhighest connected isoforms (NCIs) regardless of tissues/cell lines in the mouse. HCIs and their expression behavior in the human remain unexplored. Here we identified HCIs for 6157 multi-isoform genes using a human isoform network that we constructed by integrating a large compendium of heterogeneous genomic data. We present examples for pairs of transcript isoforms of ABCC3, RBM34, ERBB2, and ANXA7. We found that functional networks of isoforms of the same gene can show large differences. Interestingly, differential expression between HCIs and NCIs was also observed in the human on an independent set of 940 RNA-seq samples across multiple tissues, including heart, kidney, and liver. Using proteomic data from normal human retina and placenta, we showed that HCIs are a promising indicator of expressed protein isoforms exemplified by NUDFB6 and M6PR. Furthermore, we found that a significant percentage (20%, p = 0.0003) of human and mouse HCIs are homologues, suggesting their conservation between species. Our identified HCIs expand the repertoire of canonical isoforms and are expected to facilitate studying main protein products, understanding gene

  17. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides

    SciTech Connect

    Silver, Kristopher S.; Soderlund, David M. . E-mail: dms6@cornell.edu

    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{sub v}1.2a, Na{sub v}1.4, Na{sub v}1.5, and Na{sub 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 {beta}1 auxiliary subunit on the sensitivity of the Na{sub v}1.2a and Na{sub 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{sub v}1.4 > Na{sub v}1.2a > Na{sub v}1.5 > Na{sub v}1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na{sub v}1.8 isoform was most sensitive, the Na{sub v}1.4 isoform was completely insensitive, and the sensitivities of the Na{sub v}1.5 and Na{sub 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{sub 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{sub v}1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na{sub v}1.2a or Na{sub v}1.4 isoforms with the {beta}1 subunit was the modest reduction in the sensitivity of the Na{sub v}1.2a isoform to RH 3421. These results demonstrate that mammalian sodium

  18. Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

    PubMed Central

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

    2015-01-01

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

  19. Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

    PubMed Central

    Ni, W; Trelease, R N; Eising, R

    1990-01-01

    Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1695843

  20. Profiling of Biomarkers for the Exposure of Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3, Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy Number Are Identified as Universal Biomarkers

    PubMed Central

    Kim, Hwan-Young; Kim, Hye-Ran; Trang, Nguyen Thi Dai; Baek, Hee-Jo; Moon, Jae-Dong; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook; Kook, Hoon; Shin, Myung-Geun

    2014-01-01

    This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs. PMID:25114913

  1. Biochemical abnormalities in Pearson syndrome.

    PubMed

    Crippa, Beatrice Letizia; Leon, Eyby; Calhoun, Amy; Lowichik, Amy; Pasquali, Marzia; Longo, Nicola

    2015-03-01

    Pearson marrow-pancreas syndrome is a multisystem mitochondrial disorder characterized by bone marrow failure and pancreatic insufficiency. Children who survive the severe bone marrow dysfunction in childhood develop Kearns-Sayre syndrome later in life. Here we report on four new cases with this condition and define their biochemical abnormalities. Three out of four patients presented with failure to thrive, with most of them having normal development and head size. All patients had evidence of bone marrow involvement that spontaneously improved in three out of four patients. Unique findings in our patients were acute pancreatitis (one out of four), renal Fanconi syndrome (present in all patients, but symptomatic only in one), and an unusual organic aciduria with 3-hydroxyisobutyric aciduria in one patient. Biochemical analysis indicated low levels of plasma citrulline and arginine, despite low-normal ammonia levels. Regression analysis indicated a significant correlation between each intermediate of the urea cycle and the next, except between ornithine and citrulline. This suggested that the reaction catalyzed by ornithine transcarbamylase (that converts ornithine to citrulline) might not be very efficient in patients with Pearson syndrome. In view of low-normal ammonia levels, we hypothesize that ammonia and carbamylphosphate could be diverted from the urea cycle to the synthesis of nucleotides in patients with Pearson syndrome and possibly other mitochondrial disorders. PMID:25691415

  2. Semen abnormalities with SSRI antidepressants.

    PubMed

    2015-01-01

    Despite decades of widespread use, the adverse effect profile of "selective" serotonin reuptake inhibitor (SSRI) antidepressants has still not been fully elucidated. Studies in male animals have shown delayed sexual development and reduced fertility. Three prospective cohort studies conducted in over one hundred patients exposed to an SSRI for periods ranging from 5 weeks to 24 months found altered semen param-eters after as little as 3 months of exposure: reduced sperm concentration, reduced sperm motility, a higher percentage of abnormal spermatozoa, and increased levels of sperm DNA fragmentation. One clinical trial showed growth retardation in children considered depressed who were exposed to SSRls. SSRls may have endocrine disrupting properties. Dapoxetine is a short-acting serotonin reuptake inhibitor that is chemically related to fluoxetine and marketed in the European Union for men complaining of premature ejaculation. But the corresponding European summary of product characteristics does not mention any effects on fertility. In practice, based on the data available as of mid-2014, the effects of SSRI exposure on male fertility are unclear. However, it is a risk that should be taken into account and pointed out to male patients who would like to father a child or who are experiencing fertility problems. PMID:25729824

  3. The XXXXY Sex Chromosome Abnormality

    PubMed Central

    Barr, M. L.; Carr, D. H.; Pozsonyi, J.; Wilson, R. A.; Dunn, H. G.; Jacobson, T. S.; Miller, J. R.; Chown, B.

    1962-01-01

    The most common sex chromosome complex in sex chromatin-positive males with Klinefelter's syndrome is XXY. When the complex is XXYY or XXXY, the clinical findings do not seem to differ materially from those seen in XXY subjects, although more patients with these intersexual chromosome complements need to be studied to establish possible phenotypical expressions of the chromosomal variants. Two male children with an XXXXY sex chromosome abnormality are described. The data obtained from the study of these cases and five others described in the literature suggest that the XXXXY patient is likely to have congenital defects not usually seen in the common form of the Klinefelter syndrome. These include a triad of (1) skeletal anomalies (including radioulnar synostosis), (2) hypogenitalism (hypoplasia of penis and scrotum, incomplete descent of testes and defective prepubertal development of seminiferous tubules), and (3) greater risk of severe mental deficiency. That the conclusions are based on data from a small number of patients is emphasized, together with the need for a cytogenetic survey of a large control or unselected population. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10 PMID:13969480

  4. Abnormal Mitochondrial Dynamics and Neurodegenerative Diseases

    PubMed Central

    Su, Bo; Wang, Xinglong; Zheng, Ling; Perry, George; Smith, Mark A.; Zhu, Xiongwei

    2009-01-01

    Mitochondrial dysfunction is a prominent feature of various neurodegenerative diseases. A deeper understanding of the remarkably dynamic nature of mitochondria, characterized by a delicate balance of fission and fusion, has helped to fertilize a recent wave of new studies demonstrating abnormal mitochondrial dynamics in neurodegenerative diseases. This review highlights mitochondrial dysfunction and abnormal mitochondrial dynamics in Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and Huntington disease and discusses how these abnormal mitochondrial dynamics may contribute to mitochondrial and neuronal dysfunction. We propose that abnormal mitochondrial dynamics represents a key common pathway that mediates or amplifies mitochondrial dysfunction and neuronal dysfunction during the course of neurodegeneration. PMID:19799998

  5. Chromosomal abnormalities in child psychiatric patients.

    PubMed

    Hong, K E; Kim, J H; Moon, S Y; Oh, S K

    1999-08-01

    To determine the frequency of chromosomal abnormalities in a child psychiatric population, and to evaluate possible associations between types of abnormalities and patient's clinical characteristics, cytogenetic examination was performed on 604 patients. Demographic data, reasons for karyotyping, clinical signs, and other patient characteristics were assessed and correlated with the results from karyotyping. Chromosomal abnormalities were found in 69 patients (11.3%); these were structural in 49 cases and numerical in 20. Inversion of chromosome nine was found in 15 subjects, trisomy of chromosome 21 in 11, and fragile X in five patients. When karyotyping was performed because of intellectual impairment or multiple developmental delay, significantly more abnormalities were found than average; when performed because autistic disorder was suspected, the number of abnormalities was significantly fewer. There were no differences in clinical variables between structural and numerical abnormalities, nor among nine types of chromosomal abnormalities, except that numerical abnormalities and polymorphism were found at a later age, and that walking was more delayed and IQ was lower in patients with Down syndrome. Clinicians should be aware of the possible presence of chromosomal abnormalities in child psychiatric populations; the close collaboration with geneticists and the use of more defined guidelines for cytogenetic investigation are important. PMID:10485616

  6. Radiologic atlas of pulmonary abnormalities in children

    SciTech Connect

    Singleton, E.B.; Wagner, M.L.; Dutton, R.V.

    1988-01-01

    This book is an atlas about thoracic abnormalities in infants and children. The authors include computed tomographic, digital subtraction angiographic, ultrasonographic, and a few magnetic resonance (MR) images. They recognize and discuss how changes in the medical treatment of premature infants and the management of infection and pediatric tumors have altered some of the appearances and considerations in these diseases. Oriented toward all aspects of pulmonary abnormalities, the book starts with radiographic techniques and then discusses the normal chest, the newborn, infections, tumors, and pulmonary vascular diseases. There is comprehensive treatment of mediastinal abnormalities and a discussion of airway abnormalities.

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

    PubMed Central

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

    2016-01-01

    Abstract 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. PMID:26313408

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

    PubMed

    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. PMID:26313408

  9. Explication of interactions between HMGCR isoform 2 and various statins through In silico modeling and docking.

    PubMed

    Karthik, M V K; Satya Deepak, M V K N; Shukla, Pratyoosh

    2012-02-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. This study was designed to understand the mode of interactions of HMGCR isoform 2 with other statins. Hence, ligands such as Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were docked with enzymes HMGCR isoform 1 (pdb: 1DQ8) and modeled HMGCR isoform 2 (gi|196049380). Our homology modeling results were further processed to model the structure of human HMGCR isoform 2 and its accuracy was confirmed through RMS Z-scores (1.249). These interactions revealed that binding residues such as Arg515, Asp516, Tyr517 and Asn518 are found to be conserved in HMGCR isoform 2 with various statins. Our studies further concluded that Atorvastatin is most efficient inhibitor against both the isoforms of HMGCR whereas HMGCR isoform 2 shows less effectiveness with statins when compared with HMGCR isoform 1. PMID:22177940

  10. Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

    PubMed Central

    Huppe, H. C.; Turpin, D. H.

    1996-01-01

    Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-. PMID:12226271

  11. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  12. Activation of AMPK alpha and gamma-isoform complexes in the intact ischemic rat heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic alpha-subunits and regulatory beta- and gamma-subunits with multiple isoforms. Mutations in the cardiac gamma(2)-isoform have bee...

  13. Revisiting the Identification of Canonical Splice Isoforms through Integration of Functional Genomics and Proteomics Evidence

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Omenn, Gilbert S.; Guan, Yuanfang

    2014-01-01

    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 post-translational 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 non-highest connected isoforms (NCIs) 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 highest connected isoforms 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. PMID:25265570

  14. Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

    PubMed

    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. PMID:27090236

  15. A global comparison between nuclear and cytosolic transcriptomes reveals differential compartmentalization of alternative transcript isoforms

    PubMed Central

    Chen, Liang

    2010-01-01

    Transcriptome analyses have typically disregarded nucleocytoplasmic differences. This approach has ignored some post-transcriptional regulations and their effect on the ultimate protein expression levels. Despite a longstanding interest in the differences between the nuclear and cytosolic transcriptomes, it is only recently that data have become available to study such differences and their associated features on a genome-wide scale. Here, we compared the nuclear and cytosolic transcriptomes of HepG2 and HeLa cells. HepG2 and HeLa cells vary significantly in the differential compartmentalization of their transcript isoforms, indicating that nucleocytoplasmic compartmentalization is a cell-specific characteristic. The differential compartmentalization is manifested at the transcript isoform level instead of the gene level because alternative isoforms of one gene can display different nucleocytoplasmic distributions. The isoforms enriched in the cytosol tend to have more introns and longer introns in their pre-mRNAs. They have more functional RNA folds and unique exons in the 3′ regions. These isoforms are more conserved than the isoforms enriched in the nucleus. Surprisingly, the presence of microRNAs does not have a significant impact on the nucleocytoplasmic distribution of their target isoforms. In contrast, nonsense-mediated decay is significantly more associated with the isoforms enriched in the nucleus than those enriched in the cytosol. PMID:19969546

  16. Exo70 Isoform Switching upon Epithelial-Mesenchymal Transition Mediates Cancer Cell Invasion

    PubMed Central

    Lu, Hezhe; Liu, Jianglan; Liu, Shujing; Zeng, Jingwen; Ding, Deqiang; Carstens, Russ P.; Cong, Yusheng; Xu, Xiaowei; Guo, Wei

    2014-01-01

    Summary Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2, and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoforms expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal but not the epithelial isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion. PMID:24331928

  17. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

    PubMed

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L; Matin, Angabin

    2007-03-30

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  18. Differential recruitment of PKC isoforms in HeLa cells during redox stress

    PubMed Central

    Rimessi, Alessandro; Rizzuto, Rosario; Pinton, Paolo

    2007-01-01

    The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation. PMID:18229448

  19. Pharmacological targeting of PI3K isoforms as a therapeutic strategy in chronic lymphocytic leukaemia

    PubMed Central

    Blunt, Matthew D.; Steele, Andrew J.

    2015-01-01

    PI3Kδ inhibitors such as idelalisib are providing improved therapeutic options for the treatment of chronic lymphocytic leukaemia (CLL). However under certain conditions, inhibition of a single PI3K isoform can be compensated by the other PI3K isoforms, therefore PI3K inhibitors which target multiple PI3K isoforms may provide greater efficacy. The development of compounds targeting multiple PI3K isoforms (α, β, δ, and γ) in CLL cells, in vitro, resulted in sustained inhibition of BCR signalling but with enhanced cytotoxicity and the potential for improve clinical responses. This review summarises the progress of PI3K inhibitor development and describes the rationale and potential for targeting multiple PI3K isoforms. PMID:26500849

  20. Isoforms, structures, and functions of versatile spectraplakin MACF1

    PubMed Central

    Hu, Lifang; Su, Peihong; Li, Runzhi; Yin, Chong; Zhang, Yan; Shang, Peng; Yang, Tuanmin; Qian, Airong

    2016-01-01

    Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44] PMID:26521939

  1. Quantitative isoform-profiling of highly diversified recognition molecules

    PubMed Central

    Schreiner, Dietmar; Simicevic, Jovan; Ahrné, Erik; Schmidt, Alexander; Scheiffele, Peter

    2015-01-01

    Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands. DOI: http://dx.doi.org/10.7554/eLife.07794.001 PMID:25985086

  2. Extracellular rigidity sensing by talin isoform-specific mechanical linkages.

    PubMed

    Austen, Katharina; Ringer, Pia; Mehlich, Alexander; Chrostek-Grashoff, Anna; Kluger, Carleen; Klingner, Christoph; Sabass, Benedikt; Zent, Roy; Rief, Matthias; Grashoff, Carsten

    2015-12-01

    The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton forces and bear, on average, 7-10 pN during cell adhesion depending on their association with F-actin and vinculin. Disruption of talin's mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing. PMID:26523364

  3. Differential expression of two scribble isoforms during Drosophila embryogenesis.

    PubMed

    Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

    2001-10-01

    The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones. PMID:11578873

  4. Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms.

    PubMed

    Wang, Qi; Yu, Wan-Ni; Chen, Xinyu; Peng, Xiao-Ding; Jeon, Sang-Min; Birnbaum, Morris J; Guzman, Grace; Hay, Nissim

    2016-04-11

    Akt is frequently hyperactivated in human cancers and is targeted for cancer therapy. However, the physiological consequences of systemic Akt isoform inhibition were not fully explored. We showed that while combined Akt1 and Akt3 deletion in adult mice is tolerated, combined Akt1 and Akt2 deletion induced rapid mortality. Akt2(-/-) mice survived hepatic Akt1 deletion but all developed spontaneous hepatocellular carcinoma (HCC), which is associated with FoxO-dependent liver injury and inflammation. The gene expression signature of HCC-bearing livers is similar to aggressive human HCC. Consistently, neither Akt1(-/-) nor Akt2(-/-) mice are resistant to diethylnitrosamine-induced hepatocarcinogenesis, and Akt2(-/-) mice display a high incidence of lung metastasis. Thus, in contrast to other cancers, hepatic Akt inhibition induces liver injury that could promote HCC. PMID:26996309

  5. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  6. Functional Cooperativity between ABCG4 and ABCG1 Isoforms.

    PubMed

    Hegyi, Zoltán; Homolya, László

    2016-01-01

    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. PMID:27228027

  7. Isoform-selective Inhibition of Facilitative Glucose Transporters

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

    2014-01-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  8. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  9. Functional Cooperativity between ABCG4 and ABCG1 Isoforms

    PubMed Central

    Hegyi, Zoltán

    2016-01-01

    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. PMID:27228027

  10. Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers.

    PubMed

    Tokhtaeva, Elmira; Clifford, Rebecca J; Kaplan, Jack H; Sachs, George; Vagin, Olga

    2012-07-27

    To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms. PMID:22696220

  11. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. PMID:26348913

  12. An Abnormal Psychology Community Based Interview Assignment

    ERIC Educational Resources Information Center

    White, Geoffry D.

    1977-01-01

    A course option in abnormal psychology involves students in interviewing and observing the activities of individuals in the off-campus community who are concerned with some aspect of abnormal psychology. The technique generates student interest in the field when they interview people about topics such as drug abuse, transsexualism, and abuse of…

  13. Detection of Structural Abnormalities Using Neural Nets

    NASA Technical Reports Server (NTRS)

    Zak, M.; Maccalla, A.; Daggumati, V.; Gulati, S.; Toomarian, N.

    1996-01-01

    This paper describes a feed-forward neural net approach for detection of abnormal system behavior based upon sensor data analyses. A new dynamical invariant representing structural parameters of the system is introduced in such a way that any structural abnormalities in the system behavior are detected from the corresponding changes to the invariant.

  14. Immune Abnormalities in Patients with Autism.

    ERIC Educational Resources Information Center

    Warren, Reed P.; And Others

    1986-01-01

    A study of 31 autistic patients (3-28 years old) has revealed several immune-system abnormalities, including decreased numbers of T lymphocytes and an altered ratio of helper-to-suppressor T cells. Immune-system abnormalities may be directly related to underlying biologic processes of autism or an indirect reflection of the actual pathologic…

  15. Nail abnormalities in patients with vitiligo*

    PubMed Central

    Topal, Ilteris Oguz; Gungor, Sule; Kocaturk, Ozgur Emek; Duman, Hatice; Durmuscan, Mustafa

    2016-01-01

    Background Vitiligo is an acquired pigmentary skin disorder affecting 0.1-4% of the general population. The nails may be affected in patients with an autoimmune disease such as psoriasis, and in those with alopecia areata. It has been suggested that nail abnormalities should be apparent in vitiligo patients. Objective We sought to document the frequency and clinical presentation of nail abnormalities in vitiligo patients compared to healthy volunteers. We also examined the correlations between nail abnormalities and various clinical parameters. Methods This study included 100 vitiligo patients and 100 healthy subjects. Full medical histories were collected from the subjects, who underwent thorough general and nail examinations. All nail changes were noted. In the event of clinical suspicion of a fungal infection, additional mycological investigations were performed. Results Nail abnormalities were more prevalent in the patients (78%) than in the controls (55%) (p=0.001). Longitudinal ridging was the most common finding (42%), followed by (in descending order): leukonychia, an absent lunula, onycholysis, nail bed pallor, onychomycosis, splinter hemorrhage and nail plate thinning. The frequency of longitudinal ridging was significantly higher in patients than in controls (p<0.001). Conclusions Nail abnormalities were more prevalent in vitiligo patients than in controls. Systematic examination of the nails in such patients is useful because nail abnormalities are frequent. However, the causes of such abnormalities require further study. Longitudinal ridging and leukonychia were the most common abnormalities observed in this study. PMID:27579738

  16. Structural and functional differences between KRIT1A and KRIT1B isoforms: A framework for understanding CCM pathogenesis

    SciTech Connect

    Francalanci, Floriana; Avolio, Maria; De Luca, Elisa; Longo, Dario; Menchise, Valeria; Guazzi, Paolo; Sgro, Francesco; Marino, Marco; Goitre, Luca; Balzac, Fiorella; Trabalzini, Lorenza; Retta, Saverio Francesco

    2009-01-15

    KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal {beta}-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

  17. [Abnormality in bone metabolism after burn].

    PubMed

    Gong, X; Xie, W G

    2016-08-20

    Burn causes bone metabolic abnormality in most cases, including the changes in osteoblasts and osteoclasts, bone mass loss, and bone absorption, which results in decreased bone mineral density. These changes are sustainable for many years after burn and even cause growth retardation in burned children. The mechanisms of bone metabolic abnormality after burn include the increasing glucocorticoids due to stress response, a variety of cytokines and inflammatory medium due to inflammatory response, vitamin D deficiency, hypoparathyroidism, and bone loss due to long-term lying in bed. This article reviews the pathogenesis and regularity of bone metabolic abnormality after burn, the relationship between bone metabolic abnormality and burn area/depth, and the treatment of bone metabolic abnormality, etc. and discusses the research directions in the future. PMID:27562160

  18. Dysregulation of miRNA isoform level at 5' end in Alzheimer's disease.

    PubMed

    Wang, Shengqin; Xu, Yuming; Li, Musheng; Tu, Jing; Lu, Zuhong

    2016-06-15

    Alzheimer's disease (AD) is the most common form of dementia, whose mechanism is still not yet fully understood. A miRNA-based signature method, commonly according to the changes of expression levels, is widely used for AD analysis in previous studies. Recently, miRNA isoforms called as isomiR variants, which is considered to play important biological roles, have been demonstrated as the applications of high throughput sequencing platforms. Here, we presented an entropy-based model to detect the miRNA isoform level at the 5' end, and found many miRNAs with significant changes of isoform levels between the early stage and the late stage of AD by the application of this model to the public data. The statistical significance of the overlap between isoform-level changed miRNAs and AD related miRNAs extracted from HMDD2 supports that these miRNA isoforms are not degradation products. Based on the most common isomiR seed analysis of isoform-level changed AD related miRNAs, the predicted targets are also found to be enriched for genes involved in transcriptional regulation and the nervous system. After comparing with the expression level based method, we detected that changes of 5' isoform levels are more stable than those of expression levels for AD related miRNA detecting. PMID:26899870

  19. The isolation of parvalbumin isoforms from the tail muscle of the American alligator (Alligator mississipiensis).

    PubMed

    Laney, E L; Shabanowitz, J; King, G; Hunt, D F; Nelson, D J

    1997-04-01

    Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species). PMID:9076974

  20. Cloning and Characterisation of Multiple Ferritin Isoforms in the Atlantic Salmon (Salmo salar)

    PubMed Central

    Lee, Jun-Hoe; Pooley, Nicholas J.; Mohd-Adnan, Adura; Martin, Samuel A. M.

    2014-01-01

    Ferritin is a highly-conserved iron-storage protein that has also been identified as an acute phase protein within the innate immune system. The iron-storage function is mediated through complementary roles played by heavy (H)-chain subunit as well as the light (L) in mammals or middle (M)-chain in teleosts, respectively. In this study, we report the identification of five ferritin subunits (H1, H2, M1, M2, M3) in the Atlantic salmon that were supported by the presence of iron-regulatory regions, gene structure, conserved domains and phylogenetic analysis. Tissue distribution analysis across eight different tissues showed that each of these isoforms is differentially expressed. We also examined the expression of the ferritin isoforms in the liver and kidney of juvenile Atlantic salmon that was challenged with Aeromonas salmonicida as well as in muscle cell culture stimulated with interleukin-1β. We found that each isoform displayed unique expression profiles, and in certain conditions the expressions between the isoforms were completely diametrical to each other. Our study is the first report of multiple ferritin isoforms from both the H- and M-chains in a vertebrate species, as well as ferritin isoforms that showed decreased expression in response to infection. Taken together, the results of our study suggest the possibility of functional differences between the H- and M-chain isoforms in terms of tissue localisation, transcriptional response to bacterial exposure and stimulation by specific immune factors. PMID:25078784

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

    PubMed Central

    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

  2. Identification of Cardiac Myofilament Protein Isoforms Using Multiple Mass Spectrometry Based Approaches

    PubMed Central

    Kirk, Jonathan A.; Ubaida-Mohien, Ceereena; Graham, David R.; Faber, Matthijs J.; Van Eyk, Jennifer E.

    2014-01-01

    Purpose The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (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. Experimental design Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis was performed by MASPECTRAS. Results The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and sub-isoforms. Conclusion and clinical relevance We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets. PMID:24974818

  3. CK-MB isoforms for early risk stratification of emergency department patients.

    PubMed

    Green, G B; Dehlinger, E; McGrievey, T S; Li, D J; Jones, K A; Kelen, G D; Chan, D W

    2000-10-01

    The potential clinical utility of single sample CK-MB isoforms measurement for early risk stratification of Emergency Department (ED) patients with possible myocardial ischemia was evaluated among 405 patients presenting to two urban EDs. Clinical and serologic data were prospectively collected and the occurrence of adverse events (AEs) and myocardial infarction (MI) during the 14-day outcome period was recorded and utilized to calculate and compare relative risks (RR) and predictive values of isoforms and CK-MB alone. Among the 405 patients, 67 accrued 105 AEs. Both isoforms and CK-MB alone were predictive of AEs with RR of 3.32 (2.09, 5.27) and 6.28 (4.64, 8.52), respectively. Isoforms had higher sensitivity for AEs compared to CK-MB (65.7% [54.3, 77.0] vs. 14.9% [6.4, 23.5]; p<0. 01) but lower specificity (69.2% [64.3, 74.2] vs. 99.7% [99.1,100. 0]; p<0.01). Isoforms' superior sensitivity allowed identification of many high risk patients missed by CK-MB alone. Further, for the prediction of MI, isoforms had superior diagnostic sensitivity and equivalent specificity. This investigation supports the emergency department use of early, single sample CK-MB isoform testing. PMID:10958863

  4. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

    PubMed Central

    Zmora, Pawel; Moldenhauer, Anna-Sophie; Hofmann-Winkler, Heike; Pöhlmann, Stefan

    2015-01-01

    The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host. PMID:26379044

  5. Diversification of importin-α isoforms in cellular trafficking and disease states

    PubMed Central

    Pumroy, Ruth A.; Cingolani, Gino

    2015-01-01

    The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases. PMID:25656054

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

    PubMed

    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

    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

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

    PubMed

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

    2013-01-01

    Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. PMID:23152195

  8. Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

    PubMed Central

    Schweinhuber, Stefanie K.; Meßerschmidt, Tania; Hänsch, Robert; Korte, Martin; Rothkegel, Martin

    2015-01-01

    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 isoform

  9. Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

    PubMed

    Luttgeharm, Kyle D; Cahoon, Edgar B; Markham, Jonathan E

    2016-03-01

    Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism. PMID:26635357

  10. NSMAP: A method for spliced isoforms identification and quantification from RNA-Seq

    PubMed Central

    2011-01-01

    Background The development of techniques for sequencing the messenger RNA (RNA-Seq) enables it to study the biological mechanisms such as alternative splicing and gene expression regulation more deeply and accurately. Most existing methods employ RNA-Seq to quantify the expression levels of already annotated isoforms from the reference genome. However, the current reference genome is very incomplete due to the complexity of the transcriptome which hiders the comprehensive investigation of transcriptome using RNA-Seq. Novel study on isoform inference and estimation purely from RNA-Seq without annotation information is desirable. Results A Nonnegativity and Sparsity constrained Maximum APosteriori (NSMAP) model has been proposed to estimate the expression levels of isoforms from RNA-Seq data without the annotation information. In contrast to previous methods, NSMAP performs identification of the structures of expressed isoforms and estimation of the expression levels of those expressed isoforms simultaneously, which enables better identification of isoforms. In the simulations parameterized by two real RNA-Seq data sets, more than 77% expressed isoforms are correctly identified and quantified. Then, we apply NSMAP on two RNA-Seq data sets of myelodysplastic syndromes (MDS) samples and one normal sample in order to identify differentially expressed known and novel isoforms in MDS disease. Conclusions NSMAP provides a good strategy to identify and quantify novel isoforms without the knowledge of annotated reference genome which can further realize the potential of RNA-Seq technique in transcriptome analysis. NSMAP package is freely available at https://sites.google.com/site/nsmapforrnaseq. PMID:21575225

  11. Complex Interplay of the UL136 Isoforms Balances Cytomegalovirus Replication and Latency

    PubMed Central

    Caviness, Katie; Bughio, Farah; Crawford, Lindsey B.; Streblow, Daniel N.; Nelson, Jay A.; Caposio, Patrizia

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+ hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγcnull humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+ HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection. PMID:26933055

  12. PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation.

    PubMed

    Hands, Katherine J; Cuchet-Lourenco, Delphine; Everett, Roger D; Hay, Ronald T

    2014-01-15

    Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment. PMID:24190887

  13. Sleep physiology, abnormal States, and therapeutic interventions.

    PubMed

    Wickboldt, Alvah T; Bowen, Alex F; Kaye, Aaron J; Kaye, Adam M; Rivera Bueno, Franklin; Kaye, Alan D

    2012-01-01

    Sleep is essential. Unfortunately, a significant portion of the population experiences altered sleep states that often result in a multitude of health-related issues. The regulation of sleep and sleep-wake cycles is an area of intense research, and many options for treatment are available. The following review summarizes the current understanding of normal and abnormal sleep-related conditions and the available treatment options. All clinicians managing patients must recommend appropriate therapeutic interventions for abnormal sleep states. Clinicians' solid understanding of sleep physiology, abnormal sleep states, and treatments will greatly benefit patients regardless of their disease process. PMID:22778676

  14. Sleep Physiology, Abnormal States, and Therapeutic Interventions

    PubMed Central

    Wickboldt, Alvah T.; Bowen, Alex F.; Kaye, Aaron J.; Kaye, Adam M.; Rivera Bueno, Franklin; Kaye, Alan D.

    2012-01-01

    Sleep is essential. Unfortunately, a significant portion of the population experiences altered sleep states that often result in a multitude of health-related issues. The regulation of sleep and sleep-wake cycles is an area of intense research, and many options for treatment are available. The following review summarizes the current understanding of normal and abnormal sleep-related conditions and the available treatment options. All clinicians managing patients must recommend appropriate therapeutic interventions for abnormal sleep states. Clinicians' solid understanding of sleep physiology, abnormal sleep states, and treatments will greatly benefit patients regardless of their disease process. PMID:22778676

  15. Right Liver Lobe Hypoplasia and Related Abnormalities

    PubMed Central

    Alicioglu, Banu

    2015-01-01

    Summary Background Hypoplasia and agenesis of the liver lobe is a rare abnormality. It is associated with biliary system abnormalities, high location of the right kidney, and right colon interposition. These patients are prone to gallstones, portal hypertension and possible surgical complications because of anatomical disturbance. Case Report Magnetic resonance imaging features of a rare case of hypoplasia of the right lobe of the liver in a sigmoid cancer patient are presented. Conclusions Hypoplasia of the right liver should not be confused with liver atrophy; indeed, associations with other coexistent abnormalities are also possible. Awareness and familiarity with these anomalies are necessary to avoid fatal surgical and interventional complications. PMID:26634012

  16. Numerically abnormal chromosome constitutions in humans

    SciTech Connect

    1993-12-31

    Chapter 24, discusses numerically abnormal chromosome constitutions in humans. This involves abnormalities of human chromosome number, including polyploidy (when the number of sets of chromosomes increases) and aneuploidy (when the number of individual normal chromosomes changes). Chapter sections discuss the following chromosomal abnormalities: human triploids, imprinting and uniparental disomy, human tetraploids, hydatidiform moles, anomalies caused by chromosomal imbalance, 13 trisomy (D{sub 1} trisomy, Patau syndrome), 21 trisomy (Down syndrome), 18 trisomy syndrome (Edwards syndrome), other autosomal aneuploidy syndromes, and spontaneous abortions. The chapter concludes with remarks on the nonrandom participation of chromosomes in trisomy. 69 refs., 3 figs., 4 tabs.

  17. An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability

    PubMed Central

    Lima-Cabello, Elena; Garcia-Guirado, Francisco; Calvo-Medina, Rocio; el Bekay, Rajaa; Perez-Costillas, Lucia; Quintero-Navarro, Carolina; Sanchez-Salido, Lourdes

    2016-01-01

    Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome. PMID:26788253

  18. Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome.

    PubMed

    Krasnoselsky, Alexei L; Faca, Vitor M; Pitteri, Sharon J; Zhang, Qing; Hanash, Samir M

    2008-06-01

    We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states. PMID:18419151

  19. A novel alternative splicing isoform of NF2 identified in human Schwann cells

    PubMed Central

    Su, Fang; Zhou, Zhengguang; Su, Wen; Wang, Zishu; Wu, Qiong

    2016-01-01

    Vestibular schwannoma (VS) is a benign, slow-growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full-length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full-length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI-193 cell line and cell proliferation assays were performed using Cell Counting Kit-8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3′-end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild-type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein

  20. Nitric oxide synthases activation and inhibition by metallacarborane cluster-based isoform-specific affectors

    PubMed Central

    Kaplánek, Robert; Martásek, Pavel; Grüner, Bohumír; Panda, Satya; Rak, Jakub; Masters, Bettie Sue Siler; Král, Vladimír; Roman, Linda J.

    2012-01-01

    A small library of boron cluster and metallacarborane cluster-based ligands was designed, prepared and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. Based on the concept of creating a hydrophobic analog of a natural substrate, a stable and non-toxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and non-classical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms. PMID:23075390

  1. Application of denaturing gradient gel electrophoresis to detect DNA sequence differences encoding apolipoprotein E isoforms

    SciTech Connect

    Parker, S.; Angelico, M.C.; Laffel, L.; Krolewski, A.S. Harvard Medical School, Boston, MA )

    1993-04-01

    Apolipoprotein E (apoE) plays an important role in plasma lipid metabolism. Three common isoforms of this protein have been identified by the isoelectric focusing method. In this report the authors describe a new method for distinguishing these isoforms. Their method employs PCR amplification of the DNA sequence of exon 4 in the apoE gene followed by denaturing gradient gel electrophoresis (DGGE) to distinguish its different melting characteristics. Identification of the ApoE isoforms through DNA melting behavior rather than protein charge differences eliminates the problems associated with isoelectric focusing and facilitates screening for additional mutations at the apoE locus. 12 refs., 2 figs.

  2. Altered Striatal Synaptic Function and Abnormal Behaviour in Shank3 Exon4-9 Deletion Mouse Model of Autism.

    PubMed

    Jaramillo, Thomas C; Speed, Haley E; Xuan, Zhong; Reimers, Jeremy M; Liu, Shunan; Powell, Craig M

    2016-03-01

    Shank3 is a multi-domain, synaptic scaffolding protein that organizes proteins in the postsynaptic density of excitatory synapses. Clinical studies suggest that ∼ 0.5% of autism spectrum disorder (ASD) cases may involve SHANK3 mutation/deletion. Patients with SHANK3 mutations exhibit deficits in cognition along with delayed/impaired speech/language and repetitive and obsessive/compulsive-like (OCD-like) behaviors. To examine how mutation/deletion of SHANK3 might alter brain function leading to ASD, we have independently created mice with deletion of Shank3 exons 4-9, a region implicated in ASD patients. We find that homozygous deletion of exons 4-9 (Shank3(e4-9) KO) results in loss of the two highest molecular weight isoforms of Shank3 and a significant reduction in other isoforms. Behaviorally, both Shank3(e4-9) heterozygous (HET) and Shank3(e4-9) KO mice display increased repetitive grooming, deficits in novel and spatial object recognition learning and memory, and abnormal ultrasonic vocalizations. Shank3(e4-9) KO mice also display abnormal social interaction when paired with one another. Analysis of synaptosome fractions from striata of Shank3(e4-9) KO mice reveals decreased Homer1b/c, GluA2, and GluA3 expression. Both Shank3(e4-9) HET and KO demonstrated a significant reduction in NMDA/AMPA ratio at excitatory synapses onto striatal medium spiny neurons. Furthermore, Shank3(e4-9) KO mice displayed reduced hippocampal LTP despite normal baseline synaptic transmission. Collectively these behavioral, biochemical and physiological changes suggest Shank3 isoforms have region-specific roles in regulation of AMPAR subunit localization and NMDAR function in the Shank3(e4-9) mutant mouse model of autism. PMID:26559786

  3. Low-set ears and pinna abnormalities

    MedlinePlus

    Low-set ears; Microtia; "Lop" ear; Pinna abnormalities; Genetic defect-pinna; Congenital defect-pinna ... The outer ear or "pinna" forms when the baby is growing in the mother's womb. The growth of this ear part ...

  4. Pinna abnormalities and low-set ears

    MedlinePlus

    ... because they do not affect hearing. However, sometimes cosmetic surgery is recommended. Skin tags may be tied off, ... 5 years old. More severe abnormalities may require surgery for cosmetic reasons as well as for function. Surgery to ...

  5. Abnormal Uterine Bleeding (Beyond the Basics)

    MedlinePlus

    ... Approach to abnormal uterine bleeding in nonpregnant reproductive-age women Differential diagnosis of genital tract bleeding in women Postmenopausal uterine bleeding The following organizations also provide reliable health information. ● National Library of Medicine ( www.nlm.nih.gov/ ...

  6. Spontaneous occurrence of chromosome abnormality in cats.

    PubMed

    THULINE, H C; NORBY, D W

    1961-08-25

    A syndrome in male cats analogous to chromatin-positive Klinefelter's syndrome in human males has been demonstrated. The physical characteristics which suggested an abnormality of chromosome number in cats were "calico" or "tortoise-shell" coat colors in a male. Buccal mucosal smears were found to have "female-type" patterns in two out of 12 such male cats screened, and these two were found to have a diploid chromosome number of 39 rather than the normal 38. Testicular biopsy performed on one revealed an abnormal pattern; no gonadal tissue was found in the other cat with an abnormal chromosome number. These findings indicate that the cat, in addition to the mouse, is available for experimental study of chromosome number abnormalities. PMID:13776765

  7. Abnormal brain scan with subacute extradural haematomas

    PubMed Central

    Morley, J. Barrie; Langford, Keith H.

    1970-01-01

    Four patients are described with proven subacute extradural haematomas, each with an abnormal cerebral scan of diagnostic assistance. A possible mechanism of production of the subacute extradural haematoma is discussed, and appears to be similar to the mechanism involved in the subacute subdural haematoma. The means by which the abnormal scan results in such cases is also examined, from which it appears that non-specific meningeal membrane inflammatory reaction surrounding the haematoma is significant. Images PMID:5478950

  8. Prevalence of asymptomatic urinary abnormalities among adolescents.

    PubMed

    Fouad, Mohamed; Boraie, Maher

    2016-05-01

    To determine the prevalence of asymptomatic urinary abnormalities in adolescents, first morning clean mid-stream urine specimens were obtained from 2500 individuals and examined by dipstick and light microscopy. Adolescents with abnormal screening results were reexamined after two weeks and those who had abnormal results twice were subjected to systemic clinical examination and further clinical and laboratory investigations. Eight hundred and three (32.1%) individuals had urinary abnormalities at the first screening, which significantly decreased to 345 (13.8%) at the second screening, (P <0.001). Hematuria was the most common urinary abnormalities detected in 245 (9.8%) adolescents who had persistent urine abnormalities; 228 (9.1%) individuals had non glomerular hematuria. The hematuria was isolated in 150 (6%) individuals, combined with leukocyturia in 83 (3.3%) individuals, and combined with proteinuria in 12 (0.5%) individuals. Leukocyturia was detected in 150 (6%) of all studied adolescents; it was isolated in 39 (1.6%) individuals and combined with proteinuria in 28 (1.1%) of them. Asymptomatic bacteriuria was detected in 23 (0.9%) of all studied adolescents; all the cases were females. Proteinuria was detected in 65 (2.6%) of all the studied adolescents; 45 (1.8%) individuals had <0.5 g/day and twenty (0.8%) individuals had 0.5-3 g/day. Asymptomatic urinary abnormalities were more common in males than females and adolescents from rural than urban areas (P <0.01) and (P <0.001), respectively. The present study found a high prevalence of asymptomatic urinary abnormalities among adolescents in our population. PMID:27215241

  9. Ndt80 activates the meiotic ORC1 transcript isoform and SMA2 via a bi-directional middle sporulation element in Saccharomyces cerevisiae.

    PubMed

    Xie, Bingning; Horecka, Joe; Chu, Angela; Davis, Ronald W; Becker, Emmanuelle; Primig, Michael

    2016-09-01

    The origin of replication complex subunit ORC1 is important for DNA replication. The gene is known to encode a meiotic transcript isoform (mORC1) with an extended 5'-untranslated region (5'-UTR), which was predicted to inhibit protein translation. However, the regulatory mechanism that controls the mORC1 transcript isoform is unknown and no molecular biological evidence for a role of mORC1 in negatively regulating Orc1 protein during gametogenesis is available. By interpreting RNA profiling data obtained with growing and sporulating diploid cells, mitotic haploid cells, and a starving diploid control strain, we determined that mORC1 is a middle meiotic transcript isoform. Regulatory motif predictions and genetic experiments reveal that the activator Ndt80 and its middle sporulation element (MSE) target motif are required for the full induction of mORC1 and the divergently transcribed meiotic SMA2 locus. Furthermore, we find that the MSE-binding negative regulator Sum1 represses both mORC1 and SMA2 during mitotic growth. Finally, we demonstrate that an MSE deletion strain, which cannot induce mORC1, contains abnormally high Orc1 levels during post-meiotic stages of gametogenesis. Our results reveal the regulatory mechanism that controls mORC1, highlighting a novel developmental stage-specific role for the MSE element in bi-directional mORC1/SMA2 gene activation, and correlating mORC1 induction with declining Orc1 protein levels. Because eukaryotic genes frequently encode multiple transcripts possessing 5'-UTRs of variable length, our results are likely relevant for gene expression during development and disease in higher eukaryotes. PMID:27362276

  10. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity

    PubMed Central

    2015-01-01

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure–activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes. PMID:26555042

  11. Autophagosome-associated variant isoforms of cytosolic enzymes.

    PubMed Central

    Fengsrud, M; Raiborg, C; Berg, T O; Strømhaug, P E; Ueno, T; Erichsen, E S; Seglen, P O

    2000-01-01

    In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31 kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase. PMID:11104685

  12. RAS isoforms and mutations in cancer at a glance.

    PubMed

    Hobbs, G Aaron; Der, Channing J; Rossman, Kent L

    2016-04-01

    RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts. PMID:26985062

  13. Splice isoform estrogen receptors as integral transmembrane proteins

    PubMed Central

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R.

    2011-01-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus–truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element–luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets. PMID:21937726

  14. Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

    PubMed Central

    Rouet-Mayer, Marie-Aude; Bureau, Jean-Marc; Laurière, Christiane

    1992-01-01

    A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents. ImagesFigure 1 PMID:16668773

  15. Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms.

    PubMed

    Viollet, B; Lefrançois-Martinez, A M; Henrion, A; Kahn, A; Raymondjean, M; Martinez, A

    1996-01-19

    The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter. PMID:8576131

  16. Abnormal ferrite in hyper-eutectoid steels

    SciTech Connect

    Chairuangsri, T.; Edmonds, D.V.

    2000-04-19

    The microstructural characteristics of ultra-high carbon hyper-eutectoid Fe-C and Fe-C-Cu experimental steels have been examined after isothermal transformation in a range just beneath the eutectoid temperature. Particular attention was paid to the formation of so-called abnormal ferrite, which refers to coarse ferrite grains which can form, in hyper-eutectoid compositions, on the pro-eutectoid cementite before the pearlite reaction occurs. Thus it is confirmed that the abnormal ferrite is not a result of pearlite coarsening, but of austenite decomposition before the conditions for coupled growth of pearlite are established. The abnormal ferrite formed on both allotriomorphic and Widmanstaetten forms of pro-eutectoid cementite, and significantly, it was observed that the pro-eutectoid cementite continued to grow, despite being enclosed by the abnormal ferrite. Under certain conditions this could lead to the eventual formation of substantially reduced amounts of pearlite. Thus, a model for carbon redistribution that allows the proeutectoid cementite to thicken concurrently with the abnormal ferrite is presented. The orientation relationships between the abnormal ferrite and pro-eutectoid cementite were also determined and found to be close to those which have been reported between pearlitic ferrite and pearlitic cementite.

  17. Microheterogeneity of antithrombin III: effect of single amino acid substitutions and relationship with functional abnormalities.

    PubMed

    De Stefano, V; Leone, G; Mastrangelo, S; Lane, D A; Girolami, A; de Moerloose, P; Sas, G; Abildgaard, U; Blajchman, M; Rodeghiero, F

    1994-02-01

    Microheterogeneity of antithrombin III (AT-III) was investigated by crossed immunoelectrofocusing (CIEF) on eleven molecular variants. A normal pattern was found in five variants while two different abnormal CIEF patterns were found in the other four and two variants, respectively. Point mutations causing a major pI change (exceeding 4.0) of the amino acid substituted lead to alterations in the overall microheterogeneity. The variants thus substituted share a first type of abnormal CIEF pattern with alterations throughout the pH range, regardless of the location of the mutation (reactive site and adjacent regions or heparin binding region). Minor amino acid pI changes in these regions do not alter the AT-III overall microheterogeneity, whatever the resulting functional defect. However, if the mutation is placed in the region around positions 404 or 429, then even minor changes of the amino acid pI seem able to alter the overall charge, leading to a second type of abnormal CIEF pattern with the main alteration at pH 4.8-4.6. Neuraminidase treatment leads to disappearance of microheterogeneity except for the variants with the Arg393 to Cys substitution. Addition of thrombin induces CIEF modifications specifically related to the functional defect. A normal formation of thrombin-antithrombin complexes induces a shift towards the more acid pH range, whereas in the variants substituted at the reactive site the CIEF pattern is substantially unaffected by thrombin; variants substituted at positions 382-384 show a maximal thrombin-induced increase of the isoforms at pI 4.8-4.6. Therefore mutant antithrombins with different functional abnormalities but sharing a common CIEF pattern were well distinguished.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8180341

  18. A Synaptotagmin Isoform Switch during the Development of an Identified CNS Synapse.

    PubMed

    Kochubey, Olexiy; Babai, Norbert; Schneggenburger, Ralf

    2016-06-01

    Various Synaptotagmin (Syt) isoform genes are found in mammals, but it is unknown whether Syts can function redundantly in a given nerve terminal, or whether isoforms can be switched during the development of a nerve terminal. Here, we investigated the possibility of a developmental Syt isoform switch using the calyx of Held as a model synapse. At mature calyx synapses, fast Ca(2+)-driven transmitter release depended entirely on Syt2, but the release phenotype of Syt2 knockout (KO) mice was weaker at immature calyces, and absent at pre-calyceal synapses early postnatally. Instead, conditional genetic inactivation shows that Syt1 mediates fast release at pre-calyceal synapses, as well as a fast release component resistant to Syt2 deletion in immature calyces. This demonstrates a developmental Syt1-Syt2 isoform switch at an identified synapse, a mechanism that could fine-tune the speed, reliability, and plasticity of transmitter release at fast releasing CNS synapses. PMID:27210552

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  20. Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook.

    PubMed Central

    Hawkins, S. W.; Boudet, A. M.

    1994-01-01

    Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purified from the periderm (containing both suberized and lignified cell layers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P) were initially characterized, and the major form, CAD 2P, was resolved into three further isoforms by ion-exchange chromatography. Crude extracts contained two aliphatic alcohol dehydrogenases (ADH) and one aromatic ADH, which was later resolved into two further isoforms. Aliphatic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as substrates but with a much lower specific activity when compared with benzyl alcohol. The minor form, CAD 1P, was a monomer with a molecular weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis demonstrated that this protein was very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 and was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially combined to give the heterodimer and two homodimers. SDS-PAGE, western blots, and nondenaturing PAGE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our laboratory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Joffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indicated that the different CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin. PMID:12232063

  1. Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.

    PubMed

    Mok, Yee-Foong; Lin, Feng-Hsu; Graham, Laurie A; Celik, Yeliz; Braslavsky, Ido; Davies, Peter L

    2010-03-23

    The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP. PMID:20158269

  2. Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

    PubMed Central

    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. PMID:25335123

  3. Differential distribution of aggrecan isoforms in perineuronal nets of the human cerebral cortex

    PubMed Central

    Virgintino, Daniela; Perissinotto, Daniela; Girolamo, Francesco; Mucignat, Maria T; Montanini, Luisa; Errede, Mariella; Kaneiwa, Tomoyuki; Yamada, Shushei; Sugahara, Kazuyuki; Roncali, Luisa; Perris, Roberto

    2009-01-01

    Aggrecan is a component of the CNS extracellular matrix (ECM) and we show here that the three primary alternative spliced transcripts of the aggrecan gene found in cartilage are also present in the adult CNS. Using a unique panel of core protein-directed antibodies against human aggrecan we further show that different aggrecan isoforms are deposited in perineuronal nets (PNNs) and neuropil ECM of Brodmann’s area 6 of the human adult cerebral cortex. According to their distribution pattern, the identified cortical aggrecan isoforms were subdivided into five clusters spanning from cluster 1, comprised isoforms that appeared widespread throughout the cortex, to cluster 5, which was an aggrecan-free subset. Comparison of brain and cartilage tissues showed a different relative abundance of aggrecan isoforms, with cartilage-specific isoforms characterizing cluster 5, and PNN-associated isoforms lacking keratan sulphate chains. In the brain, isoforms of cluster 1 were disclosed in PNNs surrounding small-medium interneurons of layers II–V, small-medium pyramidal neurons of layers III and V and large interneurons of layer VI. Aggrecan PNNs enveloped both neuron bodies and neuronal processes, encompassing pre-terminal nerve fibres, synaptic boutons and terminal processes of glial cells and aggrecan was also observed in continuous ‘coats’ associated with satellite, neuron-associated cells of a putative glial nature. Immunolabelling for calcium-binding proteins and glutamate demonstrated that aggrecan PNNs were linked to defined subsets of cortical interneurons and pyramidal cells. We suggest that in the human cerebral cortex, discrete, layer-specific PNNs are assembled through the participation of selected aggrecan isoforms that characterize defined subsets of cortical neurons. PMID:19220578

  4. Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues.

    PubMed

    Li, Shuijie; Lamarche, Fredéric; Charton, Romain; Delphin, Christian; Gires, Olivier; Hubstenberger, Arnaud; Schlattner, Uwe; Rousseau, Denis

    2014-02-01

    ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human. Considering these observations, we propose the development of a uniform denomination for ATAD3 isoforms in rodent and human. PMID:24239551

  5. Sodium pump isoform specificity for the digitalis-like factor isolated from human peritoneal dialysate.

    PubMed

    Tao, Q F; Hollenberg, N K; Price, D A; Graves, S W

    1997-03-01

    We have isolated a labile, specific sodium pump inhibitor or digitalis-like factor from the peritoneal dialysate of volume-expanded renal failure patients whose levels correlated closely with volume status and blood pressure. This study characterizes the inhibitory profile of this agent compared with that of ouabain against the three alpha-isoforms of the sodium pump. We prepared microsomal Na,K-ATPase from rat tissues representing the highest proportion of one of the alpha-isoforms. Both Northern and Western blot analyses confirmed that kidney had predominantly the alpha1-isoform, skeletal muscle the alpha2-isoform, and fetal brain the alpha3-isoform. Ouabain (5 x 10(-6) mol/L) produced little inhibition of kidney Na,K-ATPase (3.4+/-2.0%) but significant inhibition of skeletal muscle (37.2+/-3.7%, P<.001) and fetal brain (38.8+/-3.5%, P<.001) activity. In contrast, the labile digitalis-like factor, causing comparable inhibition of fetal brain Na,K-ATPase activity (33.3+/-4.7%), produced markedly greater inhibition of kidney (42.5+/-5.6%, P<.001) and moderately greater inhibition of skeletal muscle pump activity (57.7+/-6.3%, P<.05). In addition, the labile digitalis-like factor produced a marked concentration-dependent inhibition of the alpha2- and alpha3-isoforms (r=.79, P=.00005). Experiments combining the labile digitalis-like factor and ouabain confirmed that digitalis-like factor, unlike ouabain, was an effective inhibitor of all three isoforms in rat, in particular alpha2. The different pattern of isoform sensitivity displayed by the labile digitalis-like factor and ouabain further differentiates the two agents and raises some interesting possibilities about the functional implications of the endogenous factor. PMID:9052901

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

    PubMed Central

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-01-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

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

    PubMed

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-12-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

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

    PubMed

    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. PMID:24671242

  9. Tau isoform regulation is region- and cell-specific in mouse brain.

    PubMed

    McMillan, Pamela; Korvatska, Elena; Poorkaj, Parvoneh; Evstafjeva, Zana; Robinson, Linda; Greenup, Lynne; Leverenz, James; Schellenberg, Gerard D; D'Souza, Ian

    2008-12-20

    Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, which are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R- and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wildtype mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R-tau was more "synaptic like," with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression. PMID:18925637

  10. Tau isoform regulation is region and cell-specific in mouse brain

    PubMed Central

    McMillan, Pamela; Korvatska, Elena; Poorkaj, Parvoneh; Evstafjeva, Zana; Robinson, Linda; Greenup, Lynne; Leverenz, James; Schellenberg, Gerard D.; D’Souza, Ian

    2008-01-01

    Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, that are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wild-type mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R tau was more “synaptic like”, with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression. PMID:18925637

  11. A novel tropomyosin isoform functions at the mitotic spindle and Golgi in Drosophila

    PubMed Central

    Goins, Lauren M.; Mullins, R. Dyche

    2015-01-01

    Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Only one nonmuscle tropomyosin (Tm1A) has previously been described in Drosophila, but developmental defects caused by insertion of P-elements near tropomyosin genes imply the existence of additional, nonmuscle isoforms. Using biochemical and molecular genetic approaches, we identified three tropomyosins expressed in Drosophila S2 cells: Tm1A, Tm1J, and Tm2A. The Tm1A isoform localizes to the cell cortex, lamellar actin networks, and the cleavage furrow of dividing cells—always together with myosin-II. Isoforms Tm1J and Tm2A colocalize around the Golgi apparatus with the formin-family protein Diaphanous, and loss of either isoform perturbs cell cycle progression. During mitosis, Tm1J localizes to the mitotic spindle, where it promotes chromosome segregation. Using chimeras, we identified the determinants of tropomyosin localization near the C-terminus. This work 1) identifies and characterizes previously unknown nonmuscle tropomyosins in Drosophila, 2) reveals a function for tropomyosin in the mitotic spindle, and 3) uncovers sequence elements that specify isoform-specific localizations and functions of tropomyosin. PMID:25971803

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

    SciTech Connect

    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.

  13. FTICR-MS analysis of 14-3-3 isoform substrate selection.

    PubMed

    Cardasis, Helene L; Sehnke, Paul C; Laughner, Beth; Eyler, John R; Powell, David H; Ferl, Robert J

    2007-07-01

    The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client. PMID:17569603

  14. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  15. PECAM-1 isoform-specific functions in PECAM-1-deficient brain microvascular endothelial cells.

    PubMed

    DiMaio, Terri A; Sheibani, Nader

    2008-03-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) is alternatively spliced generating eight isoforms that only differ in the length of their cytoplasmic domain. Multiple isoforms of PECAM-1 are present in the endothelium and their expression levels are regulated during vascular development and angiogenesis. However, the functional significance of PECAM-1 isoforms during these processes remains largely unknown. We recently showed that mouse brain endothelial (bEND) cells prepared from PECAM-1-deficient (PECAM-1-/-) mice differ in their cell adhesive and migratory properties compared to PECAM-1+/+ bEND cells. Here we demonstrate that the restoration of PECAM-1 expression in these cells affects their adhesive and migratory properties in an isoform-specific manner. Expression of Delta14&15 PECAM-1, the predominant isoform present in the mouse endothelium, in PECAM-1-/- bEND cells activated MAPK/ERKs, disrupted adherens junctions, and enhanced cell migration and capillary morphogenesis in Matrigel. In contrast, expression of Delta15 PECAM-1 in PECAM-1-/- bEND cells had minimal effects on their activation of MAPK/ERKs, migration, and capillary morphogenesis. The effects of PECAM-1 on cell adhesive and migratory properties were mediated in an isoform-specific manner, at least in part, through its interactions with intracellular signaling proteins, including SHP-2 and Src. These results suggest that the impact of PECAM-1 on EC adhesion, migration, and capillary morphogenesis is modulated by alternative splicing of its cytoplasmic domain. PMID:18029285

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

    PubMed

    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. PMID:25748451

  17. Inhibition of various isoforms of rat liver glutathione S-transferases by tannic acid and butein.

    PubMed

    Zhang, K; Mack, P; Wong, K P

    1997-07-01

    Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5. PMID:19856286

  18. Profiling alternatively spliced mRNA isoforms for prostate cancer classification

    PubMed Central

    Zhang, Chaolin; Li, Hai-Ri; Fan, Jian-Bing; Wang-Rodriguez, Jessica; Downs, Tracy; Fu, Xiang-Dong; Zhang, Michael Q

    2006-01-01

    Background Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. Results As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR) of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM) classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. Conclusion These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays. PMID:16608523

  19. Single-cell polyadenylation site mapping reveals 3′ isoform choice variability

    PubMed Central

    Velten, Lars; Anders, Simon; Pekowska, Aleksandra; Järvelin, Aino I; Huber, Wolfgang; Pelechano, Vicent; Steinmetz, Lars M

    2015-01-01

    Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3′ untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3′ isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3′ isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations. PMID:26040288

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

    PubMed Central

    Hansberg-Pastor, Valeria; Piña-Medina, Ana Gabriela; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2015-01-01

    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. PMID:26064112

  1. Myosin II isoform co-assembly and differential regulation in mammalian systems.

    PubMed

    Beach, Jordan R; Hammer, John A

    2015-05-15

    Non-muscle myosin 2 (NM2) is a major force-producing, actin-based motor in mammalian non-muscle cells, where it plays important roles in a broad range of fundamental biological processes, including cytokinesis, cell migration, and epithelial barrier function. This breadth of function at the tissue and cellular levels suggests extensive diversity and differential regulation of NM2 bipolar filaments, the major, if not sole, functional form of NM2s in vivo. Previous in vitro, cellular and animal studies indicate that some of this diversity is supported by the existence of multiple NM2 isoforms. Moreover, two recent studies have shown that these isoforms can co-assemble to form heterotypic filaments, further expanding functional diversity. In addition to isoform co-assembly, cells may differentially regulate NM2 function via isoform-specific expression, RLC phosphorylation, MHC phosphorylation or regulation via binding partners. Here, we provide a brief summary of NM2 filament assembly, summarize the recent findings regarding NM2 isoform co-assembly, consider the mechanisms cells might utilize to differentially regulate NM2 isoforms, and review the data available to support these mechanisms. PMID:25655283

  2. Palmitoylation of the three isoforms of human endothelin-converting enzyme-1.

    PubMed Central

    Schweizer, A; Löffler, B M; Rohrer, J

    1999-01-01

    Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation. PMID:10359648

  3. HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation.

    PubMed

    He, Zhicong; Aristoteli, Lina P; Kritharides, Leonard; Garner, Brett

    2006-05-01

    Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples. PMID:16546121

  4. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    PubMed

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. PMID:27105153

  5. N-Domain Isoform of Angiotensin I Converting Enzyme as a Marker of Hypertension: Populational Study

    PubMed Central

    Maluf-Meiken, Leila C. V.; Fernandes, Fernanda B.; Aragão, Danielle S.; Ronchi, Fernanda A.; Andrade, Maria C. C.; Franco, Maria C.; Febba, Andreia C. S.; Plavnik, Frida L.; Krieger, José E.; Mill, Jose G.; Sesso, Ricardo C. C.; Casarini, Dulce E.

    2012-01-01

    The aim of this paper was to investigate the presence of the urinary 90 kDa N-domain ACE in a cohort of the population from Vitoria, Brazil, to verify its association with essential hypertension since this isoform could be a possible genetic marker of hypertension. Anthropometric, clinical, and laboratory parameters of the individuals were evaluated (n = 1150) and the blood pressure (BP) was measured. The study population was divided according to ACE isoforms in urine as follows: ACE 65/90/190, presence of three ACE isoforms (n = 795), ACE 90+ (65/90) (n = 186), and ACE 90− (65/190) (n = 169) based on the presence (+) or absence (−) of the 90 kDa ACE isoform. The anthropometric parameters, lipid profile, serum levels of uric acid, glucose, and the systolic and diastolic BP were significantly greater in the ACE 90+ compared with the ACE 90− and ACE 65/90/190 individuals. We found that 98% of individuals from the ACE 90+ group and 38% from the ACE 65/90/190 group had hypertension, compared to only 1% hypertensive individuals in the ACE 90− group. There is a high presence of the 90 kDa N-domain ACE isoform (85%) in the studied population. The percentile of normotensive subjects with three isoforms was 62%. Our findings could contribute to the development of new efficient strategy to prevent and treat hypertension to avoid the development of cardiovascular disease. PMID:22666552

  6. Molecular characterization of human thyroid hormone receptor β isoform 4.

    PubMed

    Moriyama, Kenji; Yamamoto, Hiroyuki; Futawaka, Kumi; Atake, Asami; Kasahara, Masato; Tagami, Tetsuya

    2016-01-01

    Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus. PMID:26513165

  7. Abnormal Magnetic Field Effects on Electrogenerated Chemiluminescence

    NASA Astrophysics Data System (ADS)

    Pan, Haiping; Shen, Yan; Wang, Hongfeng; He, Lei; Hu, Bin

    2015-03-01

    We report abnormal magnetic field effects on electrogenerated chemiluminescence (MFEECL) based on triplet emission from the Ru(bpy)3Cl2-TPrA electrochemical system: the appearance of MFEECL after magnetic field ceases. In early studies the normal MFEECL have been observed from electrochemical systems during the application of magnetic field. Here, the abnormal MFEECL suggest that the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes may become magnetized in magnetic field and experience a long magnetic relaxation after removing magnetic field. Our analysis indicates that the magnetic relaxation can gradually increase the density of charge-transfer complexes within reaction region due to decayed magnetic interactions, leading to a positive component in the abnormal MFEECL. On the other hand, the magnetic relaxation facilitates an inverse conversion from triplets to singlets within charge-transfer complexes. The inverse triplet --> singlet conversion reduces the density of triplet light-emitting states through charge-transfer complexes and gives rise to a negative component in the abnormal MFEECL. The combination of positive and negative components can essentially lead to a non-monotonic profile in the abnormal MFEECL after ceasing magnetic field. Nevertheless, our experimental studies may reveal un-usual magnetic behaviors with long magnetic relaxation from the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes in solution at room temperature.

  8. Abnormal magnetic field effects on electrogenerated chemiluminescence.

    PubMed

    Pan, Haiping; Shen, Yan; Wang, Hongfeng; He, Lei; Hu, Bin

    2015-01-01

    We report abnormal magnetic field effects on electrogenerated chemiluminescence (MFEECL) based on triplet emission from the Ru(bpy)3Cl2-TPrA electrochemical system: the appearance of MFEECL after magnetic field ceases. In early studies the normal MFEECL have been observed from electrochemical systems during the application of magnetic field. Here, the abnormal MFEECL suggest that the activated charge-transfer [Ru(bpy)3(3+) … TPrA(•)] complexes may become magnetized in magnetic field and experience a long magnetic relaxation after removing magnetic field. Our analysis indicates that the magnetic relaxation can gradually increase the density of charge-transfer complexes within reaction region due to decayed magnetic interactions, leading to a positive component in the abnormal MFEECL. On the other hand, the magnetic relaxation facilitates an inverse conversion from triplets to singlets within charge-transfer complexes. The inverse triplet → singlet conversion reduces the density of triplet light-emitting states through charge-transfer complexes and gives rise to a negative component in the abnormal MFEECL. The combination of positive and negative components can essentially lead to a non-monotonic profile in the abnormal MFEECL after ceasing magnetic field. Nevertheless, our experimental studies may reveal un-usual magnetic behaviors with long magnetic relaxation from the activated charge-transfer [Ru(bpy)3(3+) … TPrA(•)] complexes in solution at room temperature. PMID:25772580

  9. Abnormal Magnetic Field Effects on Electrogenerated Chemiluminescence

    PubMed Central

    Pan, Haiping; Shen, Yan; Wang, Hongfeng; He, Lei; Hu, Bin

    2015-01-01

    We report abnormal magnetic field effects on electrogenerated chemiluminescence (MFEECL) based on triplet emission from the Ru(bpy)3Cl2-TPrA electrochemical system: the appearance of MFEECL after magnetic field ceases. In early studies the normal MFEECL have been observed from electrochemical systems during the application of magnetic field. Here, the abnormal MFEECL suggest that the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes may become magnetized in magnetic field and experience a long magnetic relaxation after removing magnetic field. Our analysis indicates that the magnetic relaxation can gradually increase the density of charge-transfer complexes within reaction region due to decayed magnetic interactions, leading to a positive component in the abnormal MFEECL. On the other hand, the magnetic relaxation facilitates an inverse conversion from triplets to singlets within charge-transfer complexes. The inverse triplet → singlet conversion reduces the density of triplet light-emitting states through charge-transfer complexes and gives rise to a negative component in the abnormal MFEECL. The combination of positive and negative components can essentially lead to a non-monotonic profile in the abnormal MFEECL after ceasing magnetic field. Nevertheless, our experimental studies may reveal un-usual magnetic behaviors with long magnetic relaxation from the activated charge-transfer [Ru(bpy)33+ … TPrA•] complexes in solution at room temperature. PMID:25772580

  10. Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles

    PubMed Central

    Pellegrino, M A; Canepari, M; Rossi, R; D'Antona, G; Reggiani, C; Bottinelli, R

    2003-01-01

    Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (Vf) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V0 and Vf determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres. PMID:12562996

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

    SciTech Connect

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

    2009-03-10

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

  12. Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains.

    PubMed

    Brudek, Tomasz; Winge, Kristian; Rasmussen, Nadja Bredo; Bahl, Justyna Maria Czarna; Tanassi, Julia; Agander, Tina Klitmøller; Hyde, Thomas M; Pakkenberg, Bente

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal

  13. Computational approaches for isoform detection and estimation: good and bad news

    PubMed Central

    2014-01-01

    Background The main goal of the whole transcriptome analysis is to correctly identify all expressed transcripts within a specific cell/tissue - at a particular stage and condition - to determine their structures and to measure their abundances. RNA-seq data promise to allow identification and quantification of transcriptome at unprecedented level of resolution, accuracy and low cost. Several computational methods have been proposed to achieve such purposes. However, it is still not clear which promises are already met and which challenges are still open and require further methodological developments. Results We carried out a simulation study to assess the performance of 5 widely used tools, such as: CEM, Cufflinks, iReckon, RSEM, and SLIDE. All of them have been used with default parameters. In particular, we considered the effect of the following three different scenarios: the availability of complete annotation, incomplete annotation, and no annotation at all. Moreover, comparisons were carried out using the methods in three different modes of action. In the first mode, the methods were forced to only deal with those isoforms that are present in the annotation; in the second mode, they were allowed to detect novel isoforms using the annotation as guide; in the third mode, they were operating in fully data driven way (although with the support of the alignment on the reference genome). In the latter modality, precision and recall are quite poor. On the contrary, results are better with the support of the annotation, even though it is not complete. Finally, abundance estimation error often shows a very skewed distribution. The performance strongly depends on the true real abundance of the isoforms. Lowly (and sometimes also moderately) expressed isoforms are poorly detected and estimated. In particular, lowly expressed isoforms are identified mainly if they are provided in the original annotation as potential isoforms. Conclusions Both detection and quantification

  14. Abnormal head position in infantile nystagmus syndrome.

    PubMed

    Noval, Susana; González-Manrique, Mar; Rodríguez-Del Valle, José María; Rodríguez-Sánchez, José María

    2011-01-01

    Infantile nystagmus is an involuntary, bilateral, conjugate, and rhythmic oscillation of the eyes which is present at birth or develops within the first 6 months of life. It may be pendular or jerk-like and, its intensity usually increases in lateral gaze, decreasing with convergence. Up to 64% of all patients with nystagmus also present strabismus, and even more patients have an abnormal head position. The abnormal head positions are more often horizontal, but they may also be vertical or take the form of a tilt, even though the nystagmus itself is horizontal. The aim of this article is to review available information about the origin and treatment of the abnormal head position associated to nystagmus, and to describe our treatment strategies. PMID:24533187

  15. Abnormal Grain Growth Suppression in Aluminum Alloys

    NASA Technical Reports Server (NTRS)

    Hales, Stephen J. (Inventor); Claytor, Harold Dale (Inventor); Alexa, Joel A. (Inventor)

    2015-01-01

    The present invention provides a process for suppressing abnormal grain growth in friction stir welded aluminum alloys by inserting an intermediate annealing treatment ("IAT") after the welding step on the article. The IAT may be followed by a solution heat treatment (SHT) on the article under effectively high solution heat treatment conditions. In at least some embodiments, a deformation step is conducted on the article under effective spin-forming deformation conditions or under effective superplastic deformation conditions. The invention further provides a welded article having suppressed abnormal grain growth, prepared by the process above. Preferably the article is characterized with greater than about 90% reduction in area fraction abnormal grain growth in any friction-stir-welded nugget.

  16. Parsing abnormal grain growth in specialty aluminas

    NASA Astrophysics Data System (ADS)

    Lawrence, Abigail Kremer

    Grain growth in alumina is strongly affected by the impurities present in the material. Certain impurity elements are known to have characteristic effects on abnormal grain growth in alumina. Specialty alumina powders contain multiple impurity species including MgO, CaO, SiO2, and Na 2O. In this work, sintered samples made from alumina powders containing various amounts of the impurities in question were characterized by their grain size and aspect ratio distributions. Multiple quantitative methods were used to characterize and classify samples with varying microstructures. The grain size distributions were used to partition the grain size population into subpopulations depending on the observed deviation from normal behavior. Using both grain size and aspect ratio a new visual representation for a microstructure was introduced called a morphology frequency map that gives a fingerprint for the material. The number of subpopulations within a sample and the shape of the distribution on the morphology map provided the basis for a classification scheme for different types of microstructures. Also using the two parameters a series of five metrics were calculated that describe the character of the abnormal grains in the sample, these were called abnormal character values. The abnormal character values describe the fraction of grains that are considered abnormal, the average magnitude of abnormality (including both grain size and aspect ratio), the average size, and variance in size. The final metric is the correlation between grain size and aspect ratio for the entire population of grains. The abnormal character values give a sense of how different from "normal" the sample is, given the assumption that a normal sample has a lognormal distribution of grain size and a Gaussian distribution of aspect ratios. In the second part of the work the quantified measures of abnormality were correlated with processing parameters such as composition and heat treatment conditions. A

  17. [Nutritional abnormalities in chronic obstructive pulmonary disease].

    PubMed

    Gea, Joaquim; Martínez-Llorens, Juana; Barreiro, Esther

    2014-07-22

    Nutritional abnormalities are associated with chronic obstructive pulmonary disease with a frequency ranging from 2 to 50%, depending on the geographical area and the study design. Diagnostic tools include anthropometry, bioelectrical impedance, dual energy radioabsortiometry and deuterium dilution, being the body mass and the lean mass indices the most frequently used parameters. While the most important consequences of nutritional abnormalities are muscle dysfunction and exercise limitation, factors implicated include an imbalance between caloric intake and consumption, and between anabolic and catabolic hormones, inflammation, tobacco smoking, poor physical activity, hypoxemia, some drugs and aging/comorbidities. The most important molecular mechanism for malnutrition associated with chronic obstructive pulmonary disease appears to be the mismatching between protein synthesis and breakdown. Among the therapeutic measures proposed for these nutritional abnormalities are improvements in lifestyle and nutritional support, although the use of anabolic drugs (such as secretagogues of the growth hormone) offers a new therapeutic strategy. PMID:24054776

  18. Retinal abnormalities in β-thalassemia major.

    PubMed

    Bhoiwala, Devang L; Dunaief, Joshua L

    2016-01-01

    Patients with beta (β)-thalassemia (β-TM: β-thalassemia major, β-TI: β-thalassemia intermedia) have a variety of complications that may affect all organs, including the eye. Ocular abnormalities include retinal pigment epithelial degeneration, angioid streaks, venous tortuosity, night blindness, visual field defects, decreased visual acuity, color vision abnormalities, and acute visual loss. Patients with β-thalassemia major are transfusion dependent and require iron chelation therapy to survive. Retinal degeneration may result from either retinal iron accumulation from transfusion-induced iron overload or retinal toxicity induced by iron chelation therapy. Some who were never treated with iron chelation therapy exhibited retinopathy, and others receiving iron chelation therapy had chelator-induced retinopathy. We will focus on retinal abnormalities present in individuals with β-thalassemia major viewed in light of new findings on the mechanisms and manifestations of retinal iron toxicity. PMID:26325202

  19. Echocardiographic abnormalities in the mucopolysaccharide storage diseases.

    PubMed

    Gross, D M; Williams, J C; Caprioli, C; Dominguez, B; Howell, R R

    1988-01-01

    The mucopolysaccharide storage diseases express themselves clinically with a wide variety of abnormalities, including growth and mental retardation, skeletal abnormalities, clouded corneas, nerve compression syndromes, upper airway obstruction and cardiovascular involvement, to name the most common. In most cases the cause of early death is cardiorespiratory failure secondary to cardiovascular involvement and upper airway obstruction. The findings of cardiac ultrasound examination in 29 children, adolescents and young adults are presented. In addition to the previously well-described abnormalities of the mitral and aortic valves in several types of mucopolysaccharide storage disease, we report patchy involvement in some cases, 3 instances of asymmetric septal hypertrophy not previously reported in mucopolysaccharide storage diseases, cardiac involvement in half of our patients with Sanfilippo syndrome and a lack of age-related severity of cardiac involvement even within the specific syndromes. PMID:3122547

  20. Advances in understanding paternally transmitted Chromosomal Abnormalities

    SciTech Connect

    Marchetti, F; Sloter, E; Wyrobek, A J

    2001-03-01

    Multicolor FISH has been adapted for detecting the major types of chromosomal abnormalities in human sperm including aneuploidies for clinically-relevant chromosomes, chromosomal aberrations including breaks and rearrangements, and other numerical abnormalities. The various sperm FISH assays have been used to evaluate healthy men, men of advanced age, and men who have received mutagenic cancer therapy. The mouse has also been used as a model to investigate the mechanism of paternally transmitted genetic damage. Sperm FISH for the mouse has been used to detect chromosomally abnormal mouse sperm, while the PAINT/DAPI analysis of mouse zygotes has been used to evaluate the types of chromosomal defects that can be paternally transmitted to the embryo and their effects on embryonic development.

  1. Cone photopigment bleaching abnormalities in diabetes.

    PubMed

    Elsner, A E; Burns, S A; Lobes, L A; Doft, B H

    1987-04-01

    We have used a color-matching technique to obtain estimates of the optical density of cone photopigments as a function of retinal illuminance in patients with insulin-dependent diabetes mellitus (IDDM). We found that the half-bleach illuminance of some patients is abnormally high. That is, it takes more light to bleach an equivalent amount of photopigment in these patients. Since low illuminance color matches for these patients are normal, this implies that these patients have normal amounts of photopigment, but the photopigment is not bleaching normally. This result clearly points to abnormalities in the outer retina of these diabetic patients. The most likely causes of this abnormality are either decreases in the ability of the cones to absorb light, or an increased rate of regeneration of the cone photopigments. PMID:3557875

  2. Schizophrenia and abnormal brain network hubs

    PubMed Central

    Rubinov, Mikail; Bullmore, Ed.

    2013-01-01

    Schizophrenia is a heterogeneous psychiatric disorder of unknown cause or characteristic pathology. Clinical neuroscientists increasingly postulate that schizophrenia is a disorder of brain network organization. In this article we discuss the conceptual framework of this dysconnection hypothesis, describe the predominant methodological paradigm for testing this hypothesis, and review recent evidence for disruption of central/hub brain regions, as a promising example of this hypothesis. We summarize studies of brain hubs in large-scale structural and functional brain networks and find strong evidence for network abnormalities of prefrontal hubs, and moderate evidence for network abnormalities of limbic, temporal, and parietal hubs. Future studies are needed to differentiate network dysfunction from previously observed gray- and white-matter abnormalities of these hubs, and to link endogenous network dysfunction phenotypes with perceptual, behavioral, and cognitive clinical phenotypes of schizophrenia. PMID:24174905

  3. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

    PubMed

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  4. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    PubMed Central

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  5. Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome.

    PubMed

    Kashima, Risa; Roy, Sougata; Ascano, Manuel; Martinez-Cerdeno, Veronica; Ariza-Torres, Jeanelle; Kim, Sunghwan; Louie, Justin; Lu, Yao; Leyton, Patricio; Bloch, Kenneth D; Kornberg, Thomas B; Hagerman, Paul J; Hagerman, Randi; Lagna, Giorgio; Hata, Akiko

    2016-01-01

    Epigenetic silencing of fragile X mental retardation 1 (FMR1) causes fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. FXS correlates with abnormal synapse and dendritic spine development, but the molecular link between the absence of the FMR1 product FMRP, an RNA binding protein, and the neuropathology is unclear. We found that the messenger RNA encoding bone morphogenetic protein type II receptor (BMPR2) is a target of FMRP. Depletion of FMRP increased BMPR2 abundance, especially that of the full-length isoform that bound and activated LIM domain kinase 1 (LIMK1), a component of the noncanonical BMP signal transduction pathway that stimulates actin reorganization to promote neurite outgrowth and synapse formation. Heterozygosity for BMPR2 rescued the morphological abnormalities in neurons both in Drosophila and in mouse models of FXS, as did the postnatal pharmacological inhibition of LIMK1 activity. Compared with postmortem prefrontal cortex tissue from healthy subjects, the amount of full-length BMPR2 and of a marker of LIMK1 activity was increased in this brain region from FXS patients. These findings suggest that increased BMPR2 signal transduction is linked to FXS and that the BMPR2-LIMK1 pathway is a putative therapeutic target in patients with FXS and possibly other forms of autism. PMID:27273096

  6. Abnormal carbene-silicon halide complexes.

    PubMed

    Wang, Yuzhong; Xie, Yaoming; Wei, Pingrong; Schaefer, Henry F; Robinson, Gregory H

    2016-04-14

    Reaction of the anionic N-heterocyclic dicarbene (NHDC), [:C{[N(2,6-Pr(i)2C6H3)]2CHCLi}]n (1), with SiCl4 gives the trichlorosilyl-substituted (at the C4 carbon) N-heterocyclic carbene complex (7). Abnormal carbene-SiCl4 complex (8) may be conveniently synthesized by combining 7 with HCl·NEt3. In addition, 7 may react with CH2Cl2 in warm hexane, giving the abnormal carbene-complexed SiCl3(+) cation (9). The nature of the bonding in 9 was probed with complementary DFT computations. PMID:26605692

  7. Hemorheological abnormalities in human arterial hypertension

    NASA Astrophysics Data System (ADS)

    Lo Presti, Rosalia; Hopps, Eugenia; Caimi, Gregorio

    2014-05-01

    Blood rheology is impaired in hypertensive patients. The alteration involves blood and plasma viscosity, and the erythrocyte behaviour is often abnormal. The hemorheological pattern appears to be related to some pathophysiological mechanisms of hypertension and to organ damage, in particular left ventricular hypertrophy and myocardial ischemia. Abnormalities have been observed in erythrocyte membrane fluidity, explored by fluorescence spectroscopy and electron spin resonance. This may be relevant for red cell flow in microvessels and oxygen delivery to tissues. Although blood viscosity is not a direct target of antihypertensive therapy, the rheological properties of blood play a role in the pathophysiology of arterial hypertension and its vascular complications.

  8. Ocular motor abnormalities in neurodegenerative disorders

    PubMed Central

    Antoniades, C A; Kennard, C

    2015-01-01

    Eye movements are a source of valuable information to both clinicians and scientists as abnormalities of them frequently act as clues to the localization of a disease process. Classically, they are divided into two main types: those that hold the gaze, keeping images steady on the retina (vestibulo-ocular and optokinetic reflexes) and those that shift gaze and redirect the line of sight to a new object of interest (saccades, vergence, and smooth pursuit). Here we will review some of the major ocular motor abnormalities present in neurodegenerative disorders. PMID:25412716

  9. Nonpathologizing trauma interventions in abnormal psychology courses.

    PubMed

    Hoover, Stephanie M; Luchner, Andrew F; Pickett, Rachel F

    2016-01-01

    Because abnormal psychology courses presuppose a focus on pathological human functioning, nonpathologizing interventions within these classes are particularly powerful and can reach survivors, bystanders, and perpetrators. Interventions are needed to improve the social response to trauma on college campuses. By applying psychodynamic and feminist multicultural theory, instructors can deliver nonpathologizing interventions about trauma and trauma response within these classes. We recommend class-based interventions with the following aims: (a) intentionally using nonpathologizing language, (b) normalizing trauma responses, (c) subjectively defining trauma, (d) challenging secondary victimization, and (e) questioning the delineation of abnormal and normal. The recommendations promote implications for instructor self-reflection, therapy interventions, and future research. PMID:26460794

  10. Normal and abnormal human vestibular ocular function

    NASA Technical Reports Server (NTRS)

    Peterka, R. J.; Black, F. O.

    1986-01-01

    The major motivation of this research is to understand the role the vestibular system plays in sensorimotor interactions which result in spatial disorientation and motion sickness. A second goal was to explore the range of abnormality as it is reflected in quantitative measures of vestibular reflex responses. The results of a study of vestibular reflex measurements in normal subjects and preliminary results in abnormal subjects are presented in this report. Statistical methods were used to define the range of normal responses, and determine age related changes in function.

  11. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages.

    PubMed

    Papakonstanti, Evangelia A; Zwaenepoel, Olivier; Bilancio, Antonio; Burns, Emily; Nock, Gemma E; Houseman, Benjamin; Shokat, Kevan; Ridley, Anne J; Vanhaesebroeck, Bart

    2008-12-15

    The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer. PMID:19033389

  12. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

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

    PubMed Central

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

    2015-01-01

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

  14. NADPH-diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

    PubMed Central

    MORAES, NECI; ZAGO, DOUGLAS; GAGIOTI, SONIA; HOSHIDA, MARA SANDRA; BEVILACQUA, ESTELA

    2001-01-01

    The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface. PMID:11327206

  15. Development and validation of MRM methods to quantify protein isoforms of polyphenol oxidase in loquat fruits.

    PubMed

    Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio; Bru-Martínez, Roque

    2013-12-01

    Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using

  16. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  17. Characterization of a saporin isoform with lower ribosome-inhibiting activity.

    PubMed Central

    Fabbrini, M S; Rappocciolo, E; Carpani, D; Solinas, M; Valsasina, B; Breme, U; Cavallaro, U; Nykjaer, A; Rovida, E; Legname, G; Soria, M R

    1997-01-01

    We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands. PMID:9148741

  18. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  19. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies.

    PubMed

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-10-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  1. Identification of a Novel Human LAP1 Isoform That Is Regulated by Protein Phosphorylation

    PubMed Central

    Santos, Mariana; Domingues, Sara C.; Costa, Patrícia; Muller, Thorsten; Galozzi, Sara; Marcus, Katrin; da Cruz e Silva, Edgar F.; da Cruz e Silva, Odete A.; Rebelo, Sandra

    2014-01-01

    Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation. PMID:25461922

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

    PubMed Central

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

    2012-01-01

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

  3. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

    PubMed

    Mundell, Stuart J; Jones, Matthew L; Hardy, Adam R; Barton, Johanna F; Beaucourt, Stephanie M; Conley, Pamela B; Poole, Alastair W

    2006-09-01

    ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking. PMID:16804093

  4. Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

    PubMed

    Zhang, Hai-Feng; Wang, Huan-Huan; Gao, Na; Wei, Jun-Ying; Tian, Xin; Zhao, Yan; Fang, Yan; Zhou, Jun; Wen, Qiang; Gao, Jie; Zhang, Yang-Jun; Qian, Xiao-Hong; Qiao, Hai-Ling

    2016-07-01

    Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content. PMID:27189963

  5. Scrapie prion liposomes and rods exhibit target sizes of 55,000 Da

    SciTech Connect

    Bellinger-Kawahara, C.G.; Kempner, E.; Groth, D.; Gabizon, R.; Prusiner, S.B.

    1988-06-01

    Scrapie is a degenerative neurologic disease in sheep and goats which can be experimentally transmitted to laboratory rodents. Considerable evidence suggests that the scrapie agent is composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc). Inactivation of scrapie prions by ionizing radiation exhibited single-hit kinetics and gave a target size of 55,000 +/- 9000 mol wt. The inactivation profile was independent of the form of the prion. Scrapie agent infectivity in brain homogenates, microsomal fractions, detergent-extracted microsomes, purified amyloid rods, and liposomes exhibited the same inactivation profile. Our data are consistent with the hypothesis that the infectious particle causing scrapie contains approximately 2 PrPSc molecules.

  6. Abnormal behaviors detection using particle motion model

    NASA Astrophysics Data System (ADS)

    Chen, Yutao; Zhang, Hong; Cheng, Feiyang; Yuan, Ding; You, Yuhu

    2015-03-01

    Human abnormal behaviors detection is one of the most challenging tasks in the video surveillance for the public security control. Interaction Energy Potential model is an effective and competitive method published recently to detect abnormal behaviors, but their model of abnormal behaviors is not accurate enough, so it has some limitations. In order to solve this problem, we propose a novel Particle Motion model. Firstly, we extract the foreground to improve the accuracy of interest points detection since the complex background usually degrade the effectiveness of interest points detection largely. Secondly, we detect the interest points using the graphics features. Here, the movement of each human target can be represented by the movements of detected interest points of the target. Then, we track these interest points in videos to record their positions and velocities. In this way, the velocity angles, position angles and distance between each two points can be calculated. Finally, we proposed a Particle Motion model to calculate the eigenvalue of each frame. An adaptive threshold method is proposed to detect abnormal behaviors. Experimental results on the BEHAVE dataset and online videos show that our method could detect fight and robbery events effectively and has a promising performance.

  7. Abnormally high formation pressures, Potwar Plateau, Pakistan

    USGS Publications Warehouse

    Law, B.E.; Shah, S.H.A.; Malik, M.A.

    1998-01-01

    Abnormally high formation pressures in the Potwar Plateau of north-central Pakistan are major obstacles to oil and gas exploration. Severe drilling problems associated with high pressures have, in some cases, prevented adequate evaluation of reservoirs and significantly increased drilling costs. Previous investigations of abnormal pressure in the Potwar Plateau have only identified abnormal pressures in Neogene rocks. We have identified two distinct pressure regimes in this Himalayan foreland fold and thrust belt basin: one in Neogene rocks and another in pre-Neogene rocks. Pore pressures in Neogene rocks are as high as lithostatic and are interpreted to be due to tectonic compression and compaction disequilibrium associated with high rates of sedimentation. Pore pressure gradients in pre-Neogene rocks are generally less than those in Neogene rocks, commonly ranging from 0.5 to 0.7 psi/ft (11.3 to 15.8 kPa/m) and are most likely due to a combination of tectonic compression and hydrocarbon generation. The top of abnormally high pressure is highly variable and doesn't appear to be related to any specific lithologic seal. Consequently, attempts to predict the depth to the top of overpressure prior to drilling are precluded.

  8. Esophageal motility abnormalities in gastroesophageal reflux disease.

    PubMed

    Martinucci, Irene; de Bortoli, Nicola; Giacchino, Maria; Bodini, Giorgia; Marabotto, Elisa; Marchi, Santino; Savarino, Vincenzo; Savarino, Edoardo

    2014-05-01

    Esophageal motility abnormalities are among the main factors implicated in the pathogenesis of gastroesophageal reflux disease. The recent introduction in clinical and research practice of novel esophageal testing has markedly improved our understanding of the mechanisms contributing to the development of gastroesophageal reflux disease, allowing a better management of patients with this disorder. In this context, the present article intends to provide an overview of the current literature about esophageal motility dysfunctions in patients with gastroesophageal reflux disease. Esophageal manometry, by recording intraluminal pressure, represents the gold standard to diagnose esophageal motility abnormalities. In particular, using novel techniques, such as high resolution manometry with or without concurrent intraluminal impedance monitoring, transient lower esophageal sphincter (LES) relaxations, hypotensive LES, ineffective esophageal peristalsis and bolus transit abnormalities have been better defined and strongly implicated in gastroesophageal reflux disease development. Overall, recent findings suggest that esophageal motility abnormalities are increasingly prevalent with increasing severity of reflux disease, from non-erosive reflux disease to erosive reflux disease and Barrett's esophagus. Characterizing esophageal dysmotility among different subgroups of patients with reflux disease may represent a fundamental approach to properly diagnose these patients and, thus, to set up the best therapeutic management. Currently, surgery represents the only reliable way to restore the esophagogastric junction integrity and to reduce transient LES relaxations that are considered to be the predominant mechanism by which gastric contents can enter the esophagus. On that ground, more in depth future studies assessing the pathogenetic role of dysmotility in patients with reflux disease are warranted. PMID:24868489

  9. Pancreatic abnormalities and AIDS related sclerosing cholangitis.

    PubMed Central

    Teare, J P; Daly, C A; Rodgers, C; Padley, S P; Coker, R J; Main, J; Harris, J R; Scullion, D; Bray, G P; Summerfield, J A

    1997-01-01

    OBJECTIVES: Biliary tract abnormalities are well recognised in AIDS, most frequently related to opportunistic infection with Cryptosporidium, Microsporidium, and cytomegalovirus. We noted a high frequency of pancreatic abnormalities associated with biliary tract disease. To define these further we reviewed the clinical and radiological features in these patients. METHODS: Notes and radiographs were available from two centres for 83 HIV positive patients who had undergone endoscopic retrograde cholangiopancreatography for the investigation of cholestatic liver function tests or abdominal pain. RESULTS: 56 patients had AIDS related sclerosing cholangitis (ARSC); 86% of these patients had epigastric or right upper quadrant pain and 52% had hepatomegaly. Of the patients with ARSC, 10 had papillary stenosis alone, 11 had intra- and extrahepatic sclerosing cholangitis alone, and 35 had a combination of the two. Ampullary biopsies performed in 24 patients confirmed an opportunistic infection in 16. In 15 patients, intraluminal polyps were noted on the cholangiogram. Pancreatograms were available in 34 of the 45 patients with papillary stenosis, in which 29 (81%) had associated pancreatic duct dilatation, often with associated features of chronic pancreatitis. In the remaining 27 patients, final diagnoses included drug induced liver disease, acalculous cholecystitis, gall bladder empyema, chronic B virus hepatitis, and alcoholic liver disease. CONCLUSION: Pancreatic abnormalities are commonly seen with ARSC and may be responsible for some of the pain not relieved by biliary sphincterotomy. The most frequent radiographic biliary abnormality is papillary stenosis combined with ductal sclerosis. Images PMID:9389948

  10. Teaching Abnormal Psychology in a Multimedia Classroom.

    ERIC Educational Resources Information Center

    Brewster, JoAnne

    1996-01-01

    Examines the techniques used in teaching an abnormal psychology class in a multimedia environment with two computers and a variety of audiovisual equipment. Students respond anonymously to various questions via keypads mounted on their desks, then immediately view and discuss summaries of their responses. (MJP)

  11. Psychology Faculty Perceptions of Abnormal Psychology Textbooks

    ERIC Educational Resources Information Center

    Rapport, Zachary

    2011-01-01

    The problem. The purpose of the current study was to investigate the perceptions and opinions of psychology professors regarding the accuracy and inclusiveness of abnormal psychology textbooks. It sought answers from psychology professors to the following questions: (1) What are the expectations of the psychology faculty at a private university of…

  12. Schizophrenogenic Parenting in Abnormal Psychology Textbooks.

    ERIC Educational Resources Information Center

    Wahl, Otto F.

    1989-01-01

    Considers the treatment of family causation of schizophrenia in undergraduate abnormal psychology textbooks. Reviews texts published only after 1986. Points out a number of implications for psychologists which arise from the inclusion in these texts of the idea that parents cause schizophrenia, not the least of which is the potential for…

  13. Familial Precocious Fetal Abnormal Cortical Sulcation.

    PubMed

    Frassoni, Carolina; Avagliano, Laura; Inverardi, Francesca; Spaccini, Luigina; Parazzini, Cecilia; Rustico, Maria Angela; Bulfamante, Gaetano; Righini, Andrea

    2016-08-01

    The development of the human cerebral cortex is a complex and precisely programmed process by which alterations may lead to morphological and functional neurological abnormalities. We report familial cases of prenatally diagnosed abnormal brain, characterized by aberrant symmetrical mesial oversulcation of the parietooccipital lobes, in fetuses affected by abnormal skeletal features. Fetal brain anomalies were characterized by prenatal magnetic resonance imaging at 21 weeks of gestation and histologically evaluated at 22 weeks. Histological examination added relevant information showing some focal cortical areas of micropoligyria and heterotopic extension of the cortical plate into the marginal zone beneath the cortical surface. Genetic analysis of the fetuses excluded FGFR3 mutations known to be related to skeletal dysplasia and aberrant symmetrical oversulcation in other brain areas (temporal lobes). Hence, the present report suggests the existence of a class of rare syndromes of skeleton and brain development abnormality unrelated to FGFR3 mutations or related to other not described FGFR3 gene defects. Using magnetic resonance imaging, histopathology and molecular characterization we provide an example of a translational study of a rare and unreported brain congenital malformation. PMID:27177044

  14. Abnormal Web Usage Control by Proxy Strategies.

    ERIC Educational Resources Information Center

    Yu, Hsiang-Fu; Tseng, Li-Ming

    2002-01-01

    Approaches to designing a proxy server with Web usage control and to making the proxy server effective on local area networks are proposed to prevent abnormal Web access and to prioritize Web usage. A system is implemented to demonstrate the approaches. The implementation reveals that the proposed approaches are effective, such that the abnormal…

  15. Ultrasonography of gallbladder abnormalities due to schistosomiasis.

    PubMed

    Richter, Joachim; Azoulay, Daniel; Dong, Yi; Holtfreter, Martha C; Akpata, Robert; Calderaro, Julien; El-Scheich, Tarik; Breuer, Matthias; Neumayr, Andreas; Hatz, Christoph; Kircheis, Gerald; Botelho, Monica C; Dietrich, Christoph F

    2016-08-01

    After malaria, schistosomiasis remains the most important tropical parasitic disease in large parts of the world. Schistosomiasis has recently re-emerged in Southern Europe. Intestinal schistosomiasis is caused by most Schistosoma (S.) spp. pathogenic to humans and leads to chronic inflammation and fibrosis of the colon as well as to liver fibrosis. Gallbladder abnormalities usually occur in patients with advanced hepatic portal fibrosis due to Schistosoma mansoni infection. Occasionally, gallbladder abnormalities have been seen also in children and occurring without associated overt liver abnormalities.The specific S. mansoni-induced gallbladder abnormalities detectable by ultrasound include typical hyperechogenic wall thickening with external gallbladder wall protuberances. The luminal wall surface is smooth. The condition is usually clinically silent although some cases of symptomatic cholecystitis have been described. The ultrasonographic Murphy response is negative. Gallbladder contractility is impaired but sludge and calculi occur rarely. Contrary to other trematodes such as liver flukes, S. mansoni does not obstruct the biliary tract. Advanced gallbladder fibrosis is unlikely to reverse after therapy. PMID:27169865

  16. Abnormal interhemispheric connectivity in male psychopathic offenders

    PubMed Central

    Hoppenbrouwers, Sylco S.; De Jesus, Danilo R.; Sun, Yinming; Stirpe, Tania; Hofman, Dennis; McMaster, Jeff; Hughes, Ginny; Daskalakis, Zafiris J.; Schutter, Dennis J.L.G.

    2014-01-01

    Background Psychopathic offenders inevitably violate interpersonal norms and frequently resort to aggressive and criminal behaviour. The affective and cognitive deficits underlying these behaviours have been linked to abnormalities in functional interhemispheric connectivity. However, direct neurophysiological evidence for dysfunctional connectivity in psychopathic offenders is lacking. Methods We used transcranial magnetic stimulation combined with electroencephalography to examine interhemispheric connectivity in the dorsolateral and motor cortex in a sample of psychopathic offenders and healthy controls. We also measured intracortical inhibition and facilitation over the left and right motor cortex to investigate the effects of local cortical processes on interhemispheric connectivity. Results We enrolled 17 psychopathic offenders and 14 controls in our study. Global abnormalities in right to left functional connectivity were observed in psychopathic offenders compared with controls. Furthermore, in contrast to controls, psychopathic offenders showed increased intracortical inhibition in the right, but not the left, hemisphere. Limitations The relatively small sample size limited the sensitivity to show that the abnormalities in interhemispheric connectivity were specifically related to the dorsolateral prefrontal cortex in psychopathic offenders. Conclusion To our knowledge, this study provides the first neurophysiological evidence for abnormal interhemispheric connectivity in psychopathic offenders and may further our understanding of the disruptive antisocial behaviour of these offenders. PMID:23937798

  17. Craniofacial abnormalities among patients with Edwards Syndrome

    PubMed Central

    Rosa, Rafael Fabiano M.; Rosa, Rosana Cardoso M.; Lorenzen, Marina Boff; Zen, Paulo Ricardo G.; Graziadio, Carla; Paskulin, Giorgio Adriano

    2013-01-01

    OBJECTIVE To determine the frequency and types of craniofacial abnormalities observed in patients with trisomy 18 or Edwards syndrome (ES). METHODS This descriptive and retrospective study of a case series included all patients diagnosed with ES in a Clinical Genetics Service of a reference hospital in Southern Brazil from 1975 to 2008. The results of the karyotypic analysis, along with clinical data, were collected from medical records. RESULTS: The sample consisted of 50 patients, of which 66% were female. The median age at first evaluation was 14 days. Regarding the karyotypes, full trisomy of chromosome 18 was the main alteration (90%). Mosaicism was observed in 10%. The main craniofacial abnormalities were: microretrognathia (76%), abnormalities of the ear helix/dysplastic ears (70%), prominent occiput (52%), posteriorly rotated (46%) and low set ears (44%), and short palpebral fissures/blepharophimosis (46%). Other uncommon - but relevant - abnormalities included: microtia (18%), orofacial clefts (12%), preauricular tags (10%), facial palsy (4%), encephalocele (4%), absence of external auditory canal (2%) and asymmetric face (2%). One patient had an initial suspicion of oculo-auriculo-vertebral spectrum (OAVS) or Goldenhar syndrome. CONCLUSIONS: Despite the literature description of a characteristic clinical presentation for ES, craniofacial alterations may be variable among these patients. The OAVS findings in this sample are noteworthy. The association of ES with OAVS has been reported once in the literature. PMID:24142310

  18. Abnormal Saccadic Eye Movements in Autistic Children.

    ERIC Educational Resources Information Center

    Kemner, C.; Verbaten, M. N.; Cuperus, J. M.; Camfferman, G.; van Engeland, H.

    1998-01-01

    The saccadic eye movements, generated during a visual oddball task, were compared for 10 autistic children, 10 children with attention deficit hyperactivity disorder, 10 dyslexic children, and 10 typically developing children. Several abnormal patterns of saccades were found in the autistic group. (DB)

  19. Abnormal Cervical Cancer Screening Test Results

    MedlinePlus

    ... LEEP) —A thin wire loop that carries an electric current is used to remove abnormal areas of the ... the cervix using a thin wire loop and electric energy. Pap ... this document sets forth current information and opinions related to women’s health. The ...

  20. Dynamic Abnormal Grain Growth in Refractory Metals

    NASA Astrophysics Data System (ADS)

    Noell, Philip J.; Taleff, Eric M.

    2015-11-01

    High-temperature plastic deformation of the body-centered cubic (BCC) refractory metals Mo and Ta can initiate and propagate abnormal grains at significantly lower temperatures and faster rates than is possible by static annealing alone. This discovery reveals a new and potentially important aspect of abnormal grain growth (AGG) phenomena. The process of AGG during plastic deformation at elevated temperatures, termed dynamic abnormal grain growth (DAGG), was observed at homologous temperatures between 0.52 and 0.72 in both Mo and Ta sheet materials; these temperatures are much lower than those for previous observations of AGG in these materials during static annealing. DAGG was used to repeatedly grow single crystals several centimeters in length. Investigations to date have produced a basic understanding of the conditions that lead to DAGG and how DAGG is affected by microstructure in BCC refractory metals. The current state of understanding for DAGG is reviewed in this paper. Attention is given to the roles of temperature, plastic strain, boundary mobility and preexisting microstructure. DAGG is considered for its potential useful applications in solid-state crystal growth and its possibly detrimental role in creating undesired abnormal grains during thermomechanical processing.

  1. On (ab)normality: Einstein's fusiform gyrus.

    PubMed

    Weiner, Kevin S

    2015-03-01

    Recently, Hines (2014) wrote an evocative paper challenging findings from both histological and morphological studies of Einstein's brain. In this discussion paper, I extend Hines' theoretical point and further discuss how best to determine 'abnormal' morphology. To do so, I assess the sulcal patterning of Einstein's fusiform gyrus (FG) for the first time. The sulcal patterning of the FG was unconsidered in prior studies because the morphological features of the mid-fusiform sulcus have only been clarified recently. On the one hand, the sulcal patterning of Einstein's FG is abnormal relative to averages of 'normal' brains generated from two independent datasets (N = 39 and N = 15, respectively). On the other hand, within the 108 hemispheres used to make these average brains, it is not impossible to find FG sulcal patterns that resemble those of Einstein. Thus, concluding whether a morphological pattern is normal or abnormal heavily depends on the chosen analysis method (e.g. group average vs. individual). Such findings question the functional meaning of morphological 'abnormalities' when determined by comparing an individual to an average brain or average frequency characteristics. These observations are not only important for analyzing a rare brain such as that of Einstein, but also for comparing macroanatomical features between typical and atypical populations. PMID:25562419

  2. Behavioral abnormalities in captive nonhuman primates.

    PubMed

    Mallapur, Avanti; Choudhury, B C

    2003-01-01

    In this study, we dealt with 11 species of nonhuman primates across 10 zoos in India. We recorded behavior as instantaneous scans between 9 a.m. and 5 p.m. In the study, we segregated behaviors for analyses into abnormal, undesirable, active, and resting. The 4 types of abnormal behavior exhibited included floating limb, self-biting, self-clasping, and stereotypic pacing. In the study, we recorded 2 types of undesirable behavior: autoerotic stimulation and begging. Langurs and group-housed macaques did not exhibit undesirable behaviors. A male lion-tailed macaque and a male gibbon exhibited begging behavior. autoerotic stimulation and self-biting occurred rarely. Males exhibited higher levels of undesirable behavior than did females. Animals confiscated from touring zoos, circuses, and animal traders exhibited higher levels of abnormal behaviors than did animals reared in larger, recognized zoos. The stump-tailed macaque was the only species to exhibit floating limb, autoerotic stimulation, self-biting, and self-clasping. Our results show that rearing experience and group composition influence the proportions of abnormal behavior exhibited by nonhuman primates in captivity. The history of early social and environmental deprivation in these species of captive nonhuman primates probably is critical in the development of behavioral pathologies. Establishing this will require further research. PMID:14965782

  3. First-Trimester Detection of Surface Abnormalities

    PubMed Central

    Rousian, Melek; Koning, Anton H. J.; Bonsel, Gouke J.; Eggink, Alex J.; Cornette, Jérôme M. J.; Schoonderwaldt, Ernst M.; Husen-Ebbinge, Margreet; Teunissen, Katinka K.; van der Spek, Peter J.; Steegers, Eric A. P.; Exalto, Niek

    2014-01-01

    The aim was to determine the diagnostic performance of 3-dimensional virtual reality ultrasound (3D_VR_US) and conventional 2- and 3-dimensional ultrasound (2D/3D_US) for first-trimester detection of structural abnormalities. Forty-eight first trimester cases (gold standard available, 22 normal, 26 abnormal) were evaluated offline using both techniques by 5 experienced, blinded sonographers. In each case, we analyzed whether each organ category was correctly indicated as normal or abnormal and whether the specific diagnosis was correctly made. Sensitivity in terms of normal or abnormal was comparable for both techniques (P = .24). The general sensitivity for specific diagnoses was 62.6% using 3D_VR_US and 52.2% using 2D/3D_US (P = .075). The 3D_VR_US more often correctly diagnosed skeleton/limb malformations (36.7% vs 10%; P = .013). Mean evaluation time in 3D_VR_US was 4:24 minutes and in 2D/3D_US 2:53 minutes (P < .001). General diagnostic performance of 3D_VR_US and 2D/3D_US apparently is comparable. Malformations of skeleton and limbs are more often detected using 3D_VR_US. Evaluation time is longer in 3D_VR_US. PMID:24440996

  4. Sensory Abnormalities in Autism: A Brief Report

    ERIC Educational Resources Information Center

    Klintwall Lars; Holm, Anette; Eriksson, Mats; Carlsson, Lotta Hoglund; Olsson, Martina Barnevik; Hedvall, Asa; Gillberg, Christopher; Fernell, Elisabeth

    2011-01-01

    Sensory abnormalities were assessed in a population-based group of 208 20-54-month-old children, diagnosed with autism spectrum disorder (ASD) and referred to a specialized habilitation centre for early intervention. The children were subgrouped based upon degree of autistic symptoms and cognitive level by a research team at the centre. Parents…

  5. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin

    PubMed Central

    Fornasa, Giulia; Tsilingiri, Katerina; Caprioli, Flavio; Botti, Fiorenzo; Mapelli, Marina; Meller, Stephan; Kislat, Andreas; Homey, Bernhard; Di Sabatino, Antonio; Sonzogni, Angelica; Viale, Giuseppe; Diaferia, Giuseppe; Gori, Alessandro; Longhi, Renato; Penna, Giuseppe; Rescigno, Maria

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. Objective In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. Methods We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. Results We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. Conclusions We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform. PMID:26014813

  6. Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

    PubMed Central

    Fitzwater, Leah; Nichols, Rebekah

    2015-01-01

    ABSTRACT Clostridium difficile is a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenic C. difficile strains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation. C. difficile toxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166 was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms on C. difficile cell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166 and TcdE142 had a profound effect on cell viability compared to the TcdE140 isoform. Similarly, TcdE166 and TcdE142 facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed. IMPORTANCE C. difficile is a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains of C. difficile produce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are

  7. Sodium Kinetics of Na,K-ATPase α Isoforms in Intact Transfected HeLa Cells

    PubMed Central

    Zahler, Raphael; Zhang, Zhong-Ting; Manor, Mira; Boron, Walter F.

    1997-01-01

    By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four α isoforms of the pump, α1 is ubiquitous, α2 is predominant in skeletal muscle, and α3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat α subunits were highly ouabain resistant: the α1 isoform is naturally resistant, whereas the α2 and α3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 μM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human α1 isoform was 12 mM, which was similar to the Km of rat α1. The α2 and α3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing α3 had a high resting [Na+]i. The maximal activity of native α1 in the α3-transfected cells was only ∼56% of native α1 activity in untransfected HeLa cells, suggesting that transfection with α3 led to a compensatory decrease in endogenous α1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence α1 < α2 < α3. The α3 isoform may be suited for handling large Na+ loads in electrically active cells. PMID:9236212

  8. Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

    PubMed Central

    Tseng, Yeu-Yang; Lin, Fong-Yuan; Cheng, Sun-Fang; Chulakasian, Songkhla; Chou, Chia-Chi; Liu, Ya-Fen; Chang, Wei-Shan; Wong, Min-Liang

    2015-01-01

    ABSTRACT Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. PMID:25694596

  9. [Preparation and properties of isocitrate lyase isoforms from the cotyledons of Glycine max L].

    PubMed

    Eprintsev, A T; Diachenko, E V; Lykova, T V; Kuen, C T H; Popov, V N

    2010-01-01

    A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays. PMID:20198926

  10. Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice.

    PubMed

    Kim, Myung-Hee; de Beer, Maria C; Wroblewski, Joanne M; Charnigo, Richard J; Ji, Ailing; Webb, Nancy R; de Beer, Frederick C; van der Westhuyzen, Deneys R

    2016-06-01

    The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism. PMID:27018443

  11. iReckon: simultaneous isoform discovery and abundance estimation from RNA-seq data.

    PubMed

    Mezlini, Aziz M; Smith, Eric J M; Fiume, Marc; Buske, Orion; Savich, Gleb L; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y; Goldenberg, Anna; Brudno, Michael

    2013-03-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r(2) = 0.94) with the predicted abundances. PMID:23204306

  12. iReckon: Simultaneous isoform discovery and abundance estimation from RNA-seq data

    PubMed Central

    Mezlini, Aziz M.; Smith, Eric J.M.; Fiume, Marc; Buske, Orion; Savich, Gleb L.; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y.; Goldenberg, Anna; Brudno, Michael

    2013-01-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r2 = 0.94) with the predicted abundances. PMID:23204306

  13. Most highly expressed protein-coding genes have a single dominant isoform.

    PubMed

    Ezkurdia, Iakes; Rodriguez, Jose Manuel; Carrillo-de Santa Pau, Enrique; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-04-01

    Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level. We interrogate peptides from eight large-scale human proteomics experiments and databases and find that there is a single dominant protein isoform, irrespective of tissue or cell type, for the vast majority of the protein-coding genes in these experiments, in partial agreement with the conclusions from the most recent large-scale RNAseq study. Remarkably, the dominant isoforms from the experimental proteomics analyses coincided overwhelmingly with the reference isoforms selected by two completely orthogonal sources, the consensus coding sequence variants, which are agreed upon by separate manual genome curation teams, and the principal isoforms from the APPRIS database, predicted automatically from the conservation of protein sequence, structure, and function. PMID:25732134

  14. Differential Roles of Postsynaptic Density-93 Isoforms in Regulating Synaptic Transmission

    PubMed Central

    Krüger, Juliane M.; Favaro, Plinio D.; Liu, Mingna; Kitlińska, Agata; Huang, Xiaojie; Raabe, Monika; Akad, Derya S.; Liu, Yanling; Urlaub, Henning; Dong, Yan; Xu, Weifeng

    2013-01-01

    In the postsynaptic density of glutamatergic synapses, the discs large (DLG)-membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins coordinates a multiplicity of signaling pathways to maintain and regulate synaptic transmission. Postsynaptic density-93 (PSD-93) is the most variable paralog in this family; it exists in six different N-terminal isoforms. Probably because of the structural and functional variability of these isoforms, the synaptic role of PSD-93 remains controversial. To accurately characterize the synaptic role of PSD-93, we quantified the expression of all six isoforms in the mouse hippocampus and examined them individually in hippocampal synapses. Using molecular manipulations, including overexpression, gene knockdown, PSD-93 knock-out mice combined with biochemical assays, and slice electrophysiology both in rat and mice, we demonstrate that PSD-93 is required at different developmental synaptic states to maintain the strength of excitatory synaptic transmission. This strength is differentially regulated by the six isoforms of PSD-93, including regulations of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-active and inactive synapses, and activity-dependent modulations. Collectively, these results demonstrate that alternative combinations of N-terminal PSD-93 isoforms and DLG-MAGUK paralogs can fine-tune signaling scaffolds to adjust synaptic needs to regulate synaptic transmission. PMID:24068818

  15. Differential expression of VegT and Antipodean protein isoforms in Xenopus.

    PubMed

    Stennard, F; Zorn, A M; Ryan, K; Garrett, N; Gurdon, J B

    1999-08-01

    The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. We conclude that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors. PMID:10446268

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

    SciTech Connect

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

    2011-08-26

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

  17. Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus.

    PubMed

    Westermeier, F; Sáez, T; Arroyo, P; Toledo, F; Gutiérrez, J; Sanhueza, C; Pardo, F; Leiva, A; Sobrevia, L

    2016-05-01

    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 related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26431063

  18. Immunohistochemical localization of transforming growth factor beta isoforms in asbestos-related diseases.

    PubMed Central

    Jagirdar, J; Lee, T C; Reibman, J; Gold, L I; Aston, C; Bégin, R; Rom, W N

    1997-01-01

    Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. PMID:9400723

  19. Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

    PubMed Central

    Kurtz, Joachim; Schmucker, Dietmar; Chen, Wei

    2014-01-01

    The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens. PMID:25310676

  20. Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms.

    PubMed

    Schmidt, T R; Jaradat, S A; Goodman, M; Lomax, M I; Grossman, L I

    1997-06-01

    Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions. PMID:9190060

  1. Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

    PubMed

    Suzuki, Miwa; Wakui, Hitomi; Itou, Takuya; Segawa, Takao; Inoshima, Yasuo; Maeda, Ken; Kikuchi, Kiyoshi

    2016-04-15

    This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, ITALIC! Tursiops truncatus mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg(-1)) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body. PMID:26944501

  2. Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

    PubMed

    Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2010-04-12

    A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability. PMID:19969041

  3. Osteopontin (OPN/SPP1) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

    PubMed Central

    Lin, Jules; Myers, Amy L.; Wang, Zhuwen; Nancarrow, Derek J.; Ferrer-Torres, Daysha; Handlogten, Amy; Leverenz, Kimmy; Bao, Julia; Thomas, Dafydd G.; Wang, Thomas D.; Orringer, Mark B.; Reddy, Rishindra M.; Chang, Andrew C.; Beer, David G.; Lin, Lin

    2015-01-01

    Esophageal adenocarcinoma (EAC) is often diagnosed at an advanced stage, thus understanding the molecular basis for EAC invasion and metastasis is critical. Here we report that SPP1/OPN was highly overexpressed in primary EACs and intracellularly localized to tumor cells. We further demonstrate that all known OPN isoforms (OPNa, b, c, 4 and 5) were frequently co-overexpressed in primary EACs. Distinct pro-invasion and dissemination phenotypes of isoform-specific OPNb and OPNc stable transfectants were observed. Expression of OPNb significantly enhanced cell migration and adhesion to laminin. In contrast, OPNc cells showed significantly decreased cell migration yet increased cell detachment. Enhanced invasion, both in vitro and in vivo, was observed for OPNb- but not OPNc-expressing cells. Inhibition of RGD integrins, one family of OPN receptors, attenuated OPNb cell migration, abrogated OPNb cell adhesion and significantly reduced OPNb cell clonogenic survival but did not affect OPNc phenotypes, indicating that OPNb but not OPNc acts through integrin-dependent signaling. Differential expression of vimentin, E-cadherin and β-catenin in OPN stable cells may account for the variation in cell adhesion and detachment between these isoforms. We conclude that while all OPN isoforms are frequently co-overexpressed in primary EACs, isoforms OPNb and OPNc enhance invasion and dissemination through collective yet distinct mechanisms. PMID:26068949

  4. The ZFHX1A Gene Is Differentially Autoregulated By Its Isoforms

    PubMed Central

    Manavella, Pablo A.; Roqueiro, Gonzalo; Darling, Douglas S.; Cabanillas, Ana M.

    2009-01-01

    The Zfhx1a gene expresses two different isoforms; the full length Zfhx1a-1 and a truncated isoform termed Zfhx1a-2 lacking the first exon. Deletion analysis of the Zfhx1a-1 promoter localized cell-specific repressors, and a proximal G-string that is critically required for transactivation. Transfection of Zfhx1a-1 cDNA, but not Zfhx1a -2, downregulates Zfhx1a-1 promoter activity. Mutation of an E2-box disrupted the binding of both Zfhx1a isoforms. Consistent with this, transfected Zfhx1a-1 does not regulate the transcriptional activity of the E-box mutated Zfhx1a-1 promoter. Competitive EMSAs and transfection assays show that Zfhx1a-2 can function as a dominant negative isoform since it is able to compete and displace Zfhx1a-1 from its binding site and overcome Zfhx1a-1 induced repression of the Zfhx1a-1 promoter in cells. Hence, the Zfhx1a-1 gene is autoregulated in part by negative feedback on its own promoter which is, in turn, modified by the availability of the negative dominant isoform Zfhx1a-2. PMID:17610840

  5. The ZFHX1A gene is differentially autoregulated by its isoforms.

    PubMed

    Manavella, Pablo A; Roqueiro, Gonzalo; Darling, Douglas S; Cabanillas, Ana M

    2007-08-31

    The Zfhx1a gene expresses two different isoforms; the full length Zfhx1a-1 and a truncated isoform termed Zfhx1a-2 lacking the first exon. Deletion analysis of the Zfhx1a-1 promoter localized cell-specific repressors, and a proximal G-string that is critically required for transactivation. Transfection of Zfhx1a-1 cDNA, but not Zfhx1a-2, downregulates Zfhx1a-1 promoter activity. Mutation of an E2-box disrupted the binding of both Zfhx1a isoforms. Consistent with this, transfected Zfhx1a-1 does not regulate the transcriptional activity of the E-box mutated Zfhx1a-1 promoter. Competitive EMSAs and transfection assays show that Zfhx1a-2 can function as a dominant negative isoform since it is able to compete and displace Zfhx1a-1 from its binding site and overcome Zfhx1a-1 induced repression of the Zfhx1a-1 promoter in cells. Hence, the Zfhx1a-1 gene is autoregulated in part by negative feedback on its own promoter which is, in turn, modified by the availability of the negative dominant isoform Zfhx1a-2. PMID:17610840

  6. An innovative approach for the characterization of the isoforms of a monoclonal antibody product

    PubMed Central

    Sundaram, Shanmuuga; Matathia, Alice; Qian, Jun; Zhang, Jingming; Hsieh, Ming-Ching; Liu, Tun; Crowley, Richard

    2011-01-01

    Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS. PMID:22123057

  7. The N-terminal Set-β Protein Isoform Induces Neuronal Death.

    PubMed

    Trakhtenberg, Ephraim F; Morkin, Melina I; Patel, Karan H; Fernandez, Stephanie G; Sang, Alan; Shaw, Peter; Liu, Xiongfei; Wang, Yan; Mlacker, Gregory M; Gao, Han; Velmeshev, Dmitry; Dombrowski, Susan M; Vitek, Michael P; Goldberg, Jeffrey L

    2015-05-22

    Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death. PMID:25833944

  8. A Subtle Alternative Splicing Event Gives Rise to a Widely Expressed Human RNase k Isoform

    PubMed Central

    Karousis, Evangelos D.; Sideris, Diamantis C.

    2014-01-01

    Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms. PMID:24797913

  9. Secretion of PDGF isoforms during osteoclastogenesis and its modulation by anti-osteoclast drugs.

    PubMed

    Rahman, M Motiur; Matsuoka, Kazuhiko; Takeshita, Sunao; Ikeda, Kyoji

    2015-06-26

    In an attempt to identify secretory products of osteoclasts that mediate the coupling of bone formation to resorption, we found that along with osteoclast differentiation, PDGF-A gene expression increase occurred first, by 12 h after stimulation of bone marrow macrophages with M-CSF and RANKL, and peaked at 36 h. This was next followed by a progressive increase in PDGF-B gene expression until a peak at 60 h, when mature osteoclasts formed. Isoform-specific ELISA of the conditioned medium collected every 24 h revealed that all three of the isoforms of PDGF-AA, AB and BB were secreted, in this temporal order as differentiation proceeded. Their secretion was enhanced when osteoclasts were activated by placing them on dentin slices. The secretion of all three isoforms was decreased in cathepsin K-deficient osteoclasts compared with wild-type osteoclasts. Pharmacological inhibition of cathepsin K with odanacatib also inhibited the secretion of all three isoforms, as was also the case with alendronate treatment. The secretion of sphingosine-1-phosphate, which increased during osteoclastogenesis, was reduced from cathepsin K-deficient osteoclasts, and was inhibited by treatment with odanacatib more profoundly than with alendronate. Thus, all three isoforms of PDGF, which are secreted at distinct differentiation stages of osteoclasts, appear to have distinct roles in the cell-cell communication that takes place in the microenvironment of bone remodeling, especially from the osteoclast lineage to mesenchymal cells and vascular cells, thereby stimulating osteogenesis and angiogenesis. PMID:25951977

  10. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity

    PubMed Central

    Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W.

    2016-01-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies. PMID:26909609

  11. Tau in Alzheimer's disease and Down's syndrome is insoluble and abnormally phosphorylated.

    PubMed Central

    Hanger, D P; Brion, J P; Gallo, J M; Cairns, N J; Luthert, P J; Anderton, B H

    1991-01-01

    Some investigators have described the presence in Alzheimer's disease brain extracts of several abnormal forms of the microtubule-associated protein tau, based on their unusual mobility in SDS/PAGE. It has been proposed that these abnormal forms of tau may be the result of aberrant tau phosphorylation. In this study we show that tau in extracts of Alzheimer's disease brain can be separated into two fractions based upon its solubility (100,000 g x 1 h supernatant) in non-denaturing conditions (100 mM-Mes, pH 6.5, 0.5 mM-MgCl2, 1 mM-EGTA and 1 M-NaCl). The tau isoforms with decreased mobility in SDS/PAGE are predominantly in an insoluble fraction, whereas the soluble tau is indistinguishable by its mobility in SDS/PAGE from tau in soluble extracts of control brain. Insoluble tau displaying abnormal mobility on SDS/PAGE was only found in Alzheimer and adult Down's syndrome brains and was absent from the brains of age-matched controls and from foetal and infant Down's syndrome brains. There was a good correlation between the presence of insoluble tau in brain extracts and the abundance of neurofibrillary tangles and senile neuritic plaques. The monoclonal antibody Tau. 1 stained insoluble tau on Western blots only after treatment of the nitrocellulose transfers with alkaline phosphatase, implying that this insoluble tau is in a particular state of phosphorylation. We conclude that, in Alzheimer's disease, a fraction of tau has a modified phosphorylation state and a decreased solubility; these modifications may precede formation of the neurofibrillary tangles characteristic of Alzheimer's disease and Down's syndrome in adults. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1826835

  12. Enhancement of phagocytotic activity by prion protein in PrP-deficient macrophage cells.

    PubMed

    Uraki, Ryuta; Sakudo, Akikazu; Ando, Saeko; Kitani, Hiroshi; Onodera, Takashi

    2010-10-01

    Macrophages, especially follicular dendritic cells, contribute to the pathogenesis of prion diseases by accumulating an abnormal isoform of prion protein (PrPSc), which is converted from the cellular isoform of prion protein (PrPC). As information on the function of PrPC in macrophages is limited, we have established a prion protein (PrP) gene (Prnp)-deficient macrophage cell line from the bone marrow of ZrchI Prnp-/- mice. These cells expressed macrophage specific proteins (F4/80 and MOMA-2) and displayed phagocytotic properties. The Prnp-/- macrophage cell line (MplZ) showed shorter pseudopodium extension and less phagocytotic activity than a Prnp+/+ macrophage cell line (MWF). In addition, the MplZ cells were more sensitive to serum deprivation than the MWF cells and underwent apoptotic cell death in these conditions. These findings suggest that PrPC enhances the incorporation of materials possibly including PrPSc and decreases the sensitivity of cells to oxidative stress, which may be induced by PrPSc accumulation. PMID:20818492

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24722188

  14. Drug Delivery Innovations for Enhancing the Anticancer Potential of Vitamin E Isoforms and Their Derivatives

    PubMed Central

    Neophytou, Christiana M.; Constantinou, Andreas I.

    2015-01-01

    Vitamin E isoforms have been extensively studied for their anticancer properties. Novel drug delivery systems (DDS) that include liposomes, nanoparticles, and micelles are actively being developed to improve Vitamin E delivery. Furthermore, several drug delivery systems that incorporate Vitamin E isoforms have been synthesized in order to increase the bioavailability of chemotherapeutic agents or to provide a synergistic effect. D-alpha-tocopheryl polyethylene glycol succinate (Vitamin E TPGS or TPGS) is a synthetic derivative of natural alpha-tocopherol which is gaining increasing interest in the development of drug delivery systems and has also shown promising anticancer effect as a single agent. This review provides a summary of the properties and anticancer effects of the most potent Vitamin E isoforms and an overview of the various formulations developed to improve their efficacy, with an emphasis on the use of TPGS in drug delivery approaches. PMID:26137487

  15. Dynamic conformational ensembles regulate casein kinase-1 isoforms: Insights from molecular dynamics and molecular docking studies.

    PubMed

    Singh, Surya Pratap; Gupta, Dwijendra K

    2016-04-01

    Casein kinase-1 (CK1) isoforms actively participate in the down-regulation of canonical Wnt signaling pathway; however recent studies have shown their active roles in oncogenesis of various tissues through this pathway. Functional loss of two isoforms (CK1-α/ε) has been shown to activate the carcinogenic pathway which involves the stabilization of of cytoplasmic β-catenin. Development of anticancer therapeutics is very laborious task and depends upon the structural and conformational details of the target. This study focuses on, how the structural dynamics and conformational changes of two CK1 isoforms are synchronized in carcinogenic pathway. The conformational dynamics in kinases is the responsible for their action as has been supported by the molecular docking experiments. PMID:26788877

  16. Altered CD45 isoform expression affects lymphocyte function in CD45 Tg mice.

    PubMed

    Tchilian, Elma Z; Dawes, Ritu; Hyland, Lisa; Montoya, Maria; Le Bon, Agnes; Borrow, Persephone; Hou, Sam; Tough, David; Beverley, Peter C L

    2004-09-01

    Transgenic mice have been constructed expressing high (CD45RABC) and low (CD45R0) molecular weight CD45 isoforms on a CD45-/- background. Phenotypic analysis and in vivo challenge of these mice with influenza and lymphocytic choriomeningitis viruses shows that T cell differentiation and peripheral T cell function are related to the level of CD45 expression but not to which CD45 isoform is expressed. In contrast, B cell differentiation is not restored, irrespective of the level of expression of a single isoform. All CD45 trangenic mice have T cells with an activated phenotype and increased T cell turnover. These effects are more prominent in CD8 than CD4 cells. The transgenic mice share several properties with humans expressing variant CD45 alleles and provide a model to understand immune function in variant individuals. PMID:15302847

  17. NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase.

    PubMed

    Omairi-Nasser, Amin; Galmozzi, Carla V; Latifi, Amel; Muro-Pastor, M Isabel; Ajlani, Ghada

    2014-04-01

    In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform. PMID:24464800

  18. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  19. DJ-1 isoforms in whole blood as potential biomarkers of Parkinson disease

    NASA Astrophysics Data System (ADS)

    Lin, Xiangmin; Cook, Travis J.; Zabetian, Cyrus P.; Leverenz, James B.; Peskind, Elaine R.; Hu, Shu-Ching; Cain, Kevin C.; Pan, Catherine; Edgar, John Scott; Goodlett, David R.; Racette, Brad A.; Checkoway, Harvey; Montine, Thomas J.; Shi, Min; Zhang, Jing

    2012-12-01

    DJ-1 is a multifunctional protein that plays an important role in oxidative stress, cell death, and synucleinopathies, including Parkinson disease. Previous studies have demonstrated that total DJ-1 levels decrease in the cerebrospinal fluid, but do not change significantly in human plasma from patients with Parkinson disease when compared with controls. In this study, we measured total DJ-1 and its isoforms in whole blood of patients with Parkinson disease at various stages, Alzheimer disease, and healthy controls to identify potential peripheral biomarkers of PD. In an initial discovery study of 119 subjects, 7 DJ-1 isoforms were reliably detected, and blood levels of those with 4-hydroxy-2-nonenal modifications were discovered to be altered in late-stage Parkinson disease. This result was further confirmed in a validation study of another 114 participants, suggesting that, unlike total DJ-1 levels, post-translationally modified isoforms of DJ-1 from whole blood are candidate biomarkers of late-stage Parkinson disease.

  20. Pyridinium derivatives of histamine are potent activators of cytosolic carbonic anhydrase isoforms I, II and VII.

    PubMed

    Dave, Khyati; Scozzafava, Andrea; Vullo, Daniela; Supuran, Claudiu T; Ilies, Marc A

    2011-04-21

    A series of positively-charged derivatives has been prepared by reaction of histamine with substituted pyrylium salts. These pyridinium histamine derivatives were investigated as activators of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely the human isoforms hCA I, II and VII. Activities from the subnanomolar to the micromolar range were detected for these compounds as activators of the three isoforms, confirming the validity of current and previous designs. The substitution pattern at the pyridinium ring was the main factor influencing activity, the three isoforms showing different structural requirements for good activity, related with the number of pyridinium substituting groups and their nature, among various alkyl, phenyl and para-substituted styryl moieties. We were successful in identifying nanomolar potent and selective activators for each isozyme and also activators with a relatively good activity against all isozymes tested--valuable lead compounds for physiology and pathology studies involving these isozymes. PMID:21369613

  1. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

    PubMed

    Bogdanov, I V; Finkina, E I; Balandin, S V; Melnikova, D N; Stukacheva, E A; Ovchinnikova, T V

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity. PMID:26483961

  2. Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein

    PubMed Central

    Bogdanov, I. V.; Finkina, E. I.; Balandin, S. V.; Melnikova, D. N.; Stukacheva, E. A.; Ovchinnikova, T. V.

    2015-01-01

    The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity. PMID:26483961

  3. CpG Island Hypermethylation Frequently Silences FILIP1L Isoform 2 Expression in Prostate Cancer

    PubMed Central

    Desotelle, Joshua; Truong, Matthew; Ewald, Jonathan; Weeratunga, Pushpa; Yang, Bing; Huang, Wei; Jarrard, David

    2013-01-01

    Purpose Senescence related regulatory pathways serve as barriers to cancer immortalization and progression but they are currently not well defined. FILIP1L is a growth inhibitory gene with multiple isoforms whose expression is increased in senescent prostate and prostate cancer cells, and decreased in many cancers. We investigated whether DNA methylation regulates FILIP1L in senescence and in prostate cancer development. Materials and Methods FILIP1L mRNA expression was assessed in prostate cancer and associated normal prostate tissues using quantitative polymerase chain reaction. A tissue microarray was constructed using 95 prostate cancer specimens and 45 benign prostate specimens. Vectra™ imaging was used to quantitate nuclear and cytoplasmic FILIP1L protein expression. Bisulfite sequencing and Pyrosequencing® were used to assess methylation. Prostate cancer cell lines were treated with 2′-deoxy-5-azacytidine and mRNA expression was assessed. Results FILIP1L isoform 2 mRNA was increased in replicatively senescent human prostate epithelial cells and decreased in prostate cancer specimens. We verified a reduction in nuclear FILIP1L protein in prostate cancer using tissue microarrays (p = 0.006). A CpG island 5′ of the isoform 2 translational start site was identified that showed hypermethylation in prostate cancer cell lines and tumors compared to normal prostate cells and tissues. Pyrosequencing confirmed FILIP1L hypermethylation in all 14 tumors compared to paired normal tissues (p <0.0001). Isoform 2 expression was induced in prostate cancer cell lines using 2′-deoxy-5-azacytidine. Conclusions FILIP1L isoform 2 is one of the most commonly hypermethylated genes in prostate cancer. It may serve as an important marker of prostate cancer. Isoform 2 expression is associated with senescence and its down-regulation may represent an early important biological event in prostate cancer development. PMID:23174249

  4. CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis.

    PubMed

    Ramesh, M; Bharatam, Prasad V

    2012-02-01

    Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms. PMID:21562823

  5. Smad phospho-isoforms direct context-dependent TGF-β signaling.

    PubMed

    Matsuzaki, Koichi

    2013-08-01

    Better understanding of TGF-β signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-β receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-β change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-β signaling. PMID:23871609

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

    PubMed Central

    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

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

    PubMed

    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

  8. Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms

    PubMed Central

    Tabuchi, Mitsuaki; Tanaka, Naotaka; Nishida-Kitayama, Junko; Ohno, Hiroshi; Kishi, Fumio

    2002-01-01

    Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3′ exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3′ untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type–specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y555XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type–specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types. PMID:12475959

  9. Topographies and isoforms of the progesterone receptor in female human, rat and mouse bladder.

    PubMed

    Gevaert, Thomas; Rietjens, Roma; Voets, Thomas; Everaerts, Wouter; De Ridder, Dirk

    2016-05-01

    Steroid hormones such as progesterone are known to influence bladder function. Progesterone effects are mediated by the progesterone receptor (PR) but no detailed studies of PR in bladder exist. We have investigated the presence, topography and subtypes of PR in mouse, rat and human bladder. Fresh tissue samples were obtained from cystectomies in female humans, rats and mice (n = 7 per group). Tissue samples were processed for immunohistochemistry (IHC), immunofluorescence (IF) and western blot (WB) and, for each species, a panel of specific PR antibody clones was used. Interpretation of IHC/IF was carried out by light/fluorescent microscopy and of WB via standard WB software. IHC/IF in female human bladder showed PR on the interstitial cells in the lamina propria and between detrusor smooth muscle cells, whereas in female rat and mouse bladder, PR was only found on the urothelium. WB in human bladder showed a 78-kD and a 60-kDa band, respectively, corresponding to a modified PR isoform A and PR isoform C. WB in rat and mice bladder showed a 60 kDa band and a 37 kDa band, respectively corresponding with PR isoform C and an unknown isoform. This is the first detailed investigation of the precise location and presence of several isoforms of PR in bladder, together with a comparison of these data between human, rat and mouse. Our study has revealed complex PR families in bladders from the various species studied and demonstrates obvious inter-species differences in PR topography and isoforms. PMID:26650465

  10. The α and Δ Isoforms of CREB1 Are Required to Maintain Normal Pulmonary Vascular Resistance

    PubMed Central

    Sands, Michelle; Banahan, Mark; Frohlich, Stephen; Rowan, Simon C.; Neary, Roisín; Ryan, Donal; McLoughlin, Paul

    2013-01-01

    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 (CREBαΔ mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREBαΔ mice. CREBαΔ 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 CREBαΔ 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 CREBαΔ. CREBαΔ 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. PMID:24349008

  11. Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

    PubMed Central

    Schoffstall, Brenda; Brunet, Nicolas M; Williams, Shanedah; Miller, Victor F; Barnes, Alyson T; Wang, Fang; Compton, Lisa A; McFadden, Lori A; Taylor, Dianne W; Seavy, Margaret; Dhanarajan, Rani; Chase, P Bryant

    2006-01-01

    Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca2+ activation. To determine whether Ca2+ sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and α-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca2+ sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca2+ sensitivity. PMID:17008370

  12. Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    PubMed Central

    Shugg, Ryan P. P.; Thomson, Ashley; Tanabe, Natsuko; Kashishian, Adam; Steiner, Bart H.; Puri, Kamal D.; Pereverzev, Alexey; Lannutti, Brian J.; Jirik, Frank R.; Dixon, S. Jeffrey; Sims, Stephen M.

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics. PMID:24133210

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

    PubMed

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p < 0.0001 for LS, p < 0.01 for MS). This study is the first to reveal the dominance of glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice. PMID:26643381

  14. The coevolution of insect muscle TpnT and TpnI gene isoforms.

    PubMed

    Herranz, Raúl; Mateos, Jesús; Mas, José A; García-Zaragoza, Elena; Cervera, Margarita; Marco, Roberto

    2005-11-01

    In bilaterians, the main regulator of muscle contraction is the troponin (Tpn) complex, comprising three closely interacting subunits (C, T, and I). To understand how evolutionary forces drive molecular change in protein complexes, we have compared the gene structures and expression patterns of Tpn genes in insects. In this class, while TpnC is encoded by multiple genes, TpnT and TpnI are encoded by single genes. Their isoform expression pattern is highly conserved within the Drosophilidae, and single orthologous genes were identified in the sequenced genomes of Drosophila pseudoobscura, Anopheles gambiae, and Apis mellifera. Apis expression patterns also support the equivalence of their exon organization throughout holometabolous insects. All TpnT genes include a previously unidentified indirect flight muscle (IFM)-specific exon (10A) that has evolved an expression pattern similar to that of exon 9 in TpnI. Thus, expression patterns, sequence evolution trends, and structural data indicate that Tpn genes and their isoforms have coevolved, building species- and muscle-specific troponin complexes. Furthermore, a clear case can be made for independent evolution of the IFM-specific isoforms containing alanine/proline-rich sequences. Dipteran genomes contain one tropomyosin gene that encodes one or two high-molecular weight isoforms (TmH) incorporating APPAEGA-rich sequences, specifically expressed in IFM. Corresponding exons do not exist in the Apis tropomyosin gene, but equivalent sequences occur in a high-molecular weight Apis IFM-specific TpnI isoform (TnH). Overall, our approach to comparatively analyze supramolecular complexes reveals coevolutionary trends not only in gene families but in isoforms generated by alternative splicing. PMID:16049195

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

    PubMed

    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

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

    PubMed Central

    2011-01-01

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

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

    PubMed

    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. PMID:25666558

  18. Characterization of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability.

    PubMed

    Keown, Jeremy R; Pearce, Frederick Grant

    2014-12-15

    Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (β-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach α-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the α-isoform can act as a structural scaffold for hexamer formation by the β-isoform. Additionally, it is shown that a variant of the tobacco β-isoform acts in a similar fashion to the α-isoform of spinach, forming thermally stable hexamers in the presence of ATPγS. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface. PMID:25247706

  19. Chromosomal abnormalities in a psychiatric population

    SciTech Connect

    Lewis, K.E.; Lubetsky, M.J.; Wenger, S.L.; Steele, M.W.

    1995-02-27

    Over a 3.5 year period of time, 345 patients hospitalized for psychiatric problems were evaluated cytogenetically. The patient population included 76% males and 94% children with a mean age of 12 years. The criteria for testing was an undiagnosed etiology for mental retardation and/or autism. Cytogenetic studies identified 11, or 3%, with abnormal karyotypes, including 4 fragile X positive individuals (2 males, 2 females), and 8 with chromosomal aneuploidy, rearrangements, or deletions. While individuals with chromosomal abnormalities do not demonstrate specific behavioral, psychiatric, or developmental problems relative to other psychiatric patients, our results demonstrate the need for an increased awareness to order chromosomal analysis and fragile X testing in those individuals who have combinations of behavioral/psychiatric, learning, communication, or cognitive disturbance. 5 refs., 1 fig., 2 tabs.

  20. Abnormal grain growth in TD-nickel.

    NASA Technical Reports Server (NTRS)

    Petrovic, J. J.; Ebert, L. J.

    1972-01-01

    Characteristics of the coarse grain transformation occurring in TD-nickel 1 in. bar under certain conditions of deformation and annealing were examined. The transformation exhibits Avrami-type kinetics, with an activation energy of 250 kcal per mole. Characteristics of untransformed regions are like those of the as-received state. The transformed grain size increases with increasing deformation and decreasing annealing temperature. The coarse grain transformation is significantly different from primary recrystallization in pure nickel. Its characteristics cannot be rationalized in terms of primary recrystallization concepts, but may be explained in terms of an abnormal grain growth description. The coarse grain transformation in TD-nickel is abnormal grain growth rather than primary recrystallization. The analysis suggests an explanation for the effect of thermomechanical history on the deformation and annealing behavior of TD-nickel.

  1. Evaluation of abnormal liver function tests.

    PubMed

    Agrawal, Swastik; Dhiman, Radha K; Limdi, Jimmy K

    2016-04-01

    Incidentally detected abnormality in liver function tests is a common situation encountered by physicians across all disciplines. Many of these patients do not have primary liver disease as most of the commonly performed markers are not specific for the liver and are affected by myriad factors unrelated to liver disease. Also, many of these tests like liver enzyme levels do not measure the function of the liver, but are markers of liver injury, which is broadly of two types: hepatocellular and cholestatic. A combination of a careful history and clinical examination along with interpretation of pattern of liver test abnormalities can often identify type and aetiology of liver disease, allowing for a targeted investigation approach. Severity of liver injury is best assessed by composite scores like the Model for End Stage Liver Disease rather than any single parameter. In this review, we discuss the interpretation of the routinely performed liver tests along with the indications and utility of quantitative tests. PMID:26842972

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

    PubMed Central

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

    2014-01-01

    The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. PMID:24102585

  3. Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins*

    PubMed Central

    Mehus, Aaron A.; Muhonen, Wallace W.; Garrett, Scott H.; Somji, Seema; Sens, Donald A.; Shabb, John B.

    2014-01-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These

  4. Properties of SEPT9 isoforms and the requirement for GTP binding.

    PubMed

    Robertson, Claire; Church, Stewart W; Nagar, Hans A; Price, John; Hall, Peter A; Russell, S E Hilary

    2004-05-01

    Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions

  5. Multiple isoforms of myofibrillar proteins in crustacean muscle: evidence for two slow fiber types

    SciTech Connect

    Mykles, D.L.

    1986-01-01

    Four distinct patterns of myofibrillar proteins, extracted from fast and slow muscles of the lobster, Homarus americanus, are distinguished by different assemblages of regulatory and contractile protein variants. Multiple isoforms of troponin-T, -I, and -C, paramyosin, and myosin light chains occur in six muscles of the claws and abdomen. Analysis of glycerinated fibers from the claws of lobster and land crab, Gecarcinus lateralis, show that more than one isoform is expressed in a single fiber, forming unique assemblages by which subgroups can be discriminated within the broader categories of fast and slow fibers. 9 refs., 3 figs.

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

    PubMed Central

    2010-01-01

    Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into

  7. hMENA(11a), a hMENA isoform sending survival signals.

    PubMed

    Trono, Paola; Di Modugno, Francesca; Nisticò, Paola

    2016-03-01

    Human MENA(11a) (hMENA(11a)), an epithelial-associated isoform of the actin binding protein enabled homolog (ENAH, also known as mammalian ENA [MENA]), is upregulated and phosphorylated following the activation of human epidermal growth factor receptor (HER) 1, HER2, and HER3. Here, we reveal a novel role of this isoform in sustaining cell survival and propose hMENA(11a) as a marker of HER3 activation and resistance to phosphatidylinositol-3-kinase inhibition therapies. PMID:27308605

  8. Esophageal motility abnormalities in gastroesophageal reflux disease

    PubMed Central

    Martinucci, Irene; de Bortoli, Nicola; Giacchino, Maria; Bodini, Giorgia; Marabotto, Elisa; Marchi, Santino; Savarino, Vincenzo; Savarino, Edoardo

    2014-01-01

    Esophageal motility abnormalities are among the main factors implicated in the pathogenesis of gastroesophageal reflux disease. The recent introduction in clinical and research practice of novel esophageal testing has markedly improved our understanding of the mechanisms contributing to the development of gastroesophageal reflux disease, allowing a better management of patients with this disorder. In this context, the present article intends to provide an overview of the current literature about esophageal motility dysfunctions in patients with gastroesophageal reflux disease. Esophageal manometry, by recording intraluminal pressure, represents the gold standard to diagnose esophageal motility abnormalities. In particular, using novel techniques, such as high resolution manometry with or without concurrent intraluminal impedance monitoring, transient lower esophageal sphincter (LES) relaxations, hypotensive LES, ineffective esophageal peristalsis and bolus transit abnormalities have been better defined and strongly implicated in gastroesophageal reflux disease development. Overall, recent findings suggest that esophageal motility abnormalities are increasingly prevalent with increasing severity of reflux disease, from non-erosive reflux disease to erosive reflux disease and Barrett’s esophagus. Characterizing esophageal dysmotility among different subgroups of patients with reflux disease may represent a fundamental approach to properly diagnose these patients and, thus, to set up the best therapeutic management. Currently, surgery represents the only reliable way to restore the esophagogastric junction integrity and to reduce transient LES relaxations that are considered to be the predominant mechanism by which gastric contents can enter the esophagus. On that ground, more in depth future studies assessing the pathogenetic role of dysmotility in patients with reflux disease are warranted. PMID:24868489

  9. [TMJ morphological changes in abnormal occlusion].

    PubMed

    Volkov, S I; Bazhenov, D V; Semkin, V A; Bogdanov, A O

    2013-01-01

    TMJ dysfunction is one of the most common diseases among all disorders of the maxillofacial region. Any abnormality in synchrony or amplitude of motion of the TMJ results in the malposition of the articular disc. Researchers and clinicians were always interested in topographic anatomy of the TMJ. There is currently no consensus on matters relating to changes in anatomical features of the TMJ by occlusal disturbances. PMID:23715443

  10. Congenital anorectal abnormalities in six dogs.

    PubMed

    Prassinos, N N; Papazoglou, L G; Adamama-Moraitou, K K; Galatos, A D; Gouletsou, P; Rallis, T S

    2003-07-19

    Congenital anorectal abnormalities were diagnosed in three male and three female dogs. One dog had anal stenosis, three had a persistent anal membrane, and the other two had an imperforate anus associated with a rectovaginal fistula. Five of the dogs were treated surgically, and four of them which were followed up for periods ranging from one to five years continued to pass faeces normally. PMID:12892267

  11. Practice and Educational Gaps in Abnormal Pigmentation.

    PubMed

    Mohammad, Tasneem F; Hamzavi, Iltefat H

    2016-07-01

    Dyschromia refers to abnormal pigmentation and is one of the most common diagnoses in dermatology. However, there are many educational and practice gaps in this area, specifically in melasma, postinflammatory hyperpigmentation, and vitiligo. This article aims to review the gold standard of care for these conditions as well as highlight common educational and practice gaps in these areas. Finally, possible solutions to these gaps are addressed. PMID:27363886

  12. CT of trauma to the abnormal kidney

    SciTech Connect

    Rhyner, P.; Federle, M.P.; Jeffrey, R.B.

    1984-04-01

    Traumatic injuries to already abnormal kidneys are difficult to assess by excretory urography and clinical evaluation. Bleeding and urinary extravasation may accompany minor trauma; conversely, underlying tumors, perirenal hemorrhage, and extravasation may be missed on urography. Computed tomography (CT) was performed in eight cases including three neoplasms, one adult polycystic disease, one simple renal cyst, two hydronephrotic kidneys, and one horseshoe kidney. CT provided specific and clinically useful information in each case that was not apparent on excretory urography.

  13. Chromosome abnormalities in chronic active hepatitis

    PubMed Central

    Stefanescu, D. T.; Moanga, M.; Teodorescu, M.; Brucher, J.

    1972-01-01

    An investigation on human peripheral blood lymphocyte chromosomes in chronic active hepatitis was carried out. A higher percentage of chromatid and chromosome lesions was recorded in all patients studied as compared with control groups—normal individuals, healthy subjects who had suffered from acute viral hepatitis, patients with alcoholic liver disease, and patients with mechanical jaundice due to cancer. The possible origin of these abnormalities is discussed. PMID:5076805

  14. Varenicline and Abnormal Sleep Related Events

    PubMed Central

    Savage, Ruth L.; Zekarias, Alem; Caduff-Janosa, Pia

    2015-01-01

    Study Objectives: To assess adverse drug reaction reports of “abnormal sleep related events” associated with varenicline, a partial agonist to the α4β2 subtype of nicotinic acetylcholine receptors on neurones, indicated for smoking cessation. Design: Twenty-seven reports of “abnormal sleep related events” often associated with abnormal dreams, nightmares, or somnambulism, which are known to be associated with varenicline use, were identified in the World Health Organisation (WHO) Global Individual Case Safety Reports Database. Original anonymous reports were obtained from the four national pharmacovigilance centers that submitted these reports and assessed for reaction description and causality. Measurements and Results: These 27 reports include 10 of aggressive activity occurring during sleep and seven of other sleep related harmful or potentially harmful activities, such as apparently deliberate self-harm, moving a child or a car, or lighting a stove or a cigarette. Assessment of these 17 reports of aggression or other actual or potential harm showed that nine patients recovered or were recovering on varenicline withdrawal and there were no consistent alternative explanations. Thirteen patients experienced single events, and two had multiple events. Frequency was not stated for the remaining two patients. Conclusions: The descriptions of the reports of aggression during sleep with violent dreaming are similar to those of rapid eye movement sleep behavior disorder and also nonrapid eye movement (NREM) sleep parasomnias in some adults. Patients who experience somnambulism or dreams of a violent nature while taking varenicline should be advised to consult their health providers. Consideration should be given to clarifying the term sleep disorders in varenicline product information and including sleep related harmful and potentially harmful events. Citation: Savage RL, Zekarias A, Caduff-Janosa P. Varenicline and abnormal sleep related events. SLEEP 2015

  15. Abnormal Activity Detection Using Pyroelectric Infrared Sensors

    PubMed Central

    Luo, Xiaomu; Tan, Huoyuan; Guan, Qiuju; Liu, Tong; Zhuo, Hankz Hankui; Shen, Baihua

    2016-01-01

    Healthy aging is one of the most important social issues. In this paper, we propose a method for abnormal activity detection without any manual labeling of the training samples. By leveraging the Field of View (FOV) modulation, the spatio-temporal characteristic of human activity is encoded into low-dimension data stream generated by the ceiling-mounted Pyroelectric Infrared (PIR) sensors. The similarity between normal training samples are measured based on Kullback-Leibler (KL) divergence of each pair of them. The natural clustering of normal activities is discovered through a self-tuning spectral clustering algorithm with unsupervised model selection on the eigenvectors of a modified similarity matrix. Hidden Markov Models (HMMs) are employed to model each cluster of normal activities and form feature vectors. One-Class Support Vector Machines (OSVMs) are used to profile the normal activities and detect abnormal activities. To validate the efficacy of our method, we conducted experiments in real indoor environments. The encouraging results show that our method is able to detect abnormal activities given only the normal training samples, which aims to avoid the laborious and inconsistent data labeling process. PMID:27271632

  16. Autism and chromosome abnormalities-A review.

    PubMed

    Bergbaum, Anne; Ogilvie, Caroline Mackie

    2016-07-01

    The neuro-behavioral disorder of autism was first described in the 1940s and was predicted to have a biological basis. Since that time, with the growth of genetic investigations particularly in the area of pediatric development, an increasing number of children with autism and related disorders (autistic spectrum disorders, ASD) have been the subject of genetic studies both in the clinical setting and in the wider research environment. However, a full understanding of the biological basis of ASDs has yet to be achieved. Early observations of children with chromosomal abnormalities detected by G-banded chromosome analysis (karyotyping) and in situ hybridization revealed, in some cases, ASD associated with other features arising from such an abnormality. The introduction of higher resolution techniques for whole genome screening, such as array comparative genome hybridization (aCGH), allowed smaller imbalances to be detected, some of which are now considered to represent autism susceptibility loci. In this review, we describe some of the work underpinning the conclusion that ASDs have a genetic basis; a brief history of the developments in genetic analysis tools over the last 50 years; and the most common chromosome abnormalities found in association with ASDs. Introduction of next generation sequencing (NGS) into the clinical diagnostic setting is likely to provide further insights into this complex field but will not be covered in this review. Clin. Anat. 29:620-627, 2016. © 2016 Wiley Periodicals, Inc. PMID:27012322

  17. Apparent Ruvalcaba syndrome with genitourinary abnormalities.

    PubMed

    Bialer, M G; Wilson, W G; Kelly, T E

    1989-07-01

    The Ruvalcaba syndrome is a rare malformation syndrome characterized by skeletal dysplasia, facial anomalies, and mental retardation. We report on a 22-year-old woman with severe growth and mental retardation and numerous manifestations characteristic of the Ruvalcaba syndrome. In addition, she has several anomalies not previously described in the Ruvalcaba syndrome, including upslanting palpebral fissures, torus palatinus, hiatal hernia with gastroesophageal reflux, recurrent respiratory infections, pectus excavatum, equinovarous deformity, hypotonia, unilateral renal hypoplasia, an accessory ovary, and atretic fallopian tube. Review of published reports of Ruvalcaba syndrome confirms variability of the clinical and radiographic changes. Findings present in at least 50% of reported patients include mental retardation, short stature, pubertal delay, an abnormal nose (usually beaked) with hypoplastic nasal alae, microstomia with narrow maxilla, thin upper lip vermilion, broad hips, small hands, joint limitation, short fingers and toes, and vertebral abnormalities. Because 5 of the reported patients had renal abnormalities, a renal ultrasound or contrast study is indicated in the evaluation of these patients. Additional reports, particular from multiplex families, will be important to better characterize this syndrome. PMID:2679089

  18. Abnormal Activity Detection Using Pyroelectric Infrared Sensors.

    PubMed

    Luo, Xiaomu; Tan, Huoyuan; Guan, Qiuju; Liu, Tong; Zhuo, Hankz Hankui; Shen, Baihua

    2016-01-01

    Healthy aging is one of the most important social issues. In this paper, we propose a method for abnormal activity detection without any manual labeling of the training samples. By leveraging the Field of View (FOV) modulation, the spatio-temporal characteristic of human activity is encoded into low-dimension data stream generated by the ceiling-mounted Pyroelectric Infrared (PIR) sensors. The similarity between normal training samples are measured based on Kullback-Leibler (KL) divergence of each pair of them. The natural clustering of normal activities is discovered through a self-tuning spectral clustering algorithm with unsupervised model selection on the eigenvectors of a modified similarity matrix. Hidden Markov Models (HMMs) are employed to model each cluster of normal activities and form feature vectors. One-Class Support Vector Machines (OSVMs) are used to profile the normal activities and detect abnormal activities. To validate the efficacy of our method, we conducted experiments in real indoor environments. The encouraging results show that our method is able to detect abnormal activities given only the normal training samples, which aims to avoid the laborious and inconsistent data labeling process. PMID:27271632

  19. Abnormalities in Hippocampal Functioning with Persistent Pain

    PubMed Central

    Mutso, Amelia A.; Radzicki, Daniel; Baliki, Marwan N.; Huang, Lejian; Banisadr, Ghazal; Centeno, Maria Virginia; Radulovic, Jelena; Martina, Marco; Miller, Richard J.; Apkarian, A. Vania

    2012-01-01

    Chronic pain patients exhibit increased anxiety, depression, and deficits in learning and memory. Yet how persistent pain affects the key brain area regulating these behaviors, the hippocampus, has remained minimally explored. In this study we investigated the impact of spared nerve injury (SNI) neuropathic pain in mice on hippocampal-dependent behavior and underlying cellular and molecular changes. In parallel, we measured the hippocampal volume of three groups of chronic pain patients. We found that SNI animals were unable to extinguish to contextual fear and showed increased anxiety-like behavior. Additionally, SNI mice in comparison to sham animals exhibited hippocampal 1) reduced extracellular signal-regulated kinase (ERK) expression and phosphorylation, 2) decreased neurogenesis and 3) altered short-term synaptic plasticity. In order to relate the observed hippocampal abnormalities with human chronic pain, we measured the volume of human hippocampus in chronic back pain (CBP), complex regional pain syndrome (CRPS), and osteoarthritis patients (OA). Compared to controls, CBP and CRPS, but not OA, had significantly less bilateral hippocampal volume. These results indicate that hippocampus-mediated behavior, synaptic plasticity and neurogenesis are abnormal in neuropathic rodents. The changes may be related to the reduction in hippocampal volume we see in chronic pain patients, and these abnormalities may underlie learning and emotional deficits commonly observed in such patients. PMID:22539837

  20. Persistent Pain and Sensory Abnormalities after Abdominoplasty

    PubMed Central

    Finnerup, Kenneth; Andresen, Sven R.; Nikolajsen, Lone; Finnerup, Nanna B.

    2015-01-01

    Background: Persistent postsurgical pain is a well-recognized problem after a number of common surgical procedures, such as amputation, thoracotomy, and inguinal hernia repair. Less is known about persistent pain after cosmetic surgical procedures. We, therefore, decided to study the incidence and characteristics of persistent pain after abdominoplasty, which is one of the most frequent cosmetic surgical procedures. Methods: In September 2014, a link to a web-based questionnaire was mailed to 217 patients who had undergone abdominoplasty between 2006 and 2014 at the Department of Plastic Surgery, Aalborg University Hospital, Denmark. The questionnaire included questions about pain and sensory abnormalities located to the abdominal skin, and physical and psychological function; patient satisfaction with surgery was rated on a 4-point scale. Results: One hundred seventy patients answered the questionnaire. Fourteen patients (8.2%) reported pain within the past 7 days related to the abdominoplasty. Abnormal abdominal skin sensation was common and reported by 138 patients (81%). Sensory hypersensitivity was associated with the presence of persistent pain. Satisfaction with the procedure was reported by 149 (88%) patients. The majority of patients reported improvement on all physical and psychological factors. Patients with pain were more often disappointed with the surgery and unwilling to recommend the surgery. Conclusions: Overall, patients were satisfied with the procedure, although abnormal abdominal skin sensation was common. However, there is a risk of developing persistent neuropathic pain after abdominoplasty, and patients should be informed about this before surgery. PMID:26893986

  1. Abnormal calcium homeostasis in peripheral neuropathies

    PubMed Central

    Fernyhough, Paul; Calcutt, Nigel A.

    2010-01-01

    Abnormal neuronal calcium (Ca2+) homeostasis has been implicated in numerous diseases of the nervous system. The pathogenesis of two increasingly common disorders of the peripheral nervous system, namely neuropathic pain and diabetic polyneuropathy, has been associated with aberrant Ca2+ channel expression and function. Here we review the current state of knowledge regarding the role of Ca2+ dyshomeostasis and associated mitochondrial dysfunction in painful and diabetic neuropathies. The central impact of both alterations of Ca2+ signalling at the plasma membrane and also intracellular Ca2+ handling on sensory neuron function is discussed and related to abnormal endoplasmic reticulum performance. We also present new data highlighting sub-optimal axonal Ca 2+ signalling in diabetic neuropathy and discuss the putative role for this abnormality in the induction of axonal degeneration in peripheral neuropathies. The accumulating evidence implicating Ca2+ dysregulation with both painful and degenerative neuropathies, along with recent advances in understanding of regional variations in Ca2+ channel and pump structures, makes modulation of neuronal Ca2+ handling an increasingly viable approach for therapeutic interventions against the painful and degenerative aspects of many peripheral neuropathies. PMID:20034667

  2. Dynamic Abnormal Grain Growth in Molybdenum

    NASA Astrophysics Data System (ADS)

    Worthington, Daniel L.; Pedrazas, Nicholas A.; Noell, Philip J.; Taleff, Eric M.

    2013-11-01

    A new abnormal grain growth phenomenon that occurs only during continuous plastic straining, termed dynamic abnormal grain growth (DAGG), was observed in molybdenum (Mo) at elevated temperature. DAGG was produced in two commercial-purity molybdenum sheets and in a commercial-purity molybdenum wire. Single crystals, centimeters in length, were created in these materials through the DAGG process. DAGG was observed only at temperatures of 1713 K (1440 °C) and above and occurred across the range of strain rates investigated, ~10-5 to 10-4 s-1. DAGG initiates only after a critical plastic strain, which decreases with increasing temperature but is insensitive to strain rate. Following initiation of an abnormal grain, the rate of boundary migration during DAGG is on the order of 10 mm/min. This rapid growth provides a convenient means of producing large single crystals in the solid state. When significant normal grain growth occurs prior to DAGG, island grains result. DAGG was observed in sheet materials with two very different primary recrystallization textures. DAGG grains in Mo favor boundary growth along the tensile axis in a <110> direction, preferentially producing single crystals with orientations from an approximately <110> fiber family of orientations. A mechanism of boundary unpinning is proposed to explain the dependence of boundary migration on plastic straining during DAGG.

  3. Electrocardiographic abnormalities in centenarians: impact on survival

    PubMed Central

    2012-01-01

    Background The centenarian population is gradually increasing, so it is becoming more common to see centenarians in clinical practice. Electrocardiogram abnormalities in the elderly have been reported, but several methodological biases have been detected that limit the validity of their results. The aim of this study is to analyse the ECG abnormalities in a prospective study of the centenarian population and to assess their impact on survival. Method We performed a domiciliary visit, where a medical history, an ECG and blood analysis were obtained. Barthel index (BI), cognitive mini-exam (CME) and Charlson index (ChI) were all determined. Patients were followed up by telephone up until their death. Results A total of 80 centenarians were studied, 26 men and 64 women, mean age 100.8 (SD 1.3). Of these, 81% had been admitted to the hospital at least once in the past, 81.3% were taking drugs (mean 3.3, rank 0–11). ChI was 1.21 (SD 1.19). Men had higher scores both for BI (70 -SD 34.4- vs. 50.4 -SD 36.6-, P = .005) and CME (16.5 -SD 9.1- vs. 9.1 –SD 11.6-, P = .008); 40.3% of the centenarians had anaemia, 67.5% renal failure, 13% hyperglycaemia, 22.1% hypoalbuminaemia and 10.7% dyslipidaemia, without statistically significant differences regarding sex. Only 7% had a normal ECG; 21 (26.3%) had atrial fibrillation (AF), 30 (37.5%) conduction defects and 31 (38.8%) abnormalities suggestive of ischemia, without sex-related differences. A history of heart disease was significantly associated with the presence of AF (P = .002, OR 5.2, CI 95% 1.8 to 15.2) and changes suggestive of ischemia (P = .019, OR 3.2, CI 95% 1.2-8.7). Mean survival was 628 days (SD 578.5), median 481 days. Mortality risk was independently associated with the presence of AF (RR 2.0, P = .011), hyperglycaemia (RR 2.2, P = .032), hypoalbuminaemia (RR 3.5, P < .001) and functional dependence assessed by BI (RR 1.8, P = .024). Conclusion Although ECG abnormalities are

  4. Perceived functional impact of abnormal facial appearance.

    PubMed

    Rankin, Marlene; Borah, Gregory L

    2003-06-01

    Functional facial deformities are usually described as those that impair respiration, eating, hearing, or speech. Yet facial scars and cutaneous deformities have a significant negative effect on social functionality that has been poorly documented in the scientific literature. Insurance companies are declining payments for reconstructive surgical procedures for facial deformities caused by congenital disabilities and after cancer or trauma operations that do not affect mechanical facial activity. The purpose of this study was to establish a large, sample-based evaluation of the perceived social functioning, interpersonal characteristics, and employability indices for a range of facial appearances (normal and abnormal). Adult volunteer evaluators (n = 210) provided their subjective perceptions based on facial physical appearance, and an analysis of the consequences of facial deformity on parameters of preferential treatment was performed. A two-group comparative research design rated the differences among 10 examples of digitally altered facial photographs of actual patients among various age and ethnic groups with "normal" and "abnormal" congenital deformities or posttrauma scars. Photographs of adult patients with observable congenital and posttraumatic deformities (abnormal) were digitally retouched to eliminate the stigmatic defects (normal). The normal and abnormal photographs of identical patients were evaluated by the large sample study group on nine parameters of social functioning, such as honesty, employability, attractiveness, and effectiveness, using a visual analogue rating scale. Patients with abnormal facial characteristics were rated as significantly less honest (p = 0.007), less employable (p = 0.001), less trustworthy (p = 0.01), less optimistic (p = 0.001), less effective (p = 0.02), less capable (p = 0.002), less intelligent (p = 0.03), less popular (p = 0.001), and less attractive (p = 0.001) than were the same patients with normal facial

  5. Salivary gland thrombostasin isoforms differentilally regulate blood uptake of horn flies fed on New Zealand white rabbits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report the effects of TS isoforms on blood feeding was assessed with individual flies that carried corresponding ts alleles. Laboratory studies of horn fly blood fe...

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

    SciTech Connect

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois . E-mail: Jean-Francois.Launay@inserm.u-strasbg.fr

    2005-02-15

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

  7. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

    PubMed

    Cao, Yun; Bender, Ingrid K; Konstantinidis, Athanasios K; Shin, Soon Cheon; Jewell, Christine M; Cidlowski, John A; Schleimer, Robert P; Lu, Nick Z

    2013-02-28

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  8. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans

    PubMed Central

    Cao, Yun; Bender, Ingrid K.; Konstantinidis, Athanasios K.; Shin, Soon Cheon; Jewell, Christine M.; Cidlowski, John A.; Schleimer, Robert P.

    2013-01-01

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  9. Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice.

    PubMed

    Moseley, Amy E; Williams, Michael T; Schaefer, Tori L; Bohanan, Cynthia S; Neumann, Jon C; Behbehani, Michael M; Vorhees, Charles V; Lingrel, Jerry B

    2007-01-17

    Several disorders have been associated with mutations in Na,K-ATPase alpha isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimer's disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the alpha1 isoform is found in many cell types, the alpha2 isoform is predominantly expressed in astrocytes, and the alpha3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the alpha2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the alpha3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous alpha1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the alpha2 and alpha3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders. PMID:17234593

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Salivary Gland Thrombostasin Isoforms Differentially Regulate Blood Uptake of Horn Flies Fed on New Zealand White Rabbits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thrombostasin (TS) is a previously characterized anticlotting protein with multiple isoforms found in the saliva of horn flies. In this report the effects of TS isoforms on blood feeding was assessed with individual flies that carried corresponding ts alleles. Laboratory studies of horn fly blood fe...

  13. LIGHT MODULATION OF RUBISCO ACTIVATION IN SPECIES WITHOUT A LARGER ACTIVASE ISOFORM - EXISTANCE OF AN ACTIVASE REGULATORY PROTEIN?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rubisco activase in some species like tobacco consists of only the shorter isoform. In Arabidopsis lack of the redox regulated, larger isoform results in an inability to modulate Rubisco activity in response to light intensity. However, light modulation can be observed in tobacco with characteristic...

  14. Profiling Murine Tau with 0N, 1N and 2N Isoform-Specific Antibodies in Brain and Peripheral Organs Reveals Distinct Subcellular Localization, with the 1N Isoform Being Enriched in the Nucleus

    PubMed Central

    Liu, Chang; Götz, Jürgen

    2013-01-01

    In the adult murine brain, the microtubule-associated protein tau exists as three major isoforms, which have four microtubule-binding repeats (4R), with either no (0N), one (1N) or two (2N) amino-terminal inserts. The human brain expresses three additional isoforms with three microtubule-binding repeats (3R) each. However, little is known about the role of the amino-terminal inserts and how the 0N, 1N and 2N tau species differ. In order to investigate this, we generated a series of isoform-specific antibodies and performed a profiling by Western blotting and immunohistochemical analyses using wild-type mice in three age groups: two months, two weeks and postnatal day 0 (P0). This revealed that the brain is the only organ to express tau at significant levels, with 0N4R being the predominant isoform in the two month-old adult. Subcellular fractionation of the brain showed that the 1N isoform is over-represented in the soluble nuclear fraction. This is in agreement with the immunohistochemical analysis as the 1N isoform strongly localizes to the neuronal nucleus, although it is also found in cell bodies and dendrites, but not axons. The 0N isoform is mainly found in cell bodies and axons, whereas nuclei and dendrites are only slightly stained with the 0N antibody. The 2N isoform is highly expressed in axons and in cell bodies, with a detectable expression in dendrites and a very slight expression in nuclei. The 2N isoform that was undetectable at P0, in adult brain was mainly found localized to cell bodies and dendrites. Together these findings reveal significant differences between the three murine tau isoforms that are likely to reflect different neuronal functions. PMID:24386422

  15. Bakers' cyst and tibiofemoral abnormalities are more distinctive MRI features of symptomatic osteoarthritis than patellofemoral abnormalities

    PubMed Central

    Visser, A W; Mertens, B; Reijnierse, M; Bloem, J L; de Mutsert, R; le Cessie, S; Rosendaal, F R; Kloppenburg, M

    2016-01-01

    Objective To investigate which structural MR abnormalities discriminate symptomatic knee osteoarthritis (OA), taking co-occurrence of abnormalities in all compartments into account. Methods The Netherlands Epidemiology of Obesity (NEO) study is a population-based cohort aged 45–65 years. In 1285 participants (median age 56 years, 55% women, median body mass index (BMI) 30 kg/m2), MRI of the right knee were obtained. Structural abnormalities (osteophytes, cartilage loss, bone marrow lesions (BMLs), subchondral cysts, meniscal abnormalities, effusion, Baker's cyst) at 9 patellofemoral and tibiofemoral locations were scored following the knee OA scoring system. Symptomatic OA in the imaged knee was defined following the American College of Rheumatology criteria. Logistic ridge regression analyses were used to investigate which structural abnormalities discriminate best between individuals with and without symptomatic OA, crude and adjusted for age, sex and BMI. Results Symptomatic knee OA was present in 177 individuals. Structural MR abnormalities were highly frequent both in individuals with OA and in those without. Baker's cysts showed the highest adjusted regression coefficient (0.293) for presence of symptomatic OA, followed by osteophytes and BMLs in the medial tibiofemoral compartment (0.185–0.279), osteophytes in the medial trochlear facet (0.262) and effusion (0.197). Conclusions Baker's cysts discriminate best between individuals with and without symptomatic knee OA. Structural MR abnormalities, especially in the medial side of the tibiofemoral joint and effusion, add further in discriminating symptomatic OA. Baker's cysts may present as a target for treatment. PMID:27252896

  16. Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.

    PubMed

    Aikio, Mari; Elamaa, Harri; Vicente, David; Izzi, Valerio; Kaur, Inderjeet; Seppinen, Lotta; Speedy, Helen E; Kaminska, Dorota; Kuusisto, Sanna; Sormunen, Raija; Heljasvaara, Ritva; Jones, Emma L; Muilu, Mikko; Jauhiainen, Matti; Pihlajamäki, Jussi; Savolainen, Markku J; Shoulders, Carol C; Pihlajaniemi, Taina

    2014-07-29

    Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼ 50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat

  17. Progestins reinitiate cell cycle progression in antiestrogen-arrested breast cancer cells through the B-isoform of progesterone receptor.

    PubMed

    McGowan, Eileen M; Russell, Amanda J; Boonyaratanakornkit, Viroj; Saunders, Darren N; Lehrbach, Gillian M; Sergio, C Marcelo; Musgrove, Elizabeth A; Edwards, Dean P; Sutherland, Robert L

    2007-09-15

    Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest. PMID:17875737

  18. Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I, II, IX and XII.

    PubMed

    Barresi, Elisabetta; Salerno, Silvia; Marini, Anna Maria; Taliani, Sabrina; La Motta, Concettina; Simorini, Francesca; Da Settimo, Federico; Vullo, Daniela; Supuran, Claudiu T

    2016-02-15

    Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications. PMID:26796953

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

    SciTech Connect

    Short, Stephen; Malartre, Marianne; Sharpe, Colin . E-mail: colin.sharpe@port.ac.uk

    2005-09-02

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

  20. Calcium Channel CaVα₁ Splice Isoforms - Tissue Specificity and Drug Action.

    PubMed

    Lipscombe, Diane; Andrade, Arturo

    2015-01-01

    Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cellspecific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among CaV isoforms expressed in different tissues, and such differences may be greater than those that exist across CaV channel homologs of closely related species. Here we highlight examples of CaV isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of CaV isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of CaV channel inhibitors and agonists with improved tissue-specificity. PMID:25966698