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Sample records for alpha2-delta subunit unc-36

  1. Functional properties of the CaV1.2 calcium channel activated by calmodulin in the absence of alpha2delta subunits.

    Science.gov (United States)

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M

    2009-01-01

    Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties. PMID:19106618

  2. Altered expression of the voltage-gated calcium channel subunit alpha(2)delta-1: A comparison between two experimental models of epilepsy and a sensory nerve ligation model of neuropathic pain

    Czech Academy of Sciences Publication Activity Database

    Nieto-Rostro, M.; Sandhu, G.; Bauer, C. S.; Jiruška, Přemysl; Jefferys, J. G. R.; Dolphin, A. C.

    2014-01-01

    Roč. 283, Dec (2014), s. 124-137. ISSN 0306-4522 R&D Projects: GA MZd(CZ) NT14489 Institutional support: RVO:67985823 Keywords : calcium channel * dorsal root ganglion ( DRG ) * alpha2delta subunit * epilepsy * neuropathic pain * reactive gliosis Subject RIV: FH - Neurology Impact factor: 3.357, year: 2014

  3. Does cigarette smoking affect the diagnostic reliability of hemoglobin alpha 2 delta 2 (HbA2)?

    Science.gov (United States)

    Tarazi, Issa S; Sirdah, Mahmoud M; El Jeadi, Hesham; Al Haddad, Rami M

    2008-01-01

    Quantitation of hemoglobin alpha 2 delta 2 (HbA2) is a basic and confirmatory test in diagnosing the carrier state of beta-thalassemia. The present study was designed to investigate the effect of cigarette smoking on the diagnostic reliability of HbA2. A total of 2,867 (654 smokers and 2,213 never smokers) male subjects were involved in the present study. The subjects were categorized into three groups according to their laboratory findings: beta-thalassemia minor, iron deficient, and normal groups. Complete blood count (CBC) parameters and HbA2 levels were compared between smokers and never smokers of each group according to the independent-samples t-test using the SPSS program, significance results were reported at PHct]) and Hb concentration in smokers of all groups; however, no significant differences were reported in the HbA2 level between smokers and never smokers in all groups. It was concluded that cigarette smoking does not affect the diagnostic reliability of the HbA2 test. PMID:18348310

  4. Interaction of factor XIII subunits.

    Science.gov (United States)

    Katona, Eva; Pénzes, Krisztina; Csapó, Andrea; Fazakas, Ferenc; Udvardy, Miklós L; Bagoly, Zsuzsa; Orosz, Zsuzsanna Z; Muszbek, László

    2014-03-13

    Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. PMID:24408323

  5. Nomenclature for Ion channel Subunits

    OpenAIRE

    Bradley, Jonathan; Frings, Stephan; Yau, King-Wai; Reed, Randall

    2001-01-01

    Presents the nomenclature for ion channel subunits. Role of ion channels in the mediation of visual and olfactory signal transduction; Expression of ion channels in cell types and tissues; Assessment on the nucleotide sensitivity, ion conductance and calcium modulation in heteromers.

  6. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    1987-01-01

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically rel

  7. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I; Issinger, O G

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.......cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  8. Production and delivery of recombinant subunit vaccines

    OpenAIRE

    Andersson, Christin

    2000-01-01

    Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated. Attempts to create general production strategies forrecombinant protein i...

  9. Subunit architecture of general transcription factor TFIIH

    OpenAIRE

    Gibbons, Brian J.; Brignole, Edward J; Azubel, Maia; Murakami, Kenji; Voss, Neil R; Bushnell, David A.; Asturias, Francisco J; Kornberg, Roger D.

    2012-01-01

    Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the “minimal core” subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into...

  10. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller tha...... fairness tests related directly to the problem of risk capital allocation and show that the Lorenz set satisfies all three tests in contrast to other well-known coherent methods. Finally, we discuss how to deal with non-uniqueness of the Lorenz set.......Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller than...... the sum of the risks of the individual subunits. The question is how to allocate the risk capital of the group among the subunits in a fair way. In this paper we propose to use the Lorenz set as an allocation method. We show that the Lorenz set is operational and coherent. Moreover, we propose three...

  11. Heteromeric assembly of P2X subunits

    Directory of Open Access Journals (Sweden)

    Ralf Hausmann

    2013-12-01

    Full Text Available Transcripts and/or proteins of P2X receptor (P2XR subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs

  12. Immunochemical aspects of crotoxim and its subunits

    International Nuclear Information System (INIS)

    Crotamine and crotoxin with the subunits - phospholipase A and crotapotin - were obtained by purification from Crotalus durissus terrificus venom. Interaction studies of the subunits using crotalic antiserum, indicated that: crotoxin is formed of crotapotin and phospholipase A with the molar ratio of 1 to 1; using crotapotin 125I the presence of a soluble complex was shown with the same antiserum. Immunological precipitation reactions demonstrated that crotapotin is antigenic: crotapotin and phospholipase A presented similar antigenic determinants; crotoxin antiserum reacted with each one of the submits; when the subunits are mixed to form synthetic crotoxin some antigenic determinants are masked in the process of interaction. Crotamine, interacted with crotapotin 1:1, without hidden antigenic determinants crotapotin antigenic site seems to be formed by, at least, one lysine. Enzimatical activity of phospholipase A apreared to be dependent on some reaction conditions when its arginine residues are blocked. Tyrosines of phospholipase A are more susceptible to labelling with 131I than crotapotin. Gama irradiation of aqueous solutions of the subunits produced modifications in the ultraviolet spectra. A decrease of the enzymatic activity occured as a function of radiation dosis. Immunological activities of crotapotin and phospholipase A were not altered

  13. Dynamic regulation of β1 subunit trafficking controls vascular contractility

    OpenAIRE

    Leo, M. Dennis; Bannister, John P.; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E.; Gabrick, Kyle S.; Boop, Frederick A.; Jaggar, Jonathan H.

    2014-01-01

    Plasma membrane ion channels composed of pore-forming and auxiliary subunits regulate physiological functions in virtually all cell types. A conventional view is that ion channels assemble with their auxiliary subunits prior to surface trafficking of the multiprotein complex. Arterial myocytes express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that modulate contractility and blood pressure and flow. The data here show that although most BKα subunits ar...

  14. Recent Advances in Subunit Vaccine Carriers.

    Science.gov (United States)

    Vartak, Abhishek; Sucheck, Steven J

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  15. MspA Nanopores from Subunit Dimers

    OpenAIRE

    Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated tha...

  16. Expression of Telomerase Subunits in Gastric Cancer

    Institute of Scientific and Technical Information of China (English)

    CHEN Fenghua; HU Lihua; LI Yirong; WANG Lin

    2005-01-01

    To detect the expression of telomerase subunits human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 25 %, respectively. The former was significantly higher than the latter (χ2 =26.4, P<0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7 %, respectively and no significant difference was found between them (χ2 =2.1, P>0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.

  17. Na+ channel β subunits: Overachievers of the ion channel family

    OpenAIRE

    LoriLIsom; WilliamJBrackenbury

    2011-01-01

    Voltage gated Na+ channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the...

  18. MspA nanopores from subunit dimers.

    Directory of Open Access Journals (Sweden)

    Mikhail Pavlenok

    Full Text Available Mycobacterium smegmatis porin A (MspA forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore

  19. MspA nanopores from subunit dimers.

    Science.gov (United States)

    Pavlenok, Mikhail; Derrington, Ian M; Gundlach, Jens H; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  20. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    DEFF Research Database (Denmark)

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens O L;

    2010-01-01

    In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit.......In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit....

  1. Advances in the Research of AMPK and its Subunit Genes

    Directory of Open Access Journals (Sweden)

    R.S. Jiang

    2013-01-01

    Full Text Available AMP-activated kinase (AMPK is a heterotrimeric complex composed of three subunits and is the core energy sensor of the cell. The AMPK activity is important for survival during periods of stress and starvation and also has implications in type II diabetes, obesity, metabolic syndrome, longevity and cancer, etc. The activation of AMPK is triggered through binding of Adenosine Monophosphate Activated Proteins (AMP to the Bateman domains of the gamma subunit, leading to increased phosphorylation of the threonine 172 on the alpha subunit by inducing allosteric activation and inhibiting dephosphorylation. AMPK and its subunits have been the focuses of many researchers dealing with genetic and metabolic issues. The study makes a comprehensive review on the structure, function, distribution, enzyme activity, the genetic mutation and other aspects of AMPK and its subunit genes, with the aim to outline main aspects of present researches on AMPK and its subunits in animal genetics.

  2. Comparative Analysis of Eubacterial DNA Polymerase Ⅲ Alpha Subunits

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qian Zhao; Jian-Fei Hu; Jun Yu

    2006-01-01

    DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequencebased phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers.Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.

  3. Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

    OpenAIRE

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-01-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individua...

  4. Designability of protein subunits on a lattice

    Science.gov (United States)

    Dmitry, Green; Wingreen, Ned; Tang, Chao; Miller, Jonathan

    2001-03-01

    Native protein folds are often made up of distinct sub folds. We use a two-dimensional lattice protein model based on hydrophobicity to study this feature. The "designability" of a structure is defined as the number of sequences with that structure as their unique, lowest energy configuration. A "highly designable" structure is one that is encoded by a disproportionate share of sequences. The designability of a structure encapsulates the insensitivity to mutation of the sequences that fold into it. We consider structures on a 6x6 and an 8x8 lattice that are composed of two loops, and find that their designability is correlated with the designability of the component loops. This result points to a more general principle asserting that some proteins may be built up from highly designable subunits.

  5. The Schizosaccharomyces pombe casein kinase II alpha and beta subunits: evolutionary conservation and positive role of the beta subunit.

    OpenAIRE

    Roussou, I; Draetta, G

    1994-01-01

    Casein kinase II is a key regulatory enzyme involved in many cellular processes, including the control of growth and cell division. We report the molecular cloning and sequencing of cDNAs encoding the alpha and the beta subunits of casein kinase II of Schizosaccharomyces pombe. The deduced amino acid sequence of Cka1, the alpha catalytic subunit, shows high sequence similarity to alpha subunits identified in other species. The amino acid sequence of Ckb1, the S. pombe beta subunit, is 57% ide...

  6. Subunit structure of the follitropin receptor

    International Nuclear Information System (INIS)

    Both of the α and β subunits of intact human follitropin (FSH) were radioiodinated with 125I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH α and β subunits as well as the αβ dimer changed markedly depending on the concentration of reducing agents. 125I-FSH (Ka = 1.4 x 1010 M-1), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the 125I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH αβ dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized 125I-FSH (Ka = 1.12 1010 M-1) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa

  7. Dynamic regulation of β1 subunit trafficking controls vascular contractility

    Science.gov (United States)

    Leo, M. Dennis; Bannister, John P.; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E.; Gabrick, Kyle S.; Boop, Frederick A.; Jaggar, Jonathan H.

    2014-01-01

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca2+ sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types. PMID:24464482

  8. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  9. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    Science.gov (United States)

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  10. Regulation of Glutamate Receptors by Their Auxiliary Subunits

    OpenAIRE

    Tomita, Susumu

    2010-01-01

    Glutamate receptors are major excitatory receptors in the brain. Recent findings have established auxiliary subunits of glutamate receptors as critical modulators of synaptic transmission, synaptic plasticity and neurological disorder. The elucidation of the molecular rules governing glutamate receptors and subunits will improve our understanding of synapses and of neural-circuit regulation in the brain.

  11. Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dt locus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit possessed an electrophoretic mobility that was faster than that of the Dy12 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13t gene ORF was the shortest among those of the previously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.

  12. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    Science.gov (United States)

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  13. Nonfouling hydrogels formed from charged monomer subunits.

    Science.gov (United States)

    Dobbins, Sean C; McGrath, Daniel E; Bernards, Matthew T

    2012-12-13

    A critical challenge in the field of biomaterials is the often undesirable, but immediate, coating of implants with nonspecifically adsorbed proteins upon contact with bodily fluids. Prior research has shown that overall neutral materials containing a homologous arrangement of mixed charges exhibit nonfouling properties. This has been widely demonstrated for zwitterionic materials and more recently for coatings containing an equimolar mixture of positively and negatively charged monomer subunits. In this investigation it is demonstrated that nonfouling hydrogels can be formed through this approach, and the physical properties of the resulting materials are thoroughly characterized. In particular, hydrogels were formed from mixtures of [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TM) and 3-sulfopropyl methacrylate potassium salt (SA) monomers with varying concentrations of a triethylene glycol dimethacrylate (TEGDMA) cross-linker. The swelling, weight percentage water, surface zeta potential, and compressional properties of the gels were characterized, and the nonfouling properties were demonstrated using enzyme-linked immunosorbant assays for both negatively charged fibrinogen and positively charged lysozyme. The results confirm that the TM:SA hydrogel systems have nonfouling properties that are equivalent to established nonfouling controls. Additionally, even though the gels were resistant to nonspecific protein adsorption, a composition analysis suggests that there is room to further improve the nonfouling performance because there is a slight enrichment of the SA monomer relative to the TM monomer. PMID:23189949

  14. Echinococcus granulosus antigen B structure: subunit composition and oligomeric states.

    Directory of Open Access Journals (Sweden)

    Karina M Monteiro

    Full Text Available BACKGROUND: Antigen B (AgB is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. METHODOLOGY/PRINCIPAL FINDINGS: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8 was found in one bovine sample. The exponentially modified protein abundance index (emPAI was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. CONCLUSIONS/SIGNIFICANCE: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly

  15. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    Science.gov (United States)

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  16. Telomerase Holoenzyme Proteins and Processivity Subunit in Tetrahymena thermophila

    OpenAIRE

    Min, Bosun

    2009-01-01

    Telomeres are specialized protein-DNA structures that protect the ends of linear chromosomes, and they are maintained by the telomerase ribonucleoprotein (RNP) enzyme complex. Recombinant telomerase RNP with catalytic activity contains, at a minimum, the catalytic reverse transcriptase subunit (TERT) and the telomerase RNA (TER). However, endogenous telomerase is a much larger holoenzyme complex, with telomerase-associated subunits that contribute to RNP assembly and regulation. Telomerase-as...

  17. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    OpenAIRE

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectromet...

  18. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    OpenAIRE

    Römling, Ute; Galperin, Michael Y

    2015-01-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis ...

  19. Functional diversity of complex I subunits in Candida albicans mitochondria.

    Science.gov (United States)

    Li, Dongmei; She, Xiaodong; Calderone, Richard

    2016-02-01

    Our interest in the mitochondria of Candida albicans has progressed to the identification of several proteins that are critical to complex I (CI) activity. We speculated that there should be major functional differences at the protein level between mammalian and fungal mitochondria CI. In our pursuit of this idea, we were helped by published data of CI subunit proteins from a broad diversity of species that included two subunit proteins that are not found in mammals. These subunit proteins have been designated as Nuo1p and Nuo2p (NADH-ubiquinone oxidoreductases). Since functional assignments of both C. albicans proteins were unknown, other than having a putative NADH-oxidoreductase activity, we constructed knock-out strains that could be compared to parental cells. The relevance of our research relates to the critical roles of both proteins in cell biology and pathogenesis and their absence in mammals. These features suggest they may be exploited in antifungal drug discovery. Initially, we characterized Goa1p that apparently regulates CI activity but is not a CI subunit protein. We have used the goa1∆ for comparisons to Nuo1p and Nuo2p. We have demonstrated the critical role of these proteins in maintaining CI activities, virulence, and prolonging life span. More recently, transcriptional profiling of the three mutants and an ndh51∆ (protein is a highly conserved CI subunit) has revealed that there are overlapping yet also different functional assignments that suggest subunit specificity. The differences and similarities of each are described below along with our hypotheses to explain these data. Our conclusion and perspective is that the C. albicans CI subunit proteins are highly conserved except for two that define non-mammalian functions. PMID:26373419

  20. Cytochrome c oxidase: evolution of control via nuclear subunit addition.

    Science.gov (United States)

    Pierron, Denis; Wildman, Derek E; Hüttemann, Maik; Markondapatnaikuni, Gopi Chand; Aras, Siddhesh; Grossman, Lawrence I

    2012-04-01

    According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits. PMID:21802404

  1. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  2. Subunit interactions change the heme active-site geometry in p-cresol methylhydroxylase.

    OpenAIRE

    McLendon, G L; Bagby, S; Charman, J A; Driscoll, P. C.; McIntire, W S; Mathews, F. S.; Hill, H A

    1991-01-01

    The enzyme p-cresol methylhydroxylase [4-cresol: (acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1] contains two subunits: a cytochrome c (electron transfer) subunit (cytochrome cpc) and a flavin (catalytic) subunit. When these subunits are separated by isoelectric focusing, a stable cytochrome subunit is obtained. Significant differences are observed between the one-dimensional NMR spectra of oxidized cytochrome cpc and of oxidized p-cresol methylhydroxylase. Analysis of the two...

  3. Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte; Raaf, Jennifer; Bretner, Maria; Issinger, Olaf-Georg; Niefind, Karsten

    2011-01-01

    Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement ...

  4. Dengue vaccine: an update on recombinant subunit strategies.

    Science.gov (United States)

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines. PMID:26982462

  5. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  6. Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli.

    Science.gov (United States)

    Ge, Baosheng; Sun, Haixiang; Feng, Yang; Yang, Jinying; Qin, Song

    2009-03-01

    Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g l(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. PMID:19269586

  7. Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p.

    OpenAIRE

    Ho, J. H.; Kallstrom, G; Johnson, A. W.

    2000-01-01

    Nmd3p from yeast is required for the export of the large (60S) ribosomal subunit from the nucleus (Ho et al., 2000). Here, we show that Nmd3p forms a stable complex with free 60S subunits. Using an epitope-tagged Nmd3p, we show that free 60S subunits can be coimmunoprecipitated with Nmd3p. The interaction was specific for 60S subunits; 40S subunits were not coimmunoprecipitated. Using this coprecipitation technique and pulse-chase labeling of ribosomal subunit proteins we showed that Nmd3p bo...

  8. The Essential Anatomical Subunit Approximation Unilateral Cleft Lip Repair.

    Science.gov (United States)

    Chong, David K; Swanson, Jordan W

    2016-07-01

    The anatomical subunit approximation cleft lip repair advantageously achieves a balanced lip contour, with the line of repair hidden along seams of aesthetic subunits. Dr. David Fisher's original description of the repair reflects the considerable thought that went into the evolution of his design. As his technique has gained acceptance in the intervening 10 years, the authors note several key principles embodied in it that represent a shift in the cleft lip repair paradigm. The authors believe understanding these principles is important to mastery of the anatomical subunit technique, and facilitate its teaching. First, design a plan that adheres to anatomical subunits and perform measurements precisely. Second, identify and adequately release each cleft tissue layer from the lip and nose to enable restoration of balance. Third, drive surgical approximation through inset of the lateral muscle into the superiorly backcut medial orbicularis muscle, followed by skin closure with inferior triangle interposition above the white roll. In this article, the authors present essential components of the technique, and identify several principles that enable its successful execution. PMID:27348690

  9. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David;

    2007-01-01

    to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA...

  10. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y

    2015-09-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. PMID:26077867

  11. Emergence of ion channel modal gating from independent subunit kinetics.

    Science.gov (United States)

    Bicknell, Brendan A; Goodhill, Geoffrey J

    2016-09-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca(2+) concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  12. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...

  13. Partial agonists and subunit selectivity at NMDA receptors

    DEFF Research Database (Denmark)

    Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

    2010-01-01

    Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

  14. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  15. CMF70 is a subunit of the dynein regulatory complex.

    Science.gov (United States)

    Kabututu, Zakayi P; Thayer, Michelle; Melehani, Jason H; Hill, Kent L

    2010-10-15

    Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the eukaryotic flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant eukaryotic clades, indicating that the DRC was probably present in the last common eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes. PMID:20876659

  16. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    Science.gov (United States)

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. PMID:24813157

  17. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

  18. Subunit composition and chromophore content of R-phycoerythrin from Porphyra haitanensis

    Science.gov (United States)

    Gao, Hong-Feng; Ji, Ming-Hou; Cao, Wen-Da

    1996-03-01

    R-phycoerythrin from Porphyra haitanensis exists in two aggregation states with different molecular weights. A more highly aggregated form, RPE I, was chromatographed on Bio-Rex 70 column with urea solution (pH 3.0) as eluent, and the molecular weights of the 3 subunits (α, β, γ) obtained were determined on SDS-PAGE at 18000, 19200 and 30000, respectively. α subunit carried two phycoerythrobilin (PEB); β subunit, three PEB and one phycourobilin (PUB); γ subunit, one PEB and three PUB chromophores. The molar ratio of α, β, and γ subunits of RPE I was 6: 6: 1, and their subunit composition was confired to be (αβ)6γ on account of the molecular weight of RPE I, 232000. A lower aggregated form, RPE II, contained α and β subunits similar to those of RPE I, but its subunit composition was the (αβ) monomer of RPE.

  19. Bigenomic transcriptional regulation of all thirteen cytochrome c oxidase subunit genes by specificity protein 1

    OpenAIRE

    Dhar, Shilpa S.; Johar, Kaid; Wong-Riley, Margaret T. T.

    2013-01-01

    Cytochrome c oxidase (COX) is one of only four known bigenomic proteins, with three mitochondria-encoded subunits and 10 nucleus-encoded ones derived from nine different chromosomes. The mechanism of regulating this multi-subunit, bigenomic enzyme is not fully understood. We hypothesize that specificity protein 1 (Sp1) functionally regulates the 10 nucleus-encoded COX subunit genes directly and the three mitochondrial COX subunit genes indirectly by regulating mitochondrial transcription fact...

  20. Regulation of expression of a soybean storage protein subunit gene. Progress report

    International Nuclear Information System (INIS)

    We have found that the methionine repression of the β-subunit gene expression is not due to degradation of the β-subunit but is due to an effect on synthesis of the β-subunit. The effect of methionine on the synthesis of the β-is due to an inhibition of β-subunit mRNA synthesis. 3 references, 1 figure

  1. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Issinger, Olaf-Georg; Niefind, Karsten

    2009-01-01

    The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...

  2. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne;

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development,...

  3. Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody

    OpenAIRE

    1982-01-01

    We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C- subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit- Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoe...

  4. Developments of Subunit and VLP Vaccines Against Influenza A Virus

    Institute of Scientific and Technical Information of China (English)

    Ma-ping Deng; Zhi-hong Hu; Hua-lin Wang; Fei Deng

    2012-01-01

    Influenza virus is a continuous and severe global threat to mankind.The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year,which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer,more efficient and economic way.The influenza subunit and VLP vaccines,taking the advantage of recombinant DNA technologies and expression system platforms,can be produced in such an ideal way.This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA),neuraminidase antigen (NA),Matrix 2 protein (M2) and nucleocapsid protein (NP).It would help to get insight into the current stage of influenza vaccines,and suggest the future design and development of novel influenza vaccines.

  5. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  6. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

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

    OpenAIRE

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

    2014-01-01

    Stress can increase susceptibility to developing psychiatric disorders, including depression. Understanding the neurobiological mechanisms underlying stress resilience and susceptibility is key to identifying novel targets for the development of more effective treatments for stress-related psychiatric disorders. Here we show that specific isoforms of GABAB receptor subunits differentially regulate stress resilience. Specifically, GABAB(1a)−/− mice are more susceptible whereas GABAB(1b)−/− mic...

  8. Characterisation of connective tissue cells containing factor XIII subunit a.

    OpenAIRE

    Adány, R; Glukhova, M A; Kabakov, A Y; Muszbek, L

    1988-01-01

    Paraffin embedded sections of human liver, lymph node, and placenta showed that certain connective tissue cells were positive for factor XIII subunit a. These cells were further characterised by double immunofluorescence labelling and by combined immunofluorescence and enzyme cytochemical staining on frozen sections. They were labelled by the monoclonal antibodies RFD7 and anti-Leu M3 (markers of the macrophage cell line) but gave a negative reaction for the fibroblast marker IIG10 and showed...

  9. Ribosomal small subunit domains radiate from a central core

    Science.gov (United States)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  10. Immunological Effect of Subunit Influenza Vaccine Entrapped by Liposomes

    Institute of Scientific and Technical Information of China (English)

    SHUI-HUA ZHANG; JIA-XU LIANG; SHU-YAN DAI; XIAO-LIN QIU; YAN-RONG YI; YUN PAN

    2009-01-01

    Objective To elevate the immunological effect of subunit influenza vaccine in infants and aged people (over 60) using liposomal adjuvant in the context of its relatively low immunity and to investigate the relation between vaccine antigens and liposomal characteristics. Methods Several formulations of liposomal subunit influenza vaccine were prepared. Their relevant characteristics were investigated to optimize the preparation method. Antisera obtained from immunizinged mice were used to evaluate the antibody titers of various samples by HI and ELISA. Results Liposomal trivalent influenza vaccine prepared by film evaporation in combinedation with freeze-drying significantly increased its immunological effect in SPF Balb/c mice. Liposomal vaccine stimulated the antibody titer of H3N2, H1N1, and B much stronger than conventional influenza vaccine. As a result, liposomal vaccine (mean size: 4.5-5.5 μm, entrapment efficiency: 30%-40%) significantly increased the immunological effect of subunit influenza vaccine. Conclusion The immune effect of liposomal vaccine depends on different antigens, and enhanced immunity is not positively correlated with the mean size of liposome or its entrapped efficiency.

  11. Radioimmunoassay of TSH subunits in thyroid diseases and endocrine opthalmopahty

    International Nuclear Information System (INIS)

    Highly sensitive radioimmunoassays of hTSH sub-units were developed. The hormone preparations were labelled with 125-iodine according to a modified chloramine -T method, and purified by chromatography using biogel P6 and P60. Rabbit antisera were used as antibodies. Separation of the antibody-bound and of the free antigens was carried out via the double antibody method. The antiserum required for this purpose was obtained from a goat. The sensitivity of the assay was influenced by changing the protein content of the buffer, the incubation volume, the tracer amounts, the incubation time and the incubation temperature. For hTSH-α, the lowest detectable limit was found to be 50 pg/ml, for hTSH-#betta# 20 pg/ml. Thus, the sub-units could be determined for 98% of the patients under review. The #betta#-TSH radioimmunoassay is largely specific, TSH cross-reacts to a degree of 5%. The computerized evoluation was carried out by means of Spline approximation using the Siemens 4004 computer. Precision and accurateness are in compliance with generally accpted criteria. The serum levels of α and #betta# sub-units showed no discordancy with regard to TSH. In all groups of patients examined, the levels of the hormone-specific #betta#-chain were found to be exclusively dependent upon the actual thyroid activity. (orig.)

  12. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    Science.gov (United States)

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

  13. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    Science.gov (United States)

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  14. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  15. Regulation of expression of a soybean storage protein subunit gene: Final report

    International Nuclear Information System (INIS)

    In an effort to determine why methionine decreases the level of the conglycinin β-subunit in cultured soybean seeds, we have established that: (1) methionine does not accelerate the degradation of the β-subunit; (2) that methionine is probably not acting by methylating the β-subunit gene; (3) that methionine is preventing the appearance of a translatable β-subunit mRNA; (4) that methionine is probably not accelerating the degradation of the β-subunit mRNA; and (5) methionine causes a marked increase in a small (0.7 kb) poly A+ RNA. 6 refs., 4 figs., 2 tabs

  16. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

    Science.gov (United States)

    Boettcher, B; Martinez-Carrion, M

    1975-10-01

    Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that

  17. Radioimmunoassay for determination of alpha subunit of pituitary glycoprotein hormones in patients with pituitary tumors

    International Nuclear Information System (INIS)

    A radioimmunoassay method for alpha subunit has been described and applied for serum alpha subunit determinations in normal subjects and 71 patients with pituitary tumors /45 acromegalic and 26 non-acromegalic/. The labelling of alpha subunit by the chloramine T technique yielded 125I-alpha subunit of high specific activity and high immuno-reactivity. Three purification methods of labelled 125I-alpha subunit were compared; the best separation of undamaged 125I-alpha subunit from impurities was achieved by gel filtration on Ultrogel AcA54 column, whereas gel filtration on Sephadex G-100 and adsorption chromatography on CF-11 cellulose gave less satisfactory results. Microheterogenity of 125I-alpha subunit was disclosed by chromatofocusing on PBE 94; the fractions of high immunoreactivitiy had isoelectric points of 6.0, 5.5 and 4.8. In normal subjects, radioimmunoassay of alpha subunit gave the following results /mean and SD/: 0.75 ng/ml +- 0.41 in males and 0.80 ng/ml +- 0.39 in females in reproductive age. In 9 acromegalic serum alpha subunit concentration were elevated up to 21 ng/ml, and in 8 non-acromegalic up to 30 ng/ml. One woman with acromegaly and high serum alpha subunit concentration had also elevated serum TSH associated with hyperthyroids. Our results disclosed that high serum alpha subunit concentration occurs in 25 % of patients with pituitary adenomas. (Author)

  18. Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments.

    Science.gov (United States)

    Zhu, Jiapeng; King, Martin S; Yu, Minmin; Klipcan, Liron; Leslie, Andrew G W; Hirst, Judy

    2015-09-29

    Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved "core" subunits and 31 "supernumerary" subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX9C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein-ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation. PMID:26371297

  19. Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function.

    Science.gov (United States)

    Xu, R; Thomas, E A; Gazina, E V; Richards, K L; Quick, M; Wallace, R H; Harkin, L A; Heron, S E; Berkovic, S F; Scheffer, I E; Mulley, J C; Petrou, S

    2007-08-10

    Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis. PMID:17629415

  20. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    Directory of Open Access Journals (Sweden)

    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  1. Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence...... response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative...... activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation....

  2. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    Energy Technology Data Exchange (ETDEWEB)

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  3. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a...... single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with...

  4. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    DEFF Research Database (Denmark)

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens Otto Lunde;

    2010-01-01

    when the alpha-ratios, rather than simply the alpha-subunit levels, were measured in patients with NFPAs. MATERIAL AND METHODS: Reference intervals for gonadotropin alpha-subunit serum levels and alpha-ratios were established in 231 healthy adults. The estimated cut-off limits were applied to 37...... patients with NFPAs. Gonadotropin alpha-subunit, LH and FSH levels were measured and alpha-ratios were calculated. RESULTS: In healthy adults, the cut-offs for alpha-subunit levels were significantly different between men and pre- and postmenopausal women: the cut-offs were 1.10, 0.48 and 3.76 IU....../l, respectively. Using these estimated cut-offs, increased alpha-subunit levels were identified in 10 out of 37 (27%) patients with NFPAs. By adding alpha-ratio, in combination with alpha-subunit levels, 23 patients out of 37 (62%) were identified as having elevated alpha-subunit hypersecretion, and 22 out of...

  5. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    International Nuclear Information System (INIS)

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author)

  6. Characterisation of the tryptophan synthase alpha subunit in maize

    Directory of Open Access Journals (Sweden)

    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  7. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    Science.gov (United States)

    Foged, Camilla

    2016-01-01

    In the past century, vaccines have contributed to a significant improvement in global public health by preventing a number of infectious diseases. Despite this, the vaccine field is still facing challenges related to incomplete vaccine coverage and persistent difficult vaccine targets, such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self-administrable, can be distributed independently of functioning freezers and refrigerators, and can be designed to induce mucosal and/or cell-mediated immunity, which is attractive for a number of diseases requiring stimulation of local mucosal immunity for protection. However, the design and delivery of thermostable dry powder-based vaccines represents a technological challenge: It calls for careful formulation and dosage form design, combined with cheap and efficient delivery devices, which must be engineered via a thorough understanding of the physiological barrier and the requirements for induction of mucosal immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials. PMID:26831645

  8. Molecular cloning of the human casein kinase II α subunit

    International Nuclear Information System (INIS)

    A human cDNA encoding the α subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II α subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the α subunits

  9. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

  10. Testing experimental subunit furunculosis vaccines for rainbow trout

    DEFF Research Database (Denmark)

    Marana, Moonika H.; Chettri, Jiwan Kumar; Skov, Jakob;

    2016-01-01

    Aeromonas salmonicida subsp. salmonicida (AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality. In this study we vaccinated rainbow...... response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent...

  11. Smallpox subunit vaccine produced in planta confers protection in mice

    OpenAIRE

    Golovkin, Maxim; Spitsin, Sergei; Andrianov, Vyacheslav; Smirnov, Yuriy; Xiao, Yuhong; Pogrebnyak, Natalia; Markley, Karen; Brodzik, Robert; Gleba, Yuri; Isaacs, Stuart N.; Koprowski, Hilary

    2007-01-01

    We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of sol...

  12. Posttranslational modifications in the CP43 subunit of photosystem II

    OpenAIRE

    Anderson, Lorraine B.; Maderia, Melissa; Ouellette, Anthony J. A.; Putnam-Evans, Cindy; Higgins, LeeAnn; Krick, Thomas; MacCoss, Michael J; Lim, Hanjo; Yates, John R.; Barry, Bridgette A.

    2002-01-01

    Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123–133]. The sequence A350PWLEPLR357 in luminal loop E is conserved in CP43 genes from 50 organism...

  13. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O; Pinna, L A; Issinger, O G

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the...... catalytic alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit...

  14. A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

    International Nuclear Information System (INIS)

    Eukaryotic proliferating cell nuclear antigen (PCNA), an essential accessory factor in DNA replication and repair, is a ring-shaped homotrimer. A novel nontrimeric structure of E113G-mutant PCNA protein is reported, which shows that this protein forms alternate subunit interactions. It is concluded that the charged side chain of Glu113 promotes normal trimer formation by destabilizing these alternate subunit interactions. Eukaryotic proliferating cell nuclear antigen (PCNA) is an essential replication accessory factor that interacts with a variety of proteins involved in DNA replication and repair. Each monomer of PCNA has an N-terminal domain A and a C-terminal domain B. In the structure of the wild-type PCNA protein, domain A of one monomer interacts with domain B of a neighboring monomer to form a ring-shaped trimer. Glu113 is a conserved residue at the subunit interface in domain A. Two distinct X-ray crystal structures have been determined of a mutant form of PCNA with a substitution at this position (E113G) that has previously been studied because of its effect on translesion synthesis. The first structure was the expected ring-shaped trimer. The second structure was an unanticipated nontrimeric form of the protein. In this nontrimeric form, domain A of one PCNA monomer interacts with domain A of a neighboring monomer, while domain B of this monomer interacts with domain B of a different neighboring monomer. The B–B interface is stabilized by an antiparallel β-sheet and appears to be structurally similar to the A–B interface observed in the trimeric form of PCNA. The A–A interface, in contrast, is primarily stabilized by hydrophobic interactions. Because the E113G substitution is located on this hydrophobic surface, the A–A interface should be less favorable in the case of the wild-type protein. This suggests that the side chain of Glu113 promotes trimer formation by destabilizing these possible alternate subunit interactions

  15. IMMUNOLOGICAL RESPONSE IN BOVINE LYMPH NODES STIMULATED WITH SUBUNITS VACCINES

    Directory of Open Access Journals (Sweden)

    Gabriel Andres Tafur Gomez

    2013-01-01

    Full Text Available The vaccination process belongs to the public health intervention methodologies that help prevent infections. Vaccinations performed successfully in the history of medicine reported the significance of this procedure to increase the quality of life, prevent zoonoses and improve animal production. Vaccine emergence remained without exact rules for a long time, maintaining a close relationship with pathogens. However, subunit vaccines, with a difference from the classical idea of protective immunity with microorganisms showed it is possible to trigger T-dependent responses with peptide, revealing new rules for vaccine development. This vaccination process starts by the modulation chance of adaptive immune response through peptide sequences process by APCs for immune synapse formation interceded for pMHC-TCR as a scaffold to T cells priming. In this way the immunological signal triggered by immune synapses is amplified in lymph nodes. As a consequence, T and B cells modulated by peptide activity interact between the B cell follicles region and T cell aggregates, which constitute the paracortical region of secondary lymphoid tissue to form connate unions as a prerequisite for clonal amplification and subsequent immunological memory. Indicating the knowledge of the mechanisms of immune response generated by peptides immunization is essential for understanding modulation, amplification and immune protection as demands for good subunits vaccine.

  16. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    OpenAIRE

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be...

  17. Functional consequences of Kir2.1/Kir2.2 subunit heteromerization

    OpenAIRE

    Panama, Brian K.; McLerie, Meredith; Lopatin, Anatoli N.

    2010-01-01

    Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in orde...

  18. Beta-Subunit of Human Chorionic Gonadotropin in Malignant Lymphoma : An Immunohistochemical Study

    OpenAIRE

    Senba, Masachika; Watanabe, Masami

    1991-01-01

    We present a rare case of a 77-year-old Japanese man with malignant lymphoma associated with production of beta-subunit of human chorionic gonadotropin in the cytoplasms of lymphoma cells in the lymph nodes. By immunoperoxidase staining, numerous tumor cells were reacted with beta-subunit of human chorionic gonadotropin. To the best of our knowledge, production of beta-subunit of human chorionic gonadotropin in the cytoplasm of lymphoma cells has not been reported. This patient evidences that...

  19. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells.

    Science.gov (United States)

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-01

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G(1) phase of cell cycling and decreased the proportion in the S/G(2) phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G(1) phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling. PMID:18178157

  20. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    International Nuclear Information System (INIS)

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G1 phase of cell cycling and decreased the proportion in the S/G2 phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G1 phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling

  1. Structure of Csm2 elucidates the relationship between small subunits of CRISPR-Cas effector complexes.

    Science.gov (United States)

    Venclovas, Česlovas

    2016-05-01

    Type I and type III CRISPR-Cas effector complexes share similar architecture and have homologous key subunits. However, the relationship between the so-called small subunits of these complexes remains a contentious issue. Here, it is shown that the recently solved structure of Thermotoga maritima Csm2 represents a dimer with the extensive structure swapping between monomers. Unswapping the structure generates a compact globular monomer which shares similar structure and surface properties with Cmr5, the small subunit of a related Cmr complex. Detailed analysis of available structures of small subunits reveals that they all have a common fold suggesting their common origin. PMID:27091242

  2. Genetic Diversity of High and Low Molecular Weight Glutenin Subunits in Algerian Aegilops geniculata

    Directory of Open Access Journals (Sweden)

    Asma MEDOURI

    2014-12-01

    Full Text Available Aegilops geniculata Roth is an annual grass relative to cultivated wheat and is widely distributed in North Algeria. Endosperm storage proteins of wheat and its relatives, namely glutenins and gliadins, play an important role in dough properties and bread making quality. In the present study, the different alleles encoded at the four glutenin loci (Glu-M1, Glu-U1, Glu-M3 and Glu-U3 were identified from thirty five accessions of the tetraploid wild wheat A. geniculata collected in Algeria using Sodium dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE. At Glu-M1 and Glu-U1 loci, encoding high molecular weight glutenin subunits (HMW-GS or A-subunits, 15 and 12 alleles were observed respectively, including one new subunit. B-Low molecular weight glutenin subunits zone (B-LMW-GS displayed a far greater variation, as 28 and 25 alleles were identified at loci Glu-M3 and Glu-U3 respectively. Thirty two subunits patterns were revealed at the C subunits- zone and a total of thirty four patterns resulted from the genetic combination of the two zones (B- and C-zone. The wide range of glutenin subunits variation (high molecular weight glutenin subunits and low molecular weight glutenin subunits in this species has the potential to enhance the genetic variability for improving the quality of wheat./span>

  3. Structure–Function Relationships in Fungal Large-Subunit Catalases

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W

    2009-01-01

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  4. Structure-function relationships in fungal large-subunit catalases.

    Science.gov (United States)

    Díaz, Adelaida; Valdés, Víctor-Julián; Rudiño-Piñera, Enrique; Horjales, Eduardo; Hansberg, Wilhelm

    2009-02-13

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H(2)O(2)) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H(2)O(2) to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-gamma-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H(2)O(2) concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics. PMID:19109972

  5. The phosphorylation pattern of bovine heart complex I subunits

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Sardanelli, Anna Maria; Signorile, Anna;

    2007-01-01

    The phosphoproteome of bovine heart complex I of the respiratory chain has been analysed with a procedure based on nondenaturing gel electrophoretic separation of complex I from small quantities of mitochondria samples, in-gel digestion, in combination with phosphopeptide enrichment by titanium...... dioxide and MS. The results, complemented by analyses of purified samples of complex I, showed phosphorylation of five subunits of the complex, 42 kDa (human gene NDUFA10), ESSS, B14.5a (human gene NDUFA7), B14.5b (human gene NDUFC2) and B16.6 (GRIM-19). MS also revealed the presence of phosphorylated...... programmed cell death protein 8(AIF) in native and purified samples of complex I analysed. The possible physiological relevance of these findings is discussed....

  6. Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins.

    Science.gov (United States)

    Morales-Perez, Claudio L; Noviello, Colleen M; Hibbs, Ryan E

    2016-05-01

    The ability of oligomeric membrane proteins to assemble in different functional ratios of subunits is a common feature across many systems. Recombinant expression of hetero-oligomeric proteins with defined stoichiometries facilitates detailed structural and functional analyses, but remains a major challenge. Here we present two methods for overcoming this challenge: one for rapid virus titration and another for stoichiometry determination. When these methods are coupled, they allow for efficient dissection of the heteromer stoichiometry problem and optimization of homogeneous protein expression. We demonstrate the utility of the methods in a system that to date has proved resistant to atomic-scale structural study, the nicotinic acetylcholine receptor. Leveraging these two methods, we have successfully expressed, purified, and grown diffraction-quality crystals of this challenging target. PMID:27041595

  7. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  8. Purification and subunit composition of atrial natriuretic peptide receptor

    International Nuclear Information System (INIS)

    A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4.0 X 10(-10)M. Upon treatment with 10 mM dithiothreitol, the purified receptor migrated as a single band at Mr 60,500 in NaDodSO4/polyacrylamide gel electrophoresis. These findings show that the holoreceptor for ANP in vascular tissue is composed of two subunits of identical apparent molecular weight, presumably linked by a disulfide bridge(s)

  9. Nasal reconstruction based on aesthetic subunits in Orientals.

    Science.gov (United States)

    Yotsuyanagi, T; Yamashita, K; Urushidate, S; Yokoi, K; Sawada, Y

    2000-07-01

    Reconstruction based on the aesthetic subunit principle has yielded good aesthetic outcomes in patients with moderate to severe nasal defects caused by trauma or tumor resection. However, the topographic subunits previously proposed are often unsuitable for Orientals. Compared with the nose in white patients, the nose in Orientals is low, lacks nasal muscle, and has a flat glabella; the structural features of the underlying cartilage and bone are not distinctly reflected in outward appearance. The authors devised aesthetic subunits suitable for Orientals, and they used these units to reconstruct various parts of the nose. The major difference between these units and those presented previously is the lack of soft triangles and the addition of the glabella as an independent unit. The authors divided the nose into the following five topographic units: the glabella, the nasal dorsum, the nasal tip, and the two alae. The border of the nasal dorsum unit was extended to above the maxillonasal suture. The basic reconstruction techniques use a V-Y advancement flap from the forehead to reconstruct the glabella, an island flap from the forehead to reconstruct the nasal dorsum and nasal tip, a nasolabial flap to reconstruct an ala, and a malar flap to reconstruct the cheek. A combination of flaps was used when the defect involved more than one unit. This concept was used for nasal reconstruction in 24 patients. In one patient undergoing reconstruction of the nasal dorsum and in one undergoing reconstruction of the nasal tip, the texture of the forearm flap did not match well, which resulted in a slightly unsatisfactory aesthetic outcome. In one patient in whom the glabella, nasal dorsum, and part of the cheek were reconstructed simultaneously, a web was formed at the medial ocular angle, and a secondary operation was subsequently performed using Z-plasty. In one patient undergoing reconstruction with a forehead flap, defatting was required to reduce the bulk of the

  10. Chaperonin Structure - The Large Multi-Subunit Protein Complex

    Directory of Open Access Journals (Sweden)

    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  11. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    Science.gov (United States)

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform. PMID:25484331

  12. Prokaryotic expression of Chinese bovine enterokinase catalytic subunit

    Institute of Scientific and Technical Information of China (English)

    黄鹤; 赵阳; 甘一如

    2004-01-01

    Background To express in vitro the bovine enterokinase catalytic subunit (EKL ) protein, which could be used in the future for the cleavage and purification of fusion proteins. Methods Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from the duodenal mucosa of a bovine obtained at a wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EK, was purified with His · Tag affinity chromatography, and its bioactivity was analyzed. Results Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5'terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using a NJ-IDA resin, After desalting and changing the buffer, the crude kinase was incubated at 21℃ overnight and shown to have a high autocatalytic cleavage activity. Conclusion The EKE gene from a Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.

  13. The subunit structure of the extracellular hemoglobin of Biomphalaria glabrata

    International Nuclear Information System (INIS)

    Full text. The hemoglobin of Biomphalaria glabrata was purified to homogeneity by a two step purification protocol using a gel filtration column (Superose 6 HR/Pharmacia ) followed by an anion exchange chromatography (MONO-Q Sepharose/Pharmacia). The dissociation products were analysed by a 5 - 15 % Polyacrylamide gel electrophoresis containing Sodium Dodecyl Sulfate (SDS-PAGE) giving a band of 270 K Daltons and a band of 180 K Daltons after reduction with β-mercaptoethanol. The same profile was obtained in a 3.5 % Agarose gel electrophoresis containing SDS (SDS-AGE) showing additional bands of higher molecular weight. These bands were proposed to be monomers, dimers and trimers and, after reduction in a Bidimensional SDS-AGE, the proposed monomers and dimers were decomposed in two and four bands that were interpreted as 1 - 4 chains. The hemoglobin was digested by four different proteases ( Thrombin, Trypsin, Chymotrypsin and Subtilisin ) showing several equivalent fragments with molecular weights multiples of its minimum molecular weight ( 17.7 K Daltons). The circular dichroism spectrum of the protein showed a characteristic high α-helix content. We proposed that this hemoglobin is a pentamer of approx. 360 K Daltons subunits each formed by two 180 K Daltons chains linked in pairs by disulfide bridges and each of these chains comprises ten Heme binding domains. These data were compared to other Planorbidae extracellular hemoglobins. Up to now, the quaternary structure of this hemoglobin (shape and disposition of the subunits) is unknown. It is intended to elucidate its structure by Small Angle X-Ray Scattering in Brazilian National Laboratory of Synchrotron Light (LNLS). (author)

  14. Proteomic analysis of transducin beta-subunit structural heterogeneity.

    Science.gov (United States)

    Clack, James W; Juhl, Martha; Rice, Carol A; Li, Junyu; Witzmann, Frank A

    2003-10-01

    Partially purified transducin was resolved using two-dimensional gel electrophoresis (2-DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa beta-subunit of transducin (T(beta)) was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. As many as six spots with different pI, ranging from 5.2 to 6.1, were observed when separated using 2-DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide-binding protein G(I)/G(S)/G(T) beta-subunit 1 (GNB1; T(beta1)). This suggested that post-translational modification was responsible for the differences in pI. Phosphorylation experiments showed that at least one T(beta1) spot was phosphorylated in vitro with [gamma-(32)P]ATP by an endogenous kinase. Treatment of T(beta) with alkaline phosphatase caused a large change in the spot pattern of T(beta), suggesting that phosphorylated T(beta) is a substrate for alkaline phosphatase. We conclude that T(beta1) constitutes over 99% of the T(beta) expressed in bovine rod outer segments and displays structural heterogeneity that is due to post-translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1. PMID:14595696

  15. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    International Nuclear Information System (INIS)

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing [14C]alanine and [3H] glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, [14C]alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. [3H]Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function

  16. Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

    International Nuclear Information System (INIS)

    The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of βDP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to βE (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

  17. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    Science.gov (United States)

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  18. Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper).

    Science.gov (United States)

    Polgár, J; Magnenat, E M; Peitsch, M C; Wells, T N; Saqi, M S; Clemetson, K J

    1997-04-15

    Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein. PMID:9163349

  19. Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.

    Science.gov (United States)

    Zhu, Shaotong; Canales, Alejandra; Bedair, Mai; Vik, Steven B

    2016-06-01

    Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme from Escherichia coli the C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme. PMID:26931547

  20. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels;

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We hyp...

  1. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  2. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    Science.gov (United States)

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

  3. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Johansson, Helle Wulf; Hay-Schmidt, Anders;

    2009-01-01

    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expressed...

  4. HMW glutenin subunits in multiploid Aegilops species: composition analysis and molecular cloning of coding sequences

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The Aegilops genus contains species closely related to wheat. Incommon with wheat, Aegilops species accumulate high molecular weight (HMW) glutenin subunits in their endospermic tissue. In this study, we investigated the composition of HMW glutenin subunits in four multiploid Aegilops species using SDS-PAGE analysis. Furthermore, by working with Ae. ventricosa, we established an efficient genomic PCR condition for simultaneous amplification of DNA sequences coding for either x-ory-type HMW glutenin subunits from polyploid Aegilops species. Using the genomic PCR condition, we amplified and subsequently cloned two DNA fragments that may code for HMW glutenin subunits in Ae. ventricosa. Based on an analysis of the deduced amino acid sequences, we concluded that the two cloned sequences encode one x- and one y-type of HMW glutenin subunit, respectively.

  5. Functional consequences of Kir2.1/Kir2.2 subunit heteromerization.

    Science.gov (United States)

    Panama, Brian K; McLerie, Meredith; Lopatin, Anatoli N

    2010-10-01

    Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents. PMID:20676672

  6. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Science.gov (United States)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  7. trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1 proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.

  8. Immunoradiometric assay of subunit H of human acidic isoferritin

    International Nuclear Information System (INIS)

    Acidic isoferritin (AIF) was isolated and purified from human heart muscle. Antisera against AIF were generated by immunizing rabbits and purified in affinity chromatography column which was conjugated with AIF. The immunoglobulins against AIF were made in solid-phase. 125I-monoclonal antibodies against H subunit of AIF(AIF-H) was prepared by the chloramine-T method. The AIF-H solid-phase IRMA was established and the data were processed by the automatic Spline function. The intra- and interbatch coefficients of variation were 2.443% and 10.160% respectively. The sensitivity was 0.736 μg/L and the recovery was 92.293%. The measuring range was 1.472-320 μg/L and ED50 was 23.05 μg/L. No cross-reaction was observed with AFP, CEA and LF, but there was 3.125% with ferritin. A parallel experiment demonstrated that serum matrix had no interference with this method. The level of serum AIF-H in 40 healthy men and women was 3.75 +- 1.85 μg/L and 2.92 +- 0.96 μg/L respectively while it was 8.39 +-3.87 μg/L in 10 patients with hepatoma. This method may therefore provide a valuable tool for diagnosis of tumor, especially for basic and clinical study of hepatoma

  9. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    Energy Technology Data Exchange (ETDEWEB)

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  10. Design of a hyperstable 60-subunit protein icosahedron

    Science.gov (United States)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  11. Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

    International Nuclear Information System (INIS)

    To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

  12. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  13. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    Science.gov (United States)

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations. PMID:22814267

  14. Vaults and telomerase share a common subunit, TEP1.

    Science.gov (United States)

    Kickhoefer, V A; Stephen, A G; Harrington, L; Robinson, M O; Rome, L H

    1999-11-12

    Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly. PMID:10551828

  15. The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

    OpenAIRE

    Barrera, Nelson P.; Henderson, Robert M.; Murrell-Lagnado, Ruth D.; Edwardson, J Michael

    2007-01-01

    Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing b...

  16. An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

    Directory of Open Access Journals (Sweden)

    Kevin C Miranda

    Full Text Available The vacuolar-type H(+-ATPase (V-ATPase is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

  17. Insights into the subunit in-teractions of the chloroplast ATP synthase

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.

  18. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    Science.gov (United States)

    Szuplewski, S; Terracol, R

    2001-08-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits. PMID:11514451

  19. Radioimmunodetection of choriocarcinoma in nude mouse by radiolabeled antibody to human chorionic gonadotropin. beta. -subunit

    Energy Technology Data Exchange (ETDEWEB)

    Yamanaka, Nozomu (Kobe Univ. (Japan). School of Medicine)

    1983-06-01

    Photoscans and organ radioactivity were assessed with radiolabeled antibodies to hCG or hCG-..beta.. subunit in nude mice bearing hCG-producing tumors. /sup 125/I-anti hCG or /sup 125/I-anti-hCG-..beta.. subunit was administered to nude mice bearing an hCG-producing tumor, GCH-lnu. Measurement of radioactivity revealed specific accumulation of both antibodies into the tumor. Especially the accumulation of /sup 125/I-anti hCG-..beta.. subunit on the 3rd day of administration was high, being about 4 times higher than the nonspecific accumulation in the liver. The localization of hCG in the tumor was examined by the indirect peroxidase-antiperoxidase method using anti-hCG. and anti hCG-..cap alpha.. subunit, and hCG-..beta.. subunit. The immunoperoxidase reaction was positive, and the accumulation of these radiolabeled antibodies in the tumor is suggested to be an immunologically specific phenomenon. The tumor in the tumor-bearing nude mouse injected with /sup 131/I-anti hCG or /sup 131/I-anti hCG-..beta.., subunit could be visualized as a hot area by external scintigraphy. Especially, the tumor image produced by the accumulation of anti hCG-..beta.. subunit was very clear against the background radiation.

  20. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    Science.gov (United States)

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  1. Functional Diversification of Maize RNA Polymerase IV and V Subtypes via Alternative Catalytic Subunits

    Directory of Open Access Journals (Sweden)

    Jeremy R. Haag

    2014-10-01

    Full Text Available Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two subtypes of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  2. Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus

    International Nuclear Information System (INIS)

    The two recombinant apo subunits H1 and H2 from H. americanus have been structurally characterized. Reconstitution studies with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits. Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H1 and H2 from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H1 and H2 with astaxanthin reproduced the bathochromic shift of 85–95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype–phenotype linkage

  3. A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

    Science.gov (United States)

    Tanneberger, Katrin; Kirchberger, Jürgen; Bär, Jörg; Schellenberger, Wolfgang; Rothemund, Sven; Kamprad, Manja; Otto, Henning; Schöneberg, Torsten; Edelmann, Anke

    2007-08-10

    Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources. PMID:17522059

  4. Molecular basis for differential modulation of BK channel voltage-dependent gating by auxiliary γ subunits.

    Science.gov (United States)

    Li, Qin; Fan, Fei; Kwak, Ha Rim; Yan, Jiusheng

    2015-06-01

    Large conductance Ca(2+)- and voltage-activated potassium (BK) channels are comprised of pore-forming α subunits and various regulatory auxiliary subunits. The BK channel auxiliary γ (BKγ) subunits are a newly identified class of proteins containing an extracellular leucine-rich repeat domain (LRRD), a single transmembrane (TM) segment, and a short cytoplasmic C-terminal tail (C-tail). Although each of the four BKγ proteins shifts the voltage dependence of BK channel activation in a hyperpolarizing direction, they show markedly different efficacies, mediating shifts over a range of 15-145 mV. Analyses of chimeric BKγ subunits created by swapping individual structural elements, and of BKγ deletion and substitution mutants, revealed that differential modulation of BK gating by the four BKγ subunits depends on a small region consisting of the TM segment and the adjacent intracellular cluster of positively charged amino acids. The γ1 and γ2 TM segments contributed approximately -100 mV, and the γ1 and γ3 C-tails contributed approximately -40 mV, to shifting the voltage dependence of BK channel activation, whereas the γ3 and γ4 TM segments and the γ2 and γ4 C-tails contributed much less. The large extracellular LRRDs were mainly functionally interchangeable, although the γ1 LRRD was slightly less effective at enhancing (or slightly more effective at attenuating) the shift in BK channel voltage-dependent gating toward hyperpolarizing potentials than those of the other BKγ subunits. Analysis of mutated BKγ subunits revealed that juxta-membrane clusters of positively charged amino acids determine the functions of the γ1 and γ3 C-tails. Therefore, the modulatory functions of BKγ subunits are coarse- and fine-tuned, respectively, through variations in their TM segments and in the adjacent intracellular positively charged regions. Our results suggest that BK channel modulation by auxiliary γ subunits depends on intra- and/or juxta-membrane mechanisms

  5. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    International Nuclear Information System (INIS)

    The α, β, γ subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the α chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for α and β. However, these putative homologies were not apparent when tryptic maps of the biosynthetically ([3H]leucine) labeled subunits were analyzed

  6. Transcriptional organization of the phycocyanin subunit gene clusters of the cyanobacterium Anacystis nidulans UTEX 625.

    OpenAIRE

    Kalla, S R; Lind, L K; Lidholm, J; Gustafsson, P

    1988-01-01

    The phycocyanin subunit gene cluster is duplicated on the chromosome of the cyanobacterium Anacystis nidulans UTEX 625. The two gene clusters cpcB1A1 (left) and cpcB2A2 (right) are separated by about 2,500 base pairs, and in each cluster the beta-subunit gene is located upstream from the alpha-subunit gene. Filter hybridizations with phycocyanin-specific probes to total RNA detected at least two major transcripts that were 1,300 to 1,400 nucleotides long. Besides these major mRNA species, two...

  7. zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly

    OpenAIRE

    1993-01-01

    cDNA encoding the 20-kD subunit of coatomer, zeta-COP, predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon- COP), zeta-COP exists in both coatomer bound and free pools.

  8. Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance

    OpenAIRE

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-chieh; Kim, Sunhee S.; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L.; Elpek, Kutlu G; Card, Joseph

    2014-01-01

    Activating mutations of G protein alpha subunits (Gα) occur in 4–5% of all human cancers 1 but oncogenic alterations in beta subunits (Gβ) have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors, and disrupt Gα-Gβγ interactions. Different mutations in Gβ protein...

  9. Amino acid sequence of the beta subunit of bovine lung casein kinase II.

    OpenAIRE

    Takio, K.; Kuenzel, E A; Walsh, K. A.; Krebs, E G

    1987-01-01

    The amino acid sequence of the 209-residue beta subunit of bovine lung casein kinase II has been determined. Excluding the amino-terminal blocking group, which was not identified, the molecular weight of the polypeptide chain is 24,239. A marked polarity of the beta subunit is indicated by clusters of negative charges in the amino-terminal region and of positive charges in the carboxyl-terminal region. Whereas the beta subunit shows no homology with any known protein, a segment of the sequenc...

  10. Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

    DEFF Research Database (Denmark)

    Stalter, G; Siemer, S; Becht, E; Ziegler, M; Remberger, K; Issinger, O G

    1994-01-01

    of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or...... degradation. This at least partly owing to the presence of excess enzymatically active protein kinase alpha-subunit but also to a significantly higher presence of the non-catalytic beta-subunit....

  11. Optimized polypeptide for a subunit vaccine against avian reovirus.

    Science.gov (United States)

    Goldenberg, Dana; Lublin, Avishai; Rosenbluth, Ezra; Heller, E Dan; Pitcovski, Jacob

    2016-06-01

    Avian reovirus (ARV) is a disease-causing agent. The disease is prevented by vaccination with a genotype-specific vaccine while many variants of ARV exist in the field worldwide. Production of new attenuated vaccines is a long-term process and in the case of fast-mutating viruses, an impractical one. In the era of molecular biology, vaccines may be produced by using only the relevant protein for induction of neutralizing antibodies, enabling fast adjustment to the emergence of new genetic strains. Sigma C (SC) protein of ARV is a homotrimer that facilitates host-cell attachment and induce the production and secretion of neutralizing antibodies. The aim of this study was to identify the region of SC that will elicit a protective immune response. Full-length (residues 1-326) and two partial fragments of SC (residues 122-326 and 192-326) were produced in Escherichia coli. The SC fragment of residues 122-326 include the globular head, shaft and hinge domains, while eliminating intra-capsular region. This fragment induces significantly higher levels of anti-ARV antibodies than the shorter fragment or full length SC, which neutralized embryos infection by the virulent strain to a higher extent compared with the antibodies produced in response to the whole virus vaccine. Residues 122-326 fragment is assumed to be folded correctly, exposing linear as well as conformational epitopes that are identical to those of the native protein, while possibly excluding suppressor sequences. The results of this study may serve for the development of a recombinant subunit vaccine for ARV. PMID:27155492

  12. P. berghei telomerase subunit TERT is essential for parasite survival.

    Directory of Open Access Journals (Sweden)

    Agnieszka A Religa

    Full Text Available Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA, though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT, is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further

  13. Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

    Directory of Open Access Journals (Sweden)

    Tianquan Jin

    2009-12-01

    Full Text Available Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i in a compaction of protein matrix subunit dimensions, (ii reduced conformational variability, (iii an increase in polyproline II helices, and (iv promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem

  14. Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α: ROLE OF B56α IN NUCLEAR EXPORT OF THE CATALYTIC SUBUNIT*

    OpenAIRE

    Flegg, Cameron P.; Sharma, Manisha; Medina-Palazon, Cahora; Jamieson, Cara; Galea, Melanie; Brocardo, Mariana G.; Mills, Kate; Henderson, Beric R.

    2010-01-01

    Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is depende...

  15. Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

    OpenAIRE

    Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

    2006-01-01

    The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

  16. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.

    Science.gov (United States)

    Ilca, Serban L; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M; Stuart, David I; Huiskonen, Juha T

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  17. Research and design of a production system for a Tuberculosis (TB) sub-unit vaccine

    OpenAIRE

    Hunter, Georgina

    2015-01-01

    Undergraduate research project into a new Tuberculosis vaccine for those living in endemic parts of sub-Saharan Africa through the design, process development and commercial manufacture of sub-unit vaccine process engineering and manufacturing systems.

  18. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis;

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

  19. Is There an Optimal Formulation and Delivery Strategy for Subunit Vaccines?

    Science.gov (United States)

    Bobbala, Sharan; Hook, Sarah

    2016-09-01

    Modern vaccine design has moved away from attenuated or inactivated whole-pathogen vaccines to more pure and defined subunit vaccines. However subunit antigens have poor bioavailability and stability and lack immunogenicity. To overcome these issues subunit vaccines have to be administered in a suitable delivery system in combination with immune stimulants. Many different delivery systems have been developed and investigated each having different modes of action, for example increasing delivery and/or sustaining delivery of antigen to immune cells. In addition a number of different routes of immunization are possible and these can play a crucial role in determining the fate of an immune response. In this review the different strategies for the delivery of prophylactic and therapeutic subunit vaccines along with the impact of these on the immune responses generated are discussed. PMID:27380191

  20. The subunit composition of hinokiresinol synthase controls geometrical selectivity in norlignan formation.

    Science.gov (United States)

    Suzuki, Shiro; Yamamura, Masaomi; Hattori, Takefumi; Nakatsubo, Tomoyuki; Umezawa, Toshiaki

    2007-12-26

    The selective formation of E- or Z-isomers is an important process in natural product metabolism. We show that the subunit composition of an enzyme can alter the geometrical composition of the enzymatic products. Hinokiresinol synthase, purified from Asparagus officinalis cell cultures, is responsible for the conversion of (7E,7'E)-4-coumaryl 4-coumarate to (Z)-hinokiresinol, the first step in norlignan formation. The protein is most likely a heterodimer composed of two distinct subunits, which share identity with members of the phloem protein 2 gene superfamily. Interestingly, each recombinant subunit of hinokiresinol synthase expressed in Escherichia coli solely converted (7E,7'E)-4-coumaryl 4-coumarate to the unnatural (E)-hinokiresinol, the E-isomer of (Z)-hinokiresinol. By contrast, a mixture of recombinant subunits catalyzed the formation of (Z)-hinokiresinol from the same substrate. PMID:18093914

  1. Stereocontrolled Synthesis of the C(1)-C(11) Subunit of the Iejimalides

    DEFF Research Database (Denmark)

    Mendlik, Matthew T.; Cottard, Muriel; Rein, Tobias;

    1997-01-01

    An enantioselective synthesis of the C(1)-C(11) subunit of the iejimalides has been accomplished through a combination of an asymmetric Homer-Wadsworth-Emmons condensation and a chiral pool approach. (C) 1997 Elsevier Science Ltd....

  2. Performance of information system implementation based on coupling-cohesion among subunits

    Institute of Scientific and Technical Information of China (English)

    Wang Tienan; Li Yijun; Wang Mingzhu

    2007-01-01

    The intermediate information system benefit and the coupling-cohesion of subunits are presented to study the performance of information system implementation. Based on the organizational information processing theory and the organizational behaviour theory, a theoretical model is established from the perspective of coupling-cohesion of subunits. The reliability and validity of the model are checked up with the structural equation models and the data collected with questionnaires. The results of the study give some theoretical and practical guidance.

  3. Structure and Catalysis of Acylaminoacyl Peptidase CLOSED AND OPEN SUBUNITS OF A DIMER OLIGOPEPTIDASE

    OpenAIRE

    Harmat, V.; Domokos, K.; Menyhard, D. K.; Pallo, A.; Szeltner, Z.; Szamosi, I.; Beke-Somfai, T.; Naray-Szabo, G.; Polgar, L.

    2011-01-01

    Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. ...

  4. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    International Nuclear Information System (INIS)

    Eukaryotic RNA polymerase II consists of three subspecies, designated II0, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with γ[32P]-ATP in the presence of casein kinase I and 32P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of 32P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription

  5. Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II

    Energy Technology Data Exchange (ETDEWEB)

    Cadena, D.; Dahmus, M.E.

    1986-05-01

    Eukaryotic RNA polymerase II consists of three subspecies, designated II/sub 0/, II/sub A/, and II/sub B/, which differ in the apparent M/sub r/ of their largest subunit, IIo, IIa, and IIb, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. Subunit IIa (IIo) has been shown to contain an unusual C-terminal domain composed of 52 repeats of a seven amino acid block with the consensus sequence tyr-ser-pro-thr-ser-pro-ser. In an effort to purify the C-terminal domain, purified calf thymus RNA polymerase II was cleaved with CNBr. Following SDS polyacrylamide gel electrophoresis the CNBr digests were transferred and probed with IIa mono-specific antibody. A single immunoreactive peptide with an apparent M/sub r/ of 65,000 was detected. A peptide of similar M/sub r/ was found when purified subunit IIa was digested with CNBr while no immunoreactive peptide was detected in digests of subunit IIb. Purified RNA polymerase II was phosphorylated with ..gamma..(/sup 32/P)-ATP in the presence of casein kinase I and /sup 32/P-labeled subunits IIa and IIo purified. CNBr clIIa revealed a major phosphopeptide with an apparent M/sub r/ of 65,000. Cleavage of /sup 32/P-labeled age of IIo revealed a broad phosphopeptide band of M/sub r/ 75,000-90,000. The C-terminal peptide from subunit IIa was purified by gel filtration on HPLC. Experiments are in progress to map in vivo phosphorylation sites within subunits IIo and IIa and to examine the effects of the purified C-terminal peptide on in vitro transcription.

  6. Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

    OpenAIRE

    Yan HUANG; Li, Wencheng; Yao, Xiao; Lin, Qi-jiang; Yin, Jing-wen; Liang, Yan; Heiner, Monika; Tian, Bin; HUI, JINGYI; Wang, Gang

    2012-01-01

    Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially co...

  7. Oxygen influences the subunit structure of cytochrome c oxidase in the slime mold Dictyostelium discoideum.

    OpenAIRE

    Schiavo, G; Bisson, R

    1989-01-01

    The conditions that promote the alternative expression of two nuclear-encoded subunits of cytochrome c oxidase in the slime mold Dictyostelium discoideum (Bisson, R., and Schiavo, G. (1986) J. Biol. Chem. 261, 4373-4376) have been investigated. Oxygen concentration seems to be the only factor able to cause the subunit switching. This result indicates that the polypeptide composition of the mitochondrial enzyme can be influenced by environmental conditions. The significance of this change is d...

  8. Nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties

    International Nuclear Information System (INIS)

    The structure of the nicotinic acetylcholine receptor has been highly conserved during animal evolution, and in all the species and tissues studied so far, including mammals, it is a pseudosymmetric, pentameric complex of related subunits with very similar physical properties. All subunits of these nicotinic receptors were derived from a common ancestral gene, probably by way of gene duplications occurring very early in animal evolution. 45 refs., 8 figs., 2 tabs

  9. Differential Distribution of Exosome Subunits at the Nuclear Lamina and in Cytoplasmic FociD⃞V⃞

    OpenAIRE

    Graham, Amy C.; Kiss, Daniel L.; Andrulis, Erik D.

    2006-01-01

    The exosome complex plays important roles in RNA processing and turnover. Despite significant mechanistic insight into exosome function, we still lack a basic understanding of the subcellular locales where exosome complex biogenesis and function occurs. Here, we employ a panel of Drosophila S2 stable cell lines expressing epitope-tagged exosome subunits to examine the subcellular distribution of exosome complex components. We show that tagged Drosophila exosome subunits incorporate into compl...

  10. Overexpression of ??3/??5/??4 nicotinic receptor subunits modifies impulsive-like behavior

    OpenAIRE

    Vi??als, Xavier; Molas Casacuberta, Susanna, 1985-; Gallego, Xavier; Fern??ndez Montes, Rub??n D.; Robledo, Patr??cia, 1958-; Dierssen, Mara; Maldonado, Rafael

    2012-01-01

    Recent studies have revealed that sequence variants in genes encoding the ??3/??5/??4 nicotinic acetylcholine receptor subunits are associated with nicotine dependence. In this study, we evaluated two specific aspects of executive functioning related to drug addiction (impulsivity and working memory) in transgenic mice over expressing ??3/??5/??4 nicotinic receptor subunits. Impulsivity and working memory were evaluated in an operant delayed alternation task, where mice must inhibit respondin...

  11. EFFECTS OF MUTANT DROSOPHILA K+ CHANNEL SUBUNITS ON HABITUATION OF THE OLFACTORY JUMP RESPONSE

    OpenAIRE

    Joiner, M. A.; ASZTALOS, Z.; Jones, C. J.; Tully, T.; Wu, C.-F.

    2007-01-01

    The olfactory-jump response assay was used to analyze habituation in Drosophila mutants of potassium (K+) channel subunits. As with physiological assays of the giant fiber-mediated escape reflex, mutations at loci that encode K+ channel subunits have distinct effects on habituating the olfactory-jump response. The data for slowpoke and ether à go-go indicate similar effects on habituation of the olfactory-jump response and the giant fiber-mediated escape. Habituation in the olfactory jump ass...

  12. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    OpenAIRE

    Matsuda, K; Buckingham, S D; Freeman, J.C.; Squire, M D; Baylis, H. A.; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine...

  13. Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S;

    1999-01-01

    The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) to...... produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures....

  14. Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins.

    OpenAIRE

    Plank, T; Toole, C; Anderson, L K

    1995-01-01

    Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all funct...

  15. Neutron scattering and the 30 S ribosomal subunit of E. coli

    International Nuclear Information System (INIS)

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today. 30 references, 5 figures

  16. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    Science.gov (United States)

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  17. An interaction between gramicidin and the sigma subunit of RNA polymerase.

    OpenAIRE

    Fisher, R.; Blumenthal, T

    1982-01-01

    Gramicidin, a peptide antibiotic produced by Bacillus brevis, inhibits initiation of transcription by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). We show here that the presence of gramicidin causes an increase in the rate of cleavage of the sigma subunit of Escherichia coli RNA polymerase by trypsin, although it does not alter the cleavage rate of any of the core subunits. Furthermore, whereas isolated sigma is cleaved much faster than is sigma in holoenzym...

  18. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    OpenAIRE

    Molinari, A M; Abbondanza, C; Armetta, I.; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-01-01

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl...

  19. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    Science.gov (United States)

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  20. AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

    OpenAIRE

    Nadine Harmel; Barbara Cokic; Gerd Zolles; Henrike Berkefeld; Veronika Mauric; Bernd Fakler; Valentin Stein; Nikolaj Klöcker

    2012-01-01

    Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining alpha-subunits GluA1-4 and auxiliary beta-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstit...

  1. Exposing the subunit diversity within protein complexes: a mass spectrometry approach.

    Science.gov (United States)

    Rozen, Shelly; Tieri, Alessandra; Ridner, Gabriela; Stark, Ann-Kathrin; Schmaler, Tilo; Ben-Nissan, Gili; Dubiel, Wolfgang; Sharon, Michal

    2013-03-01

    Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes. PMID:23296018

  2. A new low resolution model for the 50S subunit of Escherichia coli ribosomes

    International Nuclear Information System (INIS)

    Neutron low angle scattering studies of the 50S subunit of Escherichia coli ribosomes using the contrast variation method reveals large fluctuations in the scattering density. A region of relatively low scattering density, rich in proteins, surrounds an RNA-rich core of higher scattering density. The centres of mass of the RNA and protein parts of the 50S subunit are separated by a distance that is considerably smaller than that reported in previous studies. (orig.)

  3. Xanthan Gum as an Adjuvant in a Subunit Vaccine Preparation against Leptospirosis

    OpenAIRE

    Bacelo, Katia L.; Hartwig, Daiane D.; Seixas, Fabiana K.; Rodrigo Schuch; Angelita da S. Moreira; Marta Amaral; Tiago Collares; Vendrusculo, Claire T.; McBride, Alan J. A.; Dellagostin, Odir A.

    2014-01-01

    Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), alu...

  4. Investigation of molluscan phylogeny using large-subunit and small-subunit nuclear rRNA sequences.

    Science.gov (United States)

    Passamaneck, Yale J; Schander, Christoffer; Halanych, Kenneth M

    2004-07-01

    The Mollusca represent one of the most morphologically diverse animal phyla, prompting a variety of hypotheses on relationships between the major lineages within the phylum based upon morphological, developmental, and paleontological data. Analyses of small-ribosomal RNA (SSU rRNA) gene sequence have provided limited resolution of higher-level relationships within the Mollusca. Recent analyses suggest large-subunit (LSU) rRNA gene sequences are useful in resolving deep-level metazoan relationships, particularly when combined with SSU sequence. To this end, LSU (approximately 3.5 kb in length) and SSU (approximately 2 kb) sequences were collected for 33 taxa representing the major lineages within the Mollusca to improve resolution of intraphyletic relationships. Although the LSU and combined LSU+SSU datasets appear to hold potential for resolving branching order within the recognized molluscan classes, low bootstrap support was found for relationships between the major lineages within the Mollusca. LSU+SSU sequences also showed significant levels of rate heterogeneity between molluscan lineages. The Polyplacophora, Gastropoda, and Cephalopoda were each recovered as monophyletic clades with the LSU+SSU dataset. While the Bivalvia were not recovered as monophyletic clade in analyses of the SSU, LSU, or LSU+SSU, the Shimodaira-Hasegawa test showed that likelihood scores for these results did not differ significantly from topologies where the Bivalvia were monophyletic. Analyses of LSU sequences strongly contradict the widely accepted Diasoma hypotheses that bivalves and scaphopods are closely related to one another. The data are consistent with recent morphological and SSU analyses suggesting scaphopods are more closely related to gastropods and cephalopods than to bivalves. The dataset also presents the first published DNA sequences from a neomeniomorph aplacophoran, a group considered critical to our understanding of the origin and early radiation of the Mollusca

  5. Deletion mutants of AP-1 adaptin subunits display distinct phenotypes in fission yeast.

    Science.gov (United States)

    Ma, Yan; Takeuchi, Mai; Sugiura, Reiko; Sio, Susie O; Kuno, Takayoshi

    2009-08-01

    Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes. PMID:19624755

  6. Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone α-subunit

    International Nuclear Information System (INIS)

    We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH α-subunit, with RCXM-α as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-α. A tryptic digest of RCXM α-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM α-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency (panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-α were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of α-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit

  7. Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

    Institute of Scientific and Technical Information of China (English)

    石晓冰; 魏家绵; 沈允钢

    2000-01-01

    Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast

  8. The subunit structure of potato tuber ADPglucose pyrophosphorylase. [Solanum tuberosum L

    Energy Technology Data Exchange (ETDEWEB)

    Okita, T.W.; Nakata, P.A.; Anderson, J.M. (Washington State Univ., Pullman (USA)); Sowokinos, J. (Univ. of Minnesota, St. Paul (USA)); Morell, M.; Preiss, J. (Michigan State Univ., East Lansing (USA))

    1990-06-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.

  9. High molecular weight glutenin subunits. A molecular marker used to estimate the quality of wheat germplasm

    International Nuclear Information System (INIS)

    Recent progress made with electrophoretic separation of glutenin subunits, their genetics and their implication on breadmaking quality offers the possibility of performing indirect selection for quality using glutenin subunits whose expression is perigenetically independent. The composition of the glutenin subunits (with a high molecular weight) of 15 genotypes representative of the germplasm grown recently in Romania was analysed by electrophoresis in 15% polyacrylamide gel with SDS; eight proteinogram types were established. The average technological indices for 3 years were used to determine the correlations and linear regressions of the different indices and to score the glutenin composition. The glutenin scores are presented in Fig. 1. It should be mentioned that 11 of the 15 genotypes analyzed contained subunits that have the most important effect on breadmaking quality. For Aniversar and Lovrin 41 varieties, two to three biotypes were evident. The glutenin scores correlated significantly with the loaf volume, the farino-graphic value and the sedimentation test. It was also shown that the electrophoretic method is useful in the early breeding stages because it reveals the polymorphic composition of glutenin subunits. The role of glutenin is complex, but the variation in composition of high molecular weight glutenin subunits plays an important part in determining the differences in breadmaking quality between wheat varieties. 1 ref., 1 fig., 1 tab

  10. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    Science.gov (United States)

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites. PMID:26395202

  11. A protein residing at the subunit interface of the bacterial ribosome.

    Science.gov (United States)

    Agafonov, D E; Kolb, V A; Nazimov, I V; Spirin, A S

    1999-10-26

    Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed. PMID:10535924

  12. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  13. ATP synthase of yeast mitochondria. Isolation of subunit j and disruption of the ATP18 gene.

    Science.gov (United States)

    Arnold, I; Pfeiffer, K; Neupert, W; Stuart, R A; Schägger, H

    1999-01-01

    The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase. PMID:9867807

  14. The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.

    Directory of Open Access Journals (Sweden)

    Laurence Prunetti

    Full Text Available The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3-type two-subunit (subunits I and II enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2 of 59000 Da, subunit II (encoded by coxB(2 of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3 cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.

  15. Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms

    Science.gov (United States)

    Lewis, Kayla A.; Tollefsbol, Trygve O.

    2016-01-01

    Chromosome-shortening is characteristic of normal cells, and is known as the end replication problem. Telomerase is the enzyme responsible for extending the ends of the chromosomes in de novo synthesis, and occurs in germ cells as well as most malignant cancers. There are three subunits of telomerase: human telomerase RNA (hTERC), human telomerase associated protein (hTEP1), or dyskerin, and human telomerase reverse transcriptase (hTERT). hTERC and hTEP1 are constitutively expressed, so the enzymatic activity of telomerase is dependent on the transcription of hTERT. DNA methylation, histone methylation, and histone acetylation are basic epigenetic regulations involved in the expression of hTERT. Non-coding RNA can also serve as a form of epigenetic control of hTERT. This epigenetic-based regulation of hTERT is important in providing a mechanism for reversibility of hTERT control in various biological states. These include embryonic down-regulation of hTERT contributing to aging and the upregulation of hTERT playing a critical role in over 90% of cancers. Normal human somatic cells have a non-methylated/hypomethylated CpG island within the hTERT promoter region, while telomerase-positive cells paradoxically have at least a partially methylated promoter region that is opposite to the normal roles of DNA methylation. Histone acetylation of H3K9 within the promoter region is associated with an open chromatin state such that transcription machinery has the space to form. Histone methylation of hTERT has varied control of the gene, however. Mono- and dimethylation of H3K9 within the promoter region indicate silent euchromatin, while a trimethylated H3K9 enhances gene transcription. Non-coding RNAs can target epigenetic-modifying enzymes, as well as transcription factors involved in the control of hTERT. An epigenetics diet that can affect the epigenome of cancer cells is a recent fascination that has received much attention. By combining portions of this diet with

  16. Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Schneider Toni

    2008-03-01

    Full Text Available Abstract Background The function of voltage-gated calcium (Cav channels greatly depends on coupling to cytoplasmic accessory β subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the α1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two β subunit subtypes: a structurally conventional β subunit and a variant β subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavβ subunit. Here, we focus on the modulatory phenotype of the conventional Cavβ subunit (SmCavβ using the human Cav2.3 channel as the substrate for SmCavβ and the whole-cell patch-clamp technique. Results The conventional Schistosoma mansoni Cavβ subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavβ run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavβ lends the Cav2.3/SmCavβ complex sensitivity to Na+ ions. A mutant version of the Cavβ subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. Conclusion The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavβ subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by

  17. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    International Nuclear Information System (INIS)

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α2β2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α2β2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α2β2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α2β2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α2β2 insulin receptor subunit complex

  18. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.

    Science.gov (United States)

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N; May, Kerrie L; Kahn, Peter C; Tumer, Nilgun E

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. PMID:26483409

  19. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

    International Nuclear Information System (INIS)

    Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism

  20. Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

    Science.gov (United States)

    Gagliardi, D; Penin, F; Gautheron, D C

    1991-09-13

    A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits. PMID:1832960

  1. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli.

    Science.gov (United States)

    Locht, C; Cieplak, W; Marchitto, K S; Sato, H; Keith, J M

    1987-01-01

    The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors. Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies. Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities. Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted. The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit. Both the S1 and S2 subunits expressed in E. coli reacted with human hyperimmune serum. The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody. The different versions of S1 and S2 subunits expressed in E. coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin. These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough. Images PMID:3117686

  2. Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

    Science.gov (United States)

    Inose, Ken; Fujikawa, Masako; Yamazaki, Tomohiko; Kojima, Katsuhiro; Sode, Koji

    2003-02-21

    We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea. PMID:12573242

  3. Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine.

    OpenAIRE

    Levin, L R; Zoller, M J

    1990-01-01

    In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits. In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R. Mutation of Thr-241 to Ala in the C1 subunit of S. cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit. T...

  4. The calcium channel β2 (CACNB2 subunit repertoire in teleosts

    Directory of Open Access Journals (Sweden)

    Mueller Rachel

    2008-04-01

    Full Text Available Abstract Background Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2 indicated they are membrane-associated guanylate kinase (MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and

  5. Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

    Science.gov (United States)

    Fairlie, W D; Stanton, P G; Hearn, M T

    1995-01-01

    The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites. Images Figure 2 PMID:7538754

  6. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

    Science.gov (United States)

    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. PMID:26812300

  7. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

    Science.gov (United States)

    Li, W B; Bzik, D J; Tanaka, M; Gu, H M; Fox, B A; Inselburg, J

    1991-06-01

    We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed. PMID:1656254

  8. Prokaryotic expression of the chicken xanthine oxidase (XOD) subunit and its localization in liver and kidney.

    Science.gov (United States)

    Lin, Huayuan; Chen, Yanjun; Huang, Qiqi; Guo, Xiaoquan; Liu, Ping; Liu, Weilian; Zhang, Caiying; Cao, Huabin; Hu, Guoliang

    2016-06-01

    Xanthine oxidase (XOD) is the members of the molybdenum hydroxylase flavoprotein family and it plays a vital role in the body's purine catabolism. In this study, we cloned the XOD 37kDa subunit protein by using RT-PCR and pMD-18-T clone vector based on the total RNA extracted from chicken liver. The cloning XOD subunit protein gene was ligated into the pET-32a to construct the recombinant plasmid pET-XOD. After the pET-XOD expression vector was transformed into host cells Rosetta (DE3), the recombinant XOD subunit proteins (54.8kDa) were successfully induced by isopropy1 β-d-thiogalactoside (IPTG). Rabbit antiserums were produced by using the purification of the recombinant XOD subunit protein as antigen. The titer of the antiserum was more than 1:102,400 determined by using ELISA. The result of Western blot demonstrated that the antiserum could specifically recognize the chicken liver XOD. Immunohistochemistry and immunofluorescence showed that the XOD mainly presented in the cytoplasm of chicken hepatocytes and proximal tubular epithelial cells. Our results indicated that the XOD subunit protein polyclonal antibody prepared by this method could be used for the further researches of the biological function of the XOD in the chicken. PMID:26949113

  9. Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A; Preiss, Jack; Romero, José M

    2008-09-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  10. Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic1

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L.; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A.; Preiss, Jack; Romero, José M.

    2008-01-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  11. Molecular Characterization of Subunit G of the Vacuolar ATPase in Pathogen Dermatophyte Trichophyton rubrum

    Directory of Open Access Journals (Sweden)

    S Rezaie

    2006-06-01

    Full Text Available Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. Several properties of this fungus have been investigated so far. However, a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the subunit G of its vacuolar ATPase (V-ATPase. Pairs of 21 nt primers were designed from highly conserved regions of the V-ATPase subunit G genes in other fungi. Mentioned primers were utilized in PCR using isolated genomic DNA template as well as cytoplasmic RNA of T.rubrum and the PCR and RT-PCR fragments were then sequenced. About 469 nucleotides were sequenced which encoded a polypeptide with 119 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH for both the DNA and its deduced amino acid sequence revealed significant homology with V-ATPase subunit G genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 84% identical to the sequence of V-ATPase subunit G from other fungi. In summary, we have cloned the first V-ATPase subunit G of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells.

  12. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    International Nuclear Information System (INIS)

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer

  13. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    Energy Technology Data Exchange (ETDEWEB)

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang, E-mail: baumeist@bichem.mpg.de; Förster, Friedrich, E-mail: foerster@bichem.mpg.de

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  14. Investigation of the interaction between the large and small subunits of potato ADP-glucose pyrophosphorylase.

    Directory of Open Access Journals (Sweden)

    Ibrahim Baris

    2009-10-01

    Full Text Available ADP-glucose pyrophosphorylase (AGPase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS and small subunits (SS. Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.

  15. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals.

    Science.gov (United States)

    Dao, E Han; Sierra, Raymond G; Laksmono, Hartawan; Lemke, Henrik T; Alonso-Mori, Roberto; Coey, Aaron; Larsen, Kevin; Baxter, Elizabeth L; Cohen, Aina E; Soltis, S Michael; DeMirci, Hasan

    2015-07-01

    In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser. PMID:26798805

  16. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

    Directory of Open Access Journals (Sweden)

    E. Han Dao

    2015-07-01

    Full Text Available In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

  17. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human

    Energy Technology Data Exchange (ETDEWEB)

    Blatt, C.; Eversole-Cire, P.; Cohn, V.H.; Zollman, S.; Fournier, R.E.K.; Mohandas, L.T.; Nesbitt, M.; Lugo, T.; Jones, D.T.; Reed, R.R.; Weiner, L.P.; Sparkes, R.S.; Simon, M.I. (Weizmann Institute, Rehovoth (Israel))

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding {alpha}-subunit proteins, two different {beta} subunits, and one {gamma} subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The {beta} subunits were also assigned-GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extend of the G{alpha} gene family and may help in attempts to correlate specific genetic diseases and with genes corresponding to G proteins.

  18. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    Science.gov (United States)

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  19. Subunit composition of hinokiresinol synthase controls enantiomeric selectivity in hinokiresinol formation.

    Science.gov (United States)

    Yamamura, Masaomi; Suzuki, Shiro; Hattori, Takefumi; Umezawa, Toshiaki

    2010-03-01

    Asparagus officinalis hinokiresinol synthase (HRS) is composed of two subunits, HRSalpha and HRSbeta. Individually, each subunit forms (E)-hinokiresinol (EHR) from 4-coumaryl 4-coumarate, whereas a mixture of both subunits forms (Z)-hinokiresinol (ZHR) from the same substrate. In this study, we analyzed the enantiomeric compositions of ZHR and EHR formed after incubation of 4-coumaryl 4-coumarate with recombinant subunit proteins, recHRSalpha and/or recHRSbeta, and with naturally occurring A. officinalis ZHR. The enantiomeric composition of ZHR formed by the mixture of recHRSalpha and recHRSbeta was (+)-100% enantiomer excess (e.e.), identical to that of A. officinalis ZHR. In contrast, the enantiomeric compositions of EHR formed by recHRSalpha and recHRSbeta, individually, were (-)-20.6 and (-)-9.0% e.e., respectively. These results clearly demonstrate that the subunit composition of A. officinalis HRS controls not only cis/trans isomerism but also enantioselectivity in hinokiresinol formation. PMID:20165801

  20. [Eutopic and ectopic production of glycoprotein hormones alpha and beta subunits].

    Science.gov (United States)

    Bidart, J M; Baudin, E; Troalen, F; Bellet, D; Schlumberger, M

    1997-01-01

    Human chorionic gonadotropin (hCG) is a glycoprotein composed of two subunits, alpha and beta, linked together by a covalent bond. Ectopic production of hCG has been described in various histological types of cancer. Actually, these malignant tumors predominantly secrete the free beta subunit (hCG beta) and not hCG. Production of free hCG beta is especially found in patients with bladder, pancreas, uterine and lung tumors. In patients with neuroendocrine tumors, serum levels of free hCG beta are higher in gastrointestinal-pancreatic and lung tumors. The significance of ectopic production of hCG beta--epiphenomena or intrinsic biological role--remains unknown. Several reports on the similar structure of hCG beta and certain growth factors suggest that free hCG beta could have an effect on cell proliferation. Increased serum levels of the free alpha subunit are found mainly in patients with neuroendocrine tumors localized in the gut or lung. Serum levels may also be raised in patients with a pituitary tumor, but such production is often associated with a rise in other pituitary hormones. The free alpha subunit plays a role in embryon development and would stimulate production of prolactin by decidual cells. The free alpha subunit may also play a role in tumor growth. PMID:9239230

  1. Molecular basis of inherited calcium Channelopathies: role of mutations in pore-forming subunits

    Institute of Scientific and Technical Information of China (English)

    Lynn MCKEOWN; Philip ROBINSON; Owen T JONES

    2006-01-01

    The pore-forming alpha subunits of voltage-gated calcium channels contain the essential biophysical machinery that underlies calcium influx in response to cell depolarization.In combination with requisite auxiliary subunits,these pore subunits form calcium channel complexes that are pivotal to the physiology and pharmacology of diverse cells ranging from sperm to neurons.Not surprisingly,mutations in the pore subunits generate diverse pathologies,termed channelopathies,that range from failures in excitation-contraction coupling to night blindness.Over the last decade, major insights into the mechanisms of pathogenesis have been derived from animals showing spontaneous or induced mutations.In parallel,there has been considerable growth in our understanding of the workings of voltage-gated ion channels from a structure-function,regulation and cell biology perspective.Here we document our current understanding of the mutations underlying channelopathies involving the voltage-gated calcium channel alpha subunits in humans and other species.

  2. Morphological variation of individual Escherichia coli 50S ribosomal subunits in situ, as revealed by cryo-electron tomography

    International Nuclear Information System (INIS)

    Electron tomography (ET) has been used to reconstruct in situ individual 50S ribosomal subunits in Escherichia coli rifampicin-treated cells. Rifampicin inhibits transcription initiation. As a result, rapid degradation of preformed mRNA and dissociation of 70S ribosomes give accumulation of free subunits. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate the flexible nature of the pathway inside the large ribosomal subunit. In addition, gross morphological heterogeneity was observed in the reconstructions. Our results demonstrate a considerable structural variability among individual 50S subunits in the intracellular environment

  3. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B; Pelton, J T

    1992-01-01

    Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition......, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of...... heparin, presumably by its binding to the polylysine stretch at amino acid positions 74-77. Heat denaturation experiments (25-90 degrees C) support the notion that heparin may provide a local protective function. A similar but much larger effect was also observed in the presence of the beta subunit only...

  4. Deletion of the GABAA α2-subunit does not alter self dministration of cocaine or reinstatement of cocaine seeking

    OpenAIRE

    Dixon, C. I.; Halbout, B.; King, S.L.; Stephens, D. N.

    2014-01-01

    Rationale GABAA receptors containing α2-subunits are highly represented in brain areas that are involved in motivation and reward, and have been associated with addiction to several drugs, including cocaine. We have shown previously that a deletion of the α2-subunit results in an absence of sensitisation to cocaine. Objective We investigated the reinforcing properties of cocaine in GABAA α2-subunit knockout (KO) mice using an intravenous self-administration procedure. Methods ...

  5. Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

    OpenAIRE

    Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

    2003-01-01

    The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two sepa...

  6. Evolution of Respiratory Complex I: “SUPERNUMERARY” SUBUNITS ARE PRESENT IN THE α-PROTEOBACTERIAL ENZYME*

    OpenAIRE

    Yip, Chui-ying; Harbour, Michael E; Jayawardena, Kamburapola; Fearnley, Ian M.; Sazanov, Leonid A.

    2010-01-01

    Modern α-proteobacteria are thought to be closely related to the ancient symbiont of eukaryotes, an ancestor of mitochondria. Respiratory complex I from α-proteobacteria and mitochondria is well conserved at the level of the 14 “core” subunits, consistent with that notion. Mitochondrial complex I contains the core subunits, present in all species, and up to 31 “supernumerary” subunits, generally thought to have originated only within eukaryotic lineages. However, the full protein composition ...

  7. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    OpenAIRE

    Janssens Veerle; Goris Jozef; Louis Justin V; Zwaenepoel Karen

    2008-01-01

    Abstract Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specifici...

  8. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    OpenAIRE

    Sasaki Yukako; Takai Shinji; Watanabe Kiyotaka; Takaesu Azusa; Orino Koichi

    2008-01-01

    Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequ...

  9. Characterization of Rabbit Polyclonal Sera against Recombinant Shiga Toxin and its Subunits for Detection of Stx-Producing E. coli

    OpenAIRE

    Mana Oloomi; Saeid Bouzari

    2011-01-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera...

  10. In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.

    OpenAIRE

    Steinert, P M; Idler, W W; Cabral, F; Gottesman, M M; Goldman, R D

    1981-01-01

    This paper presents evidence that the intermediate filament (IF) subunits of muscle cells (skeletin or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IFs of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin, and two minor subunits which, on the basis of two-dimensional gel and peptide mapping c...

  11. Characterization of Rabbit Polyclonal Sera Against Recombinant Shiga Toxin and its Subunits for Detection of Stx-Producing E. Coli

    OpenAIRE

    Mana Oloomi; Saeid Bouzari

    2011-01-01

    Shiga toxin (Stx) is the principal virulence factor of Shigatoxigenic Escherichia coli (STEC), a food-born pathogen associated disease with uncomplicated diarrhea or the hemolytic-uremic syndrome. In this study, rabbit polyclonal anti recombinant A, B subunits of Shiga toxin and holotoxin antisera were raised and employed for immunological purpose. By immunoblotting, these antisera recognized respective subunit and the holotoxin antiserum recognized both subunits, equally. The raised antisera...

  12. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B;

    1999-01-01

    The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits alpha (catalytic) and beta (regulatory). The alpha' subunit was only detected in...... found for testicles and brain. The amount of CK2beta protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2-3-fold higher whereas the ratio of CK2beta between testicles and brain was estimated to be 3-4-fold. Results from the immunoprecipitation experiments...... support the notion for the existence of free CK2beta population and/or CK2beta in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2beta was observed. This is the first...

  13. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1997-01-01

    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited...... in the EMBL database under the accession numbers R08806 and Z17360, for the ribosomal protein L5 and for A-Raf kinase. All isolated clones except the one for CK2 beta showed no interaction with the catalytic alpha subunit of CK2. A-Raf kinase is a new interesting partner of CK2 beta. The isolated A...

  14. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  15. A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.

    Science.gov (United States)

    Putrenko, Igor; Zakikhani, Mahvash; Dent, Joseph A

    2005-02-25

    The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. PMID:15579462

  16. Cox26 is a novel stoichiometric subunit of the yeast cytochrome c oxidase.

    Science.gov (United States)

    Levchenko, Maria; Wuttke, Jan-Moritz; Römpler, Katharina; Schmidt, Bernhard; Neifer, Klaus; Juris, Lisa; Wissel, Mirjam; Rehling, Peter; Deckers, Markus

    2016-07-01

    The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization. PMID:27083394

  17. Kir3 channel ontogeny - the role of Gβγ subunits in channel assembly and trafficking.

    Directory of Open Access Journals (Sweden)

    Ashley M Jacobi

    2014-04-01

    Full Text Available The role of Gβγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gβγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gβγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gβ1γ2, apart from its role in channel opening that it shares with other Gβγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gβ1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gβγ effects on Kir3 maturation and trafficking.

  18. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2014-09-01

    Full Text Available The vacuolar (H+-ATPase (V-ATPase of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.

  19. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae)

    OpenAIRE

    Lina Lu; Zhijun Qi; Wenjun Wu

    2014-01-01

    The vacuolar (H+)-ATPase (V-ATPase) of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we clone...

  20. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    OpenAIRE

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M.; Fox, B.A.; Inselburg, J

    1989-01-01

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distin...

  1. The duplicated α7 subunits assemble and form functional nicotinic receptors with the full-length α7

    OpenAIRE

    Wang, Ying; Xiao, Cheng; Indersmitten, Tim; Freedman, Robert; Leonard, Sherry; Lester, Henry A.

    2014-01-01

    The α7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13-14. Exon 6 of CHRFAM7A harbors a 2 base pair deletion polymorphism, CHRFAM7AΔ2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on α7 receptors, we fused α7, dupα7, and dupΔα7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length α7 subunits in mou...

  2. Epithelial Junctions Depend on Intercellular Trans-interactions between the Na,K-ATPase β1 Subunits*

    OpenAIRE

    Tokhtaeva, Elmira; Sachs, George; Souda, Puneet; Bassilian, Sara; Whitelegge, Julian P.; Shoshani, Liora; Vagin, Olga

    2011-01-01

    N-Glycans of the Na,K-ATPase β1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β1 subunit with various YFP-linked β1 subunits expressed in Madin-Darby canine kidney cells was used to assess β1-β1 interactions. The amount of co-precipitated endogenous dog β1 was greater with dog YFP-β1 than with rat YFP...

  3. Cloning of eight Rhopalosiphum padi (Hemiptera: Aphididae) nAChR subunit genes and mutation detection of the β1 subunit in field samples from China.

    Science.gov (United States)

    Zhang, Meng; Qiao, Xianfeng; Li, Yuting; Fang, Bing; Zuo, Yayun; Chen, Maohua

    2016-09-01

    The bird cherry-oat aphid, Rhopalosiphum padi (L.), is one of the most important wheat pests. This aphid damages through direct feeding and by transmitting the Barley yellow dwarf virus (BYDV). Both types of damage significantly reduce the quality and yield of wheat crops globally. Insecticides are the primary method of controlling the bird cherry-oat aphid in China, yet this aphid species has developed resistance to different types of insecticides, especially organophosphates and carbamates. In the last decade, control of R. padi depends primarily on the spray of neonicotinoid insecticides, however, research on the resistance of R. padi to neonicotinoids has been limited. In this study, the full lengths of seven α-subunit (Rpα1, Rpα2, Rpα3, Rpα4, Rpα5, Rpα7-1, and Rpα7-2) and one β-subunit (Rpβ1) genes from R. padi were obtained with RT-PCR and RACE techniques. Sequence analysis showed that these genes had all the characteristics of the nAChR gene family and were highly homologous with the reported nAChR genes from other insects, and alternative splicing was detected in Rpα3 and Rpα5 subunits. Analysis of the cDNA sequence of the extracellular region of the nicotinic acetylcholine receptor β1 subunit gene from 120 R. padi field samples collected in 11 Provinces revealed 17 single nucleotides polymorphism (SNP) sites, of which seven were amino acid polymorphism sites (V53I, V53G, N54T, A60T, F61L, W79C, and V83I) and two were in the loop D region (W79C and V83I). The current study will facilitate further studies on the molecular mechanisms of targeted resistance of the aphid to neonicotinoid insecticides. PMID:27521918

  4. Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α

    Science.gov (United States)

    Flegg, Cameron P.; Sharma, Manisha; Medina-Palazon, Cahora; Jamieson, Cara; Galea, Melanie; Brocardo, Mariana G.; Mills, Kate; Henderson, Beric R.

    2010-01-01

    Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. PMID:20378546

  5. Three ferritin subunit analogs in Chinese giant salamander (Andrias davidianus) and their response to microbial stimulation.

    Science.gov (United States)

    You, Xiuling; Sheng, Jianghong; Liu, Liu; Nie, Dongsong; Liao, Zhiyong

    2015-10-01

    Ferritin, an evolutionarily conserved iron-binding protein, plays important roles in iron storage and detoxification and in host immune response to invading stimulus as well. In the present study, we identified three ferritin subunit analog cDNAs from Chinese giant salamander (Andrias davidianus). All the three ferritin subunit cDNAs had a putative iron responsive element in the 5'-untranslated region. Two deduced ferritin subunits (designated as cgsFerH and cgsFerM) had the highest identity of 90% to H type subunit of vertebrate ferritins, while another deduced ferritin subunit (designated as cgsFerL) had the highest identity of 84% to L type subunit of vertebrate ferritins. The Chinese giant salamander ferritin (cgsFer) was widely expressed in various tissues, with highest expression for cgsFerH and cgsFerL in liver and highest expression for cgsFerM in spleen. Infection of Chinese giant salamander with A. davidianus ranavirus showed significant induction of cgsFer expression. Both lipopolysaccharide and iron challenge drastically augmented cgsFer expression in the splenocytes and hepatocytes from Chinese giant salamander. In addition, recombinant cgsFers bound to ferrous iron in a dose-dependent manner, with significant ferroxidase activity. Furthermore, the recombinant cgsFer inhibited the growth of the pathogen Vibrio anguillarum. These results indicated that cgsFer was potential candidate of immune molecules involved in acute phase response to invading microbial pathogens in Chinese giant salamander possibly through its regulatory roles in iron homeostasis. PMID:26319314

  6. Subunit-specific sulphation of oligosaccharides relating to charge-heterogeneity in porcine lutrophin isoforms.

    Science.gov (United States)

    Ujihara, M; Yamamoto, K; Nomura, K; Toyoshima, S; Demura, H; Nakamura, Y; Ohmura, K; Osawa, T

    1992-06-01

    Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1498420

  7. Folding and stability of the b subunit of the F1F0 ATP synthase

    OpenAIRE

    Revington, Matthew; Dunn, Stanley D.; Shaw, Gary S.

    2002-01-01

    The F1F0 ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F0) and soluble (F1) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F...

  8. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Science.gov (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  9. The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

    OpenAIRE

    Szuplewski, S; Terracol, R.

    2001-01-01

    Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytoc...

  10. Cisplatin–DNA adducts inhibit translocation of the Ku subunits of DNA-PK

    OpenAIRE

    Turchi, John J.; Henkels, Karen M.; Zhou, Yun

    2000-01-01

    We have determined the effect of cisplatin–DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin–DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku–DNA complex. In addition...

  11. HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1

    OpenAIRE

    de la Salle, Henri; Zimmer, Jacques; Fricker, Dominique; Angenieux, Catherine; Cazenave, Jean-Pierre; Okubo, Mitsuo; Maeda, Hiroo; Plebani, Alessandro; Tongio, Marie-Marthe; Dormoy, Anne; Hanau, Daniel

    1999-01-01

    The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficienc...

  12. Analysis of Maxi-K alpha subunit splice variants in human myometrium

    Directory of Open Access Journals (Sweden)

    Morrison John J

    2004-09-01

    Full Text Available Abstract Background Large-conductance, calcium-activated potassium (Maxi-K channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP, and at Caesarean section, at elective (pregnant not-in-labour, PNL and intrapartum (pregnant in-labour, PL procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at

  13. The proteasome 11S regulator subunit REG alpha (PA28 alpha) is a heptamer.

    OpenAIRE

    Johnston, S.C.; Whitby, F G; Realini, C.; Rechsteiner, M; Hill, C. P.

    1997-01-01

    Activity of the 20S proteasome, which performs much of the cytosolic and nuclear proteolysis in eukaryotic cells, is controlled by regulatory complexes that bind to one or both ends of the cylindrical proteasome. One of these complexes, the 11S regulator (REG), is a complex of 28 kDa subunits that is thought to activate proteasomes toward the production of antigenic peptides. REG, purified from red blood cells, is a complex of REG alpha and REG beta subunits. We have crystallized recombinant ...

  14. Kerr constant of multi-subunit particles and semiflexible, wormlike chains

    Energy Technology Data Exchange (ETDEWEB)

    GarcIa de la Torre, J; Diaz Banos, F G; Perez Sanchez, H E, E-mail: jgt@um.e [Departamento de Quimica Fisica, Facultad de Quimica, Universidad de Murcia, 30071 Murcia (Spain)

    2010-12-15

    A formalism is developed for the Kerr constant of particles composed of subunits whose electro-optical properties have axial symmetry. A protocol is devised for the calculation of the Kerr constant from the dipole moments and the electrical and optical polarizabilities of the subunits. The conformational average is required if the particle is flexible. Based on that formalism, the Kerr constant of semiflexible, wormlike chains is calculated with the help of a discrete version to which the previous formalism is applied. The required conformational averages are evaluated by means of Monte Carlo simulation. Thus we obtain expressions for the Kerr constant of wormlike particles over the whole range of conformations.

  15. Isolation, sequencing and overexpression of the gene encoding the theta subunit of DNA polymerase III holoenzyme.

    OpenAIRE

    J.R. Carter; Franden, M A; Aebersold, R.; Kim, D.R.; McHenry, C S

    1993-01-01

    The gene encoding the theta subunit of DNA polymerase III holoenzyme, designated holE, was isolated using a strategy in which peptide sequence was used to derive a DNA hybridization probe. Sequencing of the gene, which maps to 41.43 centisomes of the chromosome, revealed a 76-codon open reading frame predicted to produce a protein of 8,846 Da. When placed in a tac promoter expression vector, the open reading frame directed expression of a protein, that comigrated with authentic theta subunit ...

  16. A Phytophthora infestans G-Protein β Subunit Is Involved in Sporangium Formation

    OpenAIRE

    Latijnhouwers, Maita; Govers, Francine

    2003-01-01

    The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes. It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans, as was concluded from previous studies on the role of the G-protein α-subunit PiGPA1 in this organism. The expression of the P. infestans G-protein β-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formation. The ...

  17. A Phytophthora infestans G-protein beta subunit is involved in sporangium formation

    OpenAIRE

    Latijnhouwers, M.; Govers, F.

    2003-01-01

    The heterotrimeric G-protein pathway regulates cellular responses to a wide range of extracellular signals in virtually all eukaryotes. It also controls various developmental processes in the oomycete plant pathogen Phytophthora infestans, as was concluded from previous studies on the role of the G-protein alpha-subunit PiGPA1 in this organism. The expression of the P. infestans G-protein beta-subunit gene Pigpb1 was induced in nutrient-starved mycelium before the onset of sporangium formatio...

  18. Molecular cloning of cDNA encoding the small subunit of Drosophila transcription initiation factor TFIIF.

    OpenAIRE

    Gong, D W; Mortin, M A; Horikoshi, M; Nakatani, Y

    1995-01-01

    Transcription initiation factor TFIIF is a tetramer consisting of two large subunits (TFIIF alpha or RAP74) and two small subunits (TFIIF beta or RAP30). We report here the molecular cloning of a Drosophila cDNA encoding TFIIF beta. The cDNA clone contains an open-reading frame encoding a 277 amino acid polypeptide having a calculated molecular mass of 32,107 Da. Comparison of the deduced amino acid sequence with the corresponding sequences from vertebrates showed only 50% identity, with four...

  19. Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit.

    OpenAIRE

    Erickson, F L; Hannig, E M

    1996-01-01

    Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type stra...

  20. Structural determinants within the subunit protein of Ty1 virus-like particles.

    Science.gov (United States)

    Martin-Rendon, E; Marfany, G; Wilson, S; Ferguson, D J; Kingsman, S M; Kingsman, A J

    1996-11-01

    The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold. PMID:8951814

  1. Crystal Structure of the P Pilus Rod Subunit PapA

    OpenAIRE

    Verger, Denis; Bullitt, Esther; Hultgren, Scott J.; Waksman, Gabriel

    2007-01-01

    P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone–usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor–strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) o...

  2. Crystal structure of the P pilus rod subunit PapA

    OpenAIRE

    Verger, D.; Bullitt, E; Hultgren, S. J.; Waksman, G

    2007-01-01

    P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G(1) beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (N...

  3. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E;

    2000-01-01

    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides...

  4. Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

    NARCIS (Netherlands)

    De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.

    2006-01-01

    It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the n

  5. Structure of the ATP synthase from chloroplasts studied by electron microscopy. Localization of the small subunits

    NARCIS (Netherlands)

    Boekema, Egbert J.; Xiao, Jianping; McCarty, Richard E.

    1990-01-01

    The structure of the hydrophilic part of the ATP synthase from chloroplasts (CF1) has been further investigated by electron microscopy and image analysis of negatively stained samples. The projections of three different types of CF1 were analyzed: the holoenzyme with five different subunits and two

  6. Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase.

    Science.gov (United States)

    Harmat, Veronika; Domokos, Klarissza; Menyhárd, Dóra K; Palló, Anna; Szeltner, Zoltán; Szamosi, Ilona; Beke-Somfai, Tamás; Náray-Szabó, Gábor; Polgár, László

    2011-01-21

    Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism. PMID:21084296

  7. epsilon, a New Subunit of RNA Polymerase Found in Gram-Positive Bacteria

    Czech Academy of Sciences Publication Activity Database

    Keller, A. N.; Yang, X.; Wiedermannová, Jana; Delumeau, O.; Krásný, Libor; Lewis, P. J.

    2014-01-01

    Roč. 196, č. 20 (2014), s. 3622-3632. ISSN 0021-9193 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:61388971 Keywords : RNA polymerase * subunit * X-ray crystallography Subject RIV: EE - Microbiology, Virology Impact factor: 2.808, year: 2014

  8. Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein

    Science.gov (United States)

    The cDNA, ArRbcS3, for the small subunit of Rubisco from Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to...

  9. Unassigned MURF1 of kinetoplastids codes for NADH dehydrogenase subunit 2

    Directory of Open Access Journals (Sweden)

    Burger Gertraud

    2008-10-01

    Full Text Available Abstract Background In a previous study, we conducted a large-scale similarity-free function prediction of mitochondrion-encoded hypothetical proteins, by which the hypothetical gene murf1 (maxicircle unidentified reading frame 1 was assigned as nad2, encoding subunit 2 of NADH dehydrogenase (Complex I of the respiratory chain. This hypothetical gene occurs in the mitochondrial genome of kinetoplastids, a group of unicellular eukaryotes including the causative agents of African sleeping sickness and leishmaniasis. In the present study, we test this assignment by using bioinformatics methods that are highly sensitive in identifying remote homologs and confront the prediction with available biological knowledge. Results Comparison of MURF1 profile Hidden Markov Model (HMM against function-known profile HMMs in Pfam, Panther and TIGR shows that MURF1 is a Complex I protein, but without specifying the exact subunit. Therefore, we constructed profile HMMs for each individual subunit, using all available sequences clustered at various identity thresholds. HMM-HMM comparison of these individual NADH subunits against MURF1 clearly identifies this hypothetical protein as NAD2. Further, we collected the relevant experimental information about kinetoplastids, which provides additional evidence in support of this prediction. Conclusion Our in silico analyses provide convincing evidence for MURF1 being a highly divergent member of NAD2.

  10. Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

    Directory of Open Access Journals (Sweden)

    Christine M Kondratick

    Full Text Available Proliferating cell nuclear antigen (PCNA plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS, a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.

  11. Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

    Science.gov (United States)

    Kondratick, Christine M; Boehm, Elizabeth M; Dieckman, Lynne M; Powers, Kyle T; Sanchez, Julio C; Mueting, Samuel R; Washington, M Todd

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process. PMID:27258147

  12. Mapping the residues of protein kinase CK2 alpha subunit responsible for responsiveness to polyanionic inhibitors

    DEFF Research Database (Denmark)

    Vaglio, P; Sarno, S; Marin, O;

    1996-01-01

    The quadruple mutation of the whole basic cluster, K74KKK77 conserved in the catalytic subunits of protein kinase CK2 and implicated in substrate recognition, not only abolishes inhibition by heparin but even induces with some peptide substrates an up to 5-fold stimulation by heparin in the 0...

  13. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A;

    1998-01-01

    The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters...

  14. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  15. Insulin binding changes the interface region between α subunits of the insulin receptor

    International Nuclear Information System (INIS)

    The homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) was used to probe the interface region between the two α subunits of the α2β2 human insulin receptor. The two α subunits formed a covalent dimer when affinity-purified receptor or membrane-bound receptor was reacted with DSS. The α2 species was detected on protein blots from SDS gels using an anti-α-subunit antibody or 125I-concanavalin A. Alternatively, iodinated receptor was reacted with DSS and the α2 species measured directly in an SDS gel. As shown by all three assay systems, more α2 was formed when insulin was bound to receptor than when insulin was absent. These data indicate that the conformational change which occurs in the α subunit response to insulin binding results in a change in the α-α interaction within the receptor complex. The results are consistent with a kinase activation mechanism involving communication between the two αβ receptor halves

  16. Expression of NR1 subunit of NMDA receptor in schizophrenic hippocampus

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Peková, S.; Klaschka, Jan; Majer, E.; Šťastný, F.; Bubeníková-Valešová, V.

    Elsevier. Roč. 23, Suppl. 2 (2008), S152. ISSN 0924-9338 R&D Projects: GA MZd(CZ) NR8792 Institutional research plan: CEZ:AV0Z10300504 Keywords : NMDA receptor * NR1 subunit * schizophrenia * post-mortem studies

  17. Changes in the expression of NR1 subunit of NMDA receptor in schizophrenic hippocampus

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Peková, S.; Klaschka, Jan; Štastný, P.

    Elsevier. Roč. 17, Suppl. 1 (2007), s237-s238. ISSN 0924-977X. [ECNP Congress /20./. 13.10.2006-17.10.2006, Vienna] Institutional research plan: CEZ:AV0Z10300504 Keywords : schizophrenia * NMDA receptor * NR1 subunit * hippocampus * gender * antipsychotics

  18. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    Science.gov (United States)

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology. PMID:27170550

  19. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    Science.gov (United States)

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  20. An immune stimulating complex (iscom) subunit rabies vaccine protects dogs and mice against street rabies challenge.

    NARCIS (Netherlands)

    M. Fekadu; J.H. Schaddock; J. Ekströ m; A.D.M.E. Osterhaus (Ab); D.W. Sanderlin; B. Sundquist; B. Morein (Bror)

    1992-01-01

    textabstractDogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two intr

  1. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    Science.gov (United States)

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  2. Immunogenicity of Mycobacterium avium subsp. paratuberculosis specific peptides for inclusion in a subunit vaccine against paratuberculosis

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Tollefsen, S.; Olsen, I.;

    efficacies. The main problem with available vaccines is their interference with surveillance and diagnosis of bovine tuberculosis and paratuberculosis. Our ultimate aim is to develop a subunit vaccine consisting of selected MAP peptides, which allow differentiation of infected from vaccinated animals. Here...

  3. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1993-01-01

    Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C...

  4. Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.

    Science.gov (United States)

    Cieplak, W; Locht, C; Mar, V L; Burnette, W N; Keith, J M

    1990-01-01

    The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity. Images Fig. 1. PMID:2363691

  5. AMPA receptors commandeer an ancient cargo exporter for use as an auxiliary subunit for signaling.

    Directory of Open Access Journals (Sweden)

    Nadine Harmel

    Full Text Available Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs. AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER. CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling.

  6. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D;

    2010-01-01

    NaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission....

  7. Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli.

    OpenAIRE

    Barbieri, J T; Rappuoli, R; Collier, R J

    1987-01-01

    The S-1 subunit of pertussis toxin was expressed as a fusion protein in a strain of Escherichia coli deficient in protein degradation. The fusion protein reacted with anti-pertussis toxin antibody, and, like authentic pertussis toxin, it ADP-ribosylated a 41,000-molecular-weight membrane protein from human erythrocytes.

  8. Chapter 7 Tracing human mitochondrial complex I assembly by use of GFP-tagged subunits.

    Science.gov (United States)

    Dieteren, Cindy E J; Koopman, Werner J H; Nijtmans, Leo G J

    2009-01-01

    Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP(1)-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP(1) tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation. PMID:19348886

  9. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...

  10. Lab-on-a-Chip method uncertanties in determination of high-molecular-weight glutenin subunits

    Directory of Open Access Journals (Sweden)

    Živančev Dragan R.

    2013-01-01

    Full Text Available Polymeric wheat endosperm proteins, especially the high-molecular-weight glutenin subunits (HMW-GS, are probably the most interesting protein fraction giving the essential information about bread-making quality of wheat flour. A relatively new method that shows a great potential for a fast, reliable and automatable analysis of protein purity, sizing and quantification is microfluidic or Lab-on-a-Chip (LoaC capillary electrophoresis. This work was aimed to explore the possibilities of implementation of LoaC method to analysis of protein samples isolated from a Serbian common wheat variety, emphasizing the steps that might bring uncertainties and affect reproducibility of obtained glutenin subunits quantitation results. A good resolution of protein bands in a molecular weight range of 14.0 to 220.0 kDa was achieved. The reproducibility of HMW-GS sizing and quantitation were good, with the average coefficient of variation values of 1.2% and 12.2%. The ratio of HMW-GS to low-molecular-weight glutenin subunits (LMW-GS was about 20%. The investigation ruled out influences of the extract solution addition and the buffer addition steps of the applied method, as well as the individual chip influence on GS quantitation results. However, there was statistically significant difference between HMW-GS quantitation results of multi-step and one-step extraction procedures applied prior to glutenin subunits extraction step.

  11. Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor

    International Nuclear Information System (INIS)

    Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a glycoprotein that circulates in blood as a tetramer (a2b2) consisting of two a and two b subunits. The primary structures of the a and b subunits of human factor XIII have been reported by a combination of cDNA cloning and amino acid sequence analysis. To establish the gene structure of the a subunit for factor XIII, several human genomic libraries were screened by using the cDNA encoding the a subunit as a probe. Among ∼5 x 107 recombinant phage, 121 have been shown to contain an insert encoding a portion of the a subunit. Twenty-five unique clones were than characterized by restriction mapping, Southern blotting, and DNA sequencing. Overlapping clones encoding the a subunit of factor XIII span >160 kilobases. DNA sequence analysis revealed that the activation peptide released by thrombin, the active site cysteine region, the two putative calcium-binding regions, and the thrombin cleavage site leading to inactivation are encoded by separate exons. This suggest that the introns may separate the a subunit into functional and structural domains. A comparison of the amino acid sequence deduced from the genomic DNA sequence with those deduced from cDNA or determined by amino acid sequence analysis of the plasma and placental proteins revealed apparent amino acid polymorphisms in six positions of the polypeptide chain of the a subunit

  12. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    Science.gov (United States)

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  13. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  14. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    Directory of Open Access Journals (Sweden)

    Torrão A.S.

    1997-01-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

  15. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor α subunit

    International Nuclear Information System (INIS)

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the α subunit of the receptor, with little or no change in the levels of γ- and δ-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of α-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of α subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of α-subunit mRNA, with little change in the amount of γ- and δ-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the α-subunit nuclear precursor

  16. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown to b...

  17. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    Science.gov (United States)

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  18. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert J.

    2000-10-04

    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  19. Dis3- and exosome subunit-responsive 3 Prime mRNA instability elements

    Energy Technology Data Exchange (ETDEWEB)

    Kiss, Daniel L.; Hou, Dezhi [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States); Gross, Robert H. [Dartmouth College, Department of Biological Sciences, Life Sciences Center 343, Hanover, NH 03755 (United States); Andrulis, Erik D., E-mail: exa32@case.edu [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are

  20. Association analysis of GABA receptor subunit genes on 5q33 with heroin dependence in a Chinese male population.

    Science.gov (United States)

    Loh, E W; Tang, N L S; Lee, D T S; Liu, S I; Stadlin, Alfreda

    2007-06-01

    GABAA receptor subunit genes clustered on 5q33 play a role in the development of alcoholism and methamphetamine use disorder without psychosis. The present study explored the possible contribution of the same subunit genes to the development of heroin dependence. Single nucleotide polymorphisms (SNPs) of the GABAA receptor subunits GABRB2, GABRA6, GABRA1, and GABRG2 were examined in 178 male Han Chinese heroin-dependent and 170 male control subjects. A significant difference in allele frequency for the SNP rs211014 in the GABAAgamma2 receptor subunit gene between cases and controls was identified (P = 0.015). A possible mechanism for the involvement of the GABA receptor subunit genes on 5q33 in the development of heroin dependence is discussed. PMID:17440936

  1. Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

    Science.gov (United States)

    Wang, Lu; Wang, Shumei; Fu, Xuejun; Sun, Li

    2015-01-01

    An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ'). PMID:25781487

  2. A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.

    Science.gov (United States)

    Sun, Bing; Chen, Linhai; Liu, Lei; Xia, Zhixiong; Pin, Jean-Philippe; Nan, Fajun; Liu, Jianfeng

    2016-03-15

    An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition. PMID:26772870

  3. Receptor-binding region in human choriogonadotropin/lutropin β subunit

    International Nuclear Information System (INIS)

    Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) β subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCGβ-(38-57) and hLHβ-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 x 10-4 and 2.03 x 10-5 M, respectively, while other peptide hormones and fragments from elsewhere in the β subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10-5 M (hCG) and 2.18 x 10-5 M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCGβ-(38-57) peptide, native hCG and β subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of β subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones

  4. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    International Nuclear Information System (INIS)

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA

  5. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    Energy Technology Data Exchange (ETDEWEB)

    Svergun, D.I.; Koch, M.H.J. [Hamburg Outstation (Germany); Pedersen, J.S. [Riso National Laboratory, Roskilde (Denmark); Serdyuk, I.N. [Inst. of Protein Research, Moscow (Russian Federation)

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  6. Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available An R-phycoerythrin (R-PE was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF. Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ', γ34.6 (γ, and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ3 γ (αβ3γ' and γ (αβ3 γ'(αβ3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ3 face-to-face with the aid of one γ subunit (γ or γ'.

  7. The subunit composition of human extracellular superoxide dismutase (EC-SOD regulate enzymatic activity

    Directory of Open Access Journals (Sweden)

    Chr Nielsen Niels

    2007-10-01

    Full Text Available Abstract Background Human extracellular superoxide dismutase (EC-SOD is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24:13875–80. One variant is enzymatically active (aEC-SOD while the other is inactive (iEC-SOD. The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist. Results The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii and a hetero-dimer (ai. Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits. Conclusion This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.

  8. β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

    Directory of Open Access Journals (Sweden)

    Ninda Syam

    2013-08-01

    Full Text Available Voltage-gated sodium channels (Navs are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V1/2 of steady-state activation and inactivation and increased Nav1.7-mediated INa density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at approximately 250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa was observed. This higher band shifted to an intermediate band (~260 kDa when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

  9. Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

    Directory of Open Access Journals (Sweden)

    Sasaki Yukako

    2008-10-01

    Full Text Available Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit. Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas. The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98 ; L: 98–100%. The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.

  10. Purification and characterization of the glycoprotein hormone α-subunit-like material secreted by HeLa cells

    International Nuclear Information System (INIS)

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the α-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 105 ng of α/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-α had a composition very similar to that of the urinary hCG α-subunit. However, comparison of hCG-α and HeLa-α demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary α-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of α-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-α hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single α-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors

  11. Complex control of GABA(A) receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Science.gov (United States)

    Mulligan, Megan K; Wang, Xusheng; Adler, Adrienne L; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal), even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer 4-100 fold

  12. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  13. Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

    OpenAIRE

    Szkudlinski, M W; Thotakura, N R; Weintraub, B D

    1995-01-01

    The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone,...

  14. Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes

    Directory of Open Access Journals (Sweden)

    Lambeth J David

    2007-09-01

    Full Text Available Abstract Background The reactive oxygen-generating NADPH oxidases (Noxes function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1 and Nox-activator 1 (NOXA1, respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits. Results Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the

  15. Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers.

    Directory of Open Access Journals (Sweden)

    Ana M Valdeolmillos

    2007-02-01

    Full Text Available The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3 appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21 (sister chromatid cohesion protein 1, SCC1 and stromal antigen protein 1 (SA1 (sister chromatid cohesion protein 3, SCC3 are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21

  16. The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

    Directory of Open Access Journals (Sweden)

    2005-04-01

    Full Text Available The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM cell-surface receptors. In 1996, we and others identified a superfamily of “regulator of G-protein signaling” (RGS proteins that accelerate the rate of GTP hydrolysis by Gα subunits (dubbed GTPase-accelerating protein or “GAP” activity. This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Gα subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs that serve as Gα effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Gα and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Gα subunits to 7TM receptors in the absence of conventional Gβγ dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Gα AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Gα subunits: namely, the guanine nucleotide dissociation inhibitor (GDI activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF activity of Ric‑8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Gα subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal

  17. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    OpenAIRE

    Zhong Tao P; Watanabe Hiroshi; Chopra Sameer S; Roden Dan M

    2007-01-01

    Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further...

  18. Association of the α2δ1 Subunit with Cav3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

    OpenAIRE

    Thompson, William R.; Majid, Amber S.; Czymmek, Kirk J; Ruff, Albert L.; García, Jesús; Duncan, Randall L.; Farach-Carson, Mary C.

    2011-01-01

    Voltage sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α1 pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs, but a relatively small amount of L-type α1 subunits. Auxiliary VSCC subunits have several functions including modulating gating kinetics, trafficking of the channel and phosphorylation events. The influence of the ...

  19. Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

    OpenAIRE

    Won, Susan; Michkov, Alexander V.; Krystofova, Svetlana; Garud, Amruta V.; Borkovich, Katherine A.

    2012-01-01

    Heterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungus Neurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge...

  20. Condensin HEAT subunits required for DNA repair, kinetochore/centromere function and ploidy maintenance in fission yeast.

    Directory of Open Access Journals (Sweden)

    Xingya Xu

    Full Text Available Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance.

  1. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units

    Science.gov (United States)

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-03-01

    We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub-unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions of an ideal flexible polymer sub-unit. We illustrate the formalism by deriving generic scattering expressions for branched structures such as stars, pom-poms, bottle-brushes, and dendrimers build out of asymmetric two-functional sub-units.

  2. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units.

    Science.gov (United States)

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-03-14

    We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub-unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions of an ideal flexible polymer sub-unit. We illustrate the formalism by deriving generic scattering expressions for branched structures such as stars, pom-poms, bottle-brushes, and dendrimers build out of asymmetric two-functional sub-units. PMID:22423826

  3. Studies of double-labeled mouse thyrotropin and free alpha-subunits to estimate relative fucose content

    International Nuclear Information System (INIS)

    The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with [35S]methionine and [3H]fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of 3H/35S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The 3H/35S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated [3H]fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones

  4. Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    International Nuclear Information System (INIS)

    The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N3-[32P]-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH2-terminal sequence was confirmed by amino acid sequencing

  5. Soy 11S Globulin Acid Subunits as the Novel Food Polymer Carrier

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2015-01-01

    Full Text Available Aflatoxins were conjugated with soy 11S globulins acid subunits and the hapten-specific monoclonal antibodies (McAbs cross-reactive with four major aflatoxins were achieved using indirect competitive ELISA screening procedure. The two antibodies (clones 1B2 and 2D3 had similar reaction efficiency with aflatoxins B1, B2, and G1 but showed a weak cross-reaction to G2. The clone 4C5 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxins B1, B2, G1, and G2 at 50% inhibition for 4C5 were 1.1, 1.2, 2.1, and 17.6 pg mL−1. The results indicated that soy 11S globulin acid subunits were suitable novel carriers for aflatoxin antigen in immunization experiments and clone 4C5 could be used for simultaneous analysis of total aflatoxins.

  6. Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

    Science.gov (United States)

    Roloff, Gabrielle A; Henry, Michael F

    2015-08-15

    Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. PMID:26108620

  7. Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

    Directory of Open Access Journals (Sweden)

    Prem Singh Kaushal

    2011-10-01

    Full Text Available Ribonucleotide reductase (RR is a crucial enzyme in de novo DNA synthesis, where it catalyses the rate determining step of dNTP synthesis. RRs consist of a large subunit called RR1 (α, that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (β, which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an anbm hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly.

  8. Structure of Rv1848 (UreA), the Mycobacterium tuberculosis urease γ subunit

    International Nuclear Information System (INIS)

    Crystal and solution structures of Rv1848 protein and their implications in the biological assembly of Mtb urease is presented. The crystal structure of the urease γ subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 Å resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (αβγ)3 composition observed for other bacterial ureases. The γ subunit may be of primary importance for the formation of the urease quaternary structure

  9. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    Science.gov (United States)

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-05-15

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID:1709742

  10. Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

    DEFF Research Database (Denmark)

    Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas;

    2015-01-01

    Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM...... patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib...... in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a...

  11. Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin βE subunit

    International Nuclear Information System (INIS)

    Activins, TGF-β superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin β subunit genes, βC and βE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin βE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells

  12. Effects of substitutions at position 180 in the Escherichia coli RNA polymerase 70 subunit

    Indian Academy of Sciences (India)

    Olga N Koroleva; Stephen Jw Busby; Valeriy L Drutsa

    2011-03-01

    In order to investigate the role of His180 residue, located in the non-conserved region of the 70 subunit of Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic acid were constructed and purified using the IMPACT system. The ability of mutant 70 subunits to interact with core RNA polymerase was investigated using native gel-electrophoresis. The properties of the corresponding reconstituted holoenzymes, as provided by gel shift analysis of their complexes with single- and double-stranded promoter-like DNA and by in vitro transcription experiments, allowed one to deduce that His180 influences several steps of transcription initiation, including core binding, promoter DNA recognition and open complex formation.

  13. Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin

    International Nuclear Information System (INIS)

    Cytolethal distending toxin subunit CdtB from E. coli strain 9142-88 was purified and crystallized. Crystals belonging to space group P212121 diffract to a resolution of 1.72 Å. Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30.5 kDa per asymmetric unit, belong to space group P212121 and diffract to 1.72 Å

  14. The TSC1-TSC2 complex consists of multiple TSC1 and TSC2 subunits

    Directory of Open Access Journals (Sweden)

    Hoogeveen-Westerveld Marianne

    2012-09-01

    Full Text Available Abstract Background Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the activity of the target of rapamycin (TOR complex 1 (TORC1 and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation. Results TSC1 and TSC2 co-eluted in high molecular weight fractions by gel filtration. Coimmunoprecipitation of distinct tagged TSC1 and TSC2 isoforms demonstrated that TSC1-TSC2 complexes contain multiple TSC1 and TSC2 subunits. Conclusions TSC1 and TSC2 interact to form large complexes containing multiple TSC1 and TSC2 subunits.

  15. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...... beta gene. Both genes contain a first untranslated exon. Also, the promoter regions from the human and murine CK2 beta gene share some common features, e.g., they contain neither a TATA nor a CAAT box, exon 1 is flanked by a cluster of CpG dinucleotides and recognition sequences for the Hpa...... has no counterpart in the murine gene. Hence, regulation of transcription of the CK2 beta gene by the catalytic CK2 alpha subunit as was described by Robitzki et al. (J. Biol. Chem. 268: 5694-5703, 1993) for the human gene cannot be considered a general regulatory mechanism....

  16. The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation.

    OpenAIRE

    Landry, S; Pettit, M T; Apolinario, E; Hoffman, C. S.

    2000-01-01

    Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gbeta subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway. While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet...

  17. Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress

    OpenAIRE

    Yadav, Dinesh Kumar; Islam, S. M. Shahinul; Tuteja, Narendra

    2012-01-01

    Heterotrimeric G-proteins (α, β and γ subunits) are primarily involved in diverse signaling processes by transducing signals from an activated transmembrane G-protein coupled receptor (GPCR) to appropriate downstream effectors within cells. The role of α and β G-protein subunits in salinity and heat stress has been reported but the regulation of γ subunit of plant G-proteins in response to abiotic stress has not heretofore been described. In the present study we report the isolation of full-l...

  18. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits

    OpenAIRE

    Tong, XiaoYong; Porter, Lisa M.; Liu, GongXin; Dhar-Chowdhury, Piyali; Srivastava, Shekhar; Pountney, David J.; Yoshida, Hidetada; Artman, Michael; Fishman, Glenn I.; Yu, Cindy; Iyer, Ramesh; Morley, Gregory E.; Gutstein, David E.; Coetzee, William A.

    2006-01-01

    Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits. Am J Physiol Heart Circ Physiol 291: H543–H551, 2006. First published February 24, 2006; doi:10.1152/ajpheart.00051.2006.—Cardiac ATP-sensitive K+ (KATP) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression ...

  19. Two quality-associated HMW glutenin subunits in a somatic hybrid line between Triticum aestivum and Agropyron elongatum

    OpenAIRE

    Feng, Deshun; Xia, Guangmin; Zhao, Shuangyi; Chen, Fanguo

    2004-01-01

    High-molecular-weight glutenin subunits (HMW-GSs) from hybrid line II-12 between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nivski were characterized with SDS-PAGE. Out of these HMW-GSs, two subunits, h1Bx and h1By, had mobilities similar to the subunits 1Bx13 and 1By16 from common wheat 4072, which was used as control. Polyclonal antibodies (pAbs) of h1Bx and h1By were prepared, and Western blotting showed that the pAbs had strong affinities for h1Bx and h1By, separately. Th...

  20. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  1. Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

    OpenAIRE

    Ma, D Q; Whitehead, P. L.; Menold, M M; Martin, E. R.; Ashley-Koch, A. E.; Mei, H; Ritchie, M. D.; Delong, G R; Abramson, R.K.; Wright, H. H.; Cuccaro, M. L.; Hussman, J. P.; Gilbert, J.R.; Pericak-Vance, M A

    2005-01-01

    Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied...

  2. Structural elements in the Girk1 subunit that potentiate G protein–gated potassium channel activity

    OpenAIRE

    Wydeven, Nicole; Young, Daniele; Mirkovic, Kelsey; Wickman, Kevin

    2012-01-01

    G protein–gated inwardly rectifying K+ (Girk/KIR3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABAB...

  3. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair

    International Nuclear Information System (INIS)

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the Pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a Pol32 deletion. Moreover, Pol3-ct shows genetic interactions, both suppression and synthetic lethality, with Pol31 mutant alleles. Structural analyses indicate that the B subunit of Pol displays a major conserved region at its surface and that Pol31 alleles interacting with Pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylo-genetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair. (authors)

  4. Crystal structure of catalytic domain of the initiation factor 2B epsilon subunit

    DEFF Research Database (Denmark)

    Boesen, Thomas; Mohammad, Sarah S.; Pavitt, Graham D.; Andersen, Gregers Rom

    CRYSTAL STRUCTURE OF CATALYTIC DOMAIN OF THE INITIATION FACTOR 2B EPSILON SUBUNIT Thomas Boesen1,Sarah S. Mohammad2, Graham Pavitt2, and Gregers R. Andersen1* 1Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Århus C, Denmark 2Department of Biomolecular Science...... of residues important for catalytic function and the location of residues for which mutations in humans give rise the the fatal brain disorder leukoencephalopathy with vanishing white matter....

  5. Positive regulation by GABA(BR1 subunit of leptin expression through gene transactivation in adipocytes.

    Directory of Open Access Journals (Sweden)

    Yukari Nakamura

    Full Text Available BACKGROUND: The view that γ-aminobutyric acid (GABA plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: GABA(B receptor 1 (GABA(BR1 subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA(BR ligands. However, no prominent expression was seen with mRNA for GABA(BR2 subunit required for heteromeric orchestration of the functional GABA(BR by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA(BR1-null mice than in wild-type mice. Knockdown by siRNA of GABA(BR1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells. CONCLUSIONS/SIGNIFICANCE: Our results indicate that GABA(BR1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(BR.

  6. Molecular characterization of the largest subunit of Plasmodium falciparum RNA polymerase I.

    Science.gov (United States)

    Fox, B A; Li, W B; Tanaka, M; Inselburg, J; Bzik, D J

    1993-09-01

    Plasmodium species possess developmentally regulated ribosomal RNA (rRNA) genes. This report describes the expression and gene structure of the largest subunit of P. falciparum RNA polymerase I (RNAPI), which is responsible for the synthesis of rRNA. The RNAPI largest subunit gene was present as a single copy gene on chromosome 9. Three exons encode the 2910-amino acid RNAPI polypeptide (340 140 Da). A comparison of Plasmodium, Trypanosoma, and Saccharomyces cerevisiae nuclear RNAP largest subunits identified conserved amino acid positions and class-specific amino acid positions. Novel amino acid insertions were found between RNAPI conserved regions A and B (region A'), D and DE1 (region D'), DE2 and E (region DE2'), and F and G (region F'). Leucine zipper domains were found within regions D', DE2, and DE2'. A novel serine-rich repeat domain, a domain with homology to the C-terminal domain of eukaryotic upstream binding factor (UBF), and 4 highly conserved casein kinase II (CKII) Ser/Thr phosphorylation motifs were found within a 127-amino acid sub-region of enlarged region F'. The novel RNAPI serine-rich repeat contained a conserved motif, Ser-X3-Ser, which was also identified in the serine-rich repeat domains of the P. falciparum RNAPII and RNAPIII largest subunits, as well as within a highly homologous serine-rich repeat from trophozoite antigen R45. The results of this molecular analysis indicate that phosphorylation and dephosphorylation mechanisms regulate the activity of P. falciparum RNAPI. PMID:8259131

  7. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    OpenAIRE

    Daley, Daniel O; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first...

  8. N-Glycosylation of cholera toxin B subunit: serendipity for novel plant-made vaccines?

    OpenAIRE

    Nobuyuki eMatoba

    2015-01-01

    The non-toxic B subunit of cholera toxin (CTB) has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as recombinant production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant ...

  9. Assessment of Sulf hydryl Group in Individual Rat Lens Protein Subunits During Galactose Cataract Development

    Institute of Scientific and Technical Information of China (English)

    HaroldI.Calvin; S.C.JosephFu

    1994-01-01

    A specific reagent DACM [N-( 7-Dimethylamino-4-methyl-3-coumarinyl) maleimide] is used to study the -SH groups in lens proteins of normal and galactose cataractous rats. DACM when reacts readily with -SH groups form strong fluorescent adducts. The two -dimensional electrophoresis with DACM pre-labeled proteins is a simple and sensitive method for detecting -SH groups of protein subunit. In the present study, based on IEF/SDS-PAGE electrophoretically characterized soluble crystallins, describes specific ...

  10. Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor.

    OpenAIRE

    Roberson, M S; Schoderbek, W E; Tremml, G; Maurer, R A

    1994-01-01

    Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for bindi...

  11. CHRNB2 Is the Second Acetylcholine Receptor Subunit Associated with Autosomal Dominant Nocturnal Frontal Lobe Epilepsy*

    OpenAIRE

    Phillips, Hilary A.; Favre, Isabelle; Kirkpatrick, Martin; Zuberi, Sameer M; Goudie, David; Heron, Sarah E.; Scheffer, Ingrid E.; Sutherland, Grant R.; Berkovic, Samuel F; Bertrand, Daniel; Mulley, John C

    2000-01-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the β2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes ...

  12. Nucleotide sequence of the gene for the b subunit of human factor XIII

    Energy Technology Data Exchange (ETDEWEB)

    Bottenus, R.E.; Ichinose, A.; Davie, E.W. (Univ. of Washington, Seattle (USA))

    1990-12-01

    Factor XIII (M{sub r} 320 000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (M{sub r} 75 000 each) and two b subunits (M{sub r} 80 000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in {lambda} phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92{percent} of the gene. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

  13. Subetta increases phosphorylation of insulin receptor β-subunit alone and in the presence of insulin

    OpenAIRE

    Gorbunov, E A; Nicoll, J; Kachaeva, E. V.; Tarasov, S A; Epstein, O. I.

    2015-01-01

    It has been previously shown that Subetta (a drug containing released-active forms of antibodies to the insulin receptor β-subunit and antibodies to endothelial nitric oxide synthase) stimulated insulin-induced adiponectin production by mature human adipocytes in the absence of insulin. Therefore, it was assumed that Subetta could activate the insulin receptor. To confirm this hypothesis, the capacity of Subetta to activate the insulin receptor in mature human adipocytes in the absence or pre...

  14. Myeloid cell–specific serine palmitoyltransferase subunit 2 haploinsufficiency reduces murine atherosclerosis

    OpenAIRE

    Chakraborty, Mahua; Lou, Caixia; Huan, Chongmin; Kuo, Ming-Shang; Park, Tae-Sik; Cao, Guoqing; Jiang, Xian-Cheng

    2013-01-01

    Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2–hapl...

  15. Phylogenetic Positions of Insectivora in Eutheria Inferred from Mitochondrial Cytochrome c Oxidase Subunit II Gene

    OpenAIRE

    Onuma, Michiko; Kusakabe, Tadashi; Kusakabe, Shinichi

    1998-01-01

    For the elucidation of the phylogenetic position of insectivora in eutheria, we have sequenced the cytochrome c oxidase subunit II (COII) gene of mitochondria for three insectivoran species [musk shrew (Suncus murinus), shrew mole (Urotrichus talpoides), Japanese mole (Mogera wogura)] and analyzed these amino acid sequences with neighbor-joining (NJ) method and maximum likelihood (ML) method. NJ analysis shows polyphyly of Insectivora and Chiroptera. Assuming that each of Primates, Ferungulat...

  16. Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions

    OpenAIRE

    Daniela Almeida; Emanuel Maldonado; Vitor Vasconcelos; Agostinho Antunes

    2015-01-01

    Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments rangi...

  17. Early postnatal switch in GABAA receptor α-subunits in the reticular thalamic nucleus.

    Science.gov (United States)

    Pangratz-Fuehrer, Susanne; Sieghart, Werner; Rudolph, Uwe; Parada, Isabel; Huguenard, John R

    2016-03-01

    The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development. PMID:26631150

  18. Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.

    Science.gov (United States)

    Hsiao, Bernard; Mihalak, Karla B; Repicky, Sarah E; Everhart, Drew; Mederos, Ana H; Malhotra, Arun; Luetje, Charles W

    2006-01-01

    We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors. PMID:16189299

  19. Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

    OpenAIRE

    Ismaya, Wangsa T.; Rozeboom, Henriette J.; Weijn, Amrah; Mes, Jurriaan J.; Fusetti, Fabrizia; Wichers, Harry J.; Dijkstra, Bauke W.

    2011-01-01

    Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode ...

  20. Chromopeptides from C-phycocyanin. Structure and linkage of a phycocyanobilin bound to the β subunit

    International Nuclear Information System (INIS)

    The smallest cyanogen bromide fragment derived from the β subunit of Synechococcus sp. 6301 C-phycocyanin, the blue heptapeptide 2, has been investigated by 360-MHz 1H NMR spectroscopy. The peptide portion, heptapeptide 3, was synthesized independently and used in comparative spectroscopic analysis. These studies have led to complete assignment of the structure of the peptide-linked phycocyanobilin and elucidation of the nature of the thioether chromophore-peptide linkage

  1. A link between the cytoplasmic engulfment protein Elmo1 and the Mediator complex subunit Med31

    OpenAIRE

    Mauldin, Joshua P.; LU, MINGJIAN; Das, Soumita; Park, Daeho; Ernst, Peter B.; Ravichandran, Kodi S.

    2012-01-01

    The cytoplasmic Elmo1:Dock180 complex acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac and functions downstream of the phagocytic receptor BAI1 during apoptotic cell clearance, and in the entry of Salmonella and Shigella into cells [1–7]. We discovered an unexpected binding between Elmo1 and Mediator complex subunit Med31. The Mediator complex is a regulatory hub for nearly all gene transcription via RNA polymerase II (Pol II), bridging the general transcription mac...

  2. Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides.

    Science.gov (United States)

    Shin, Joon-Hee; Kim, Jung-Eun; Malapi-Wight, Martha; Choi, Yoon-E; Shaw, Brian D; Shim, Won-Bo

    2013-06-01

    Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development. PMID:23452277

  3. Human acid-labile subunit deficiency: clinical, endocrine and metabolic consequences.

    NARCIS (Netherlands)

    Domene, H.M.; Hwa, V.; Argente, J.; Wit, J.M.; Camacho-Hubner, C.; Jasper, H.G.; Pozo, J.; Duyvenvoorde, H.A. van; Yakar, S.; Fofanova-Gambetti, O.V.; Rosenfeld, R.G.; Hermus, A.R.M.M.; Twickler, T.B.; Kempers, M.J.E.

    2009-01-01

    The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit (ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes.

  4. Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Hall John C

    2008-09-01

    Full Text Available Abstract Background The regulatory subunit of cAMP-dependent protein kinase (PKA exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I and type II (PKA-II. Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8. Results RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Genes specifically up-regulated in RIα cells were highly enriched for pathways involved in cell growth while genes up-regulated in RIIβ cells were enriched for pathways involved in differentiation. A large group of genes (~3600 was regulated along an axis of proliferation/differentiation between RIα, parental, and RIIβ cells. RIα/wt and RIIβ/wt gene regulation was shown by two separate and distinct gene set analytical methods to be strongly cross-correlated with a generic model of cellular differentiation. Conclusion Overexpression of PKA regulatory subunits in an ovarian cancer cell line dramatically influences the cell phenotype. The proliferation phenotype is strongly correlated with recently identified clinical biomarkers predictive of poor prognosis in ovarian cancer suggesting a possible pivotal role for PKA regulation in disease progression.

  5. Rapid evolution of the human gene for cytochrome c oxidase subunit IV.

    OpenAIRE

    Lomax, M I; Hewett-Emmett, D; Yang, T L; Grossman, L I

    1992-01-01

    We have compared the DNA sequences of nine mammalian genes for cytochrome c oxidase subunit IV (COX4 genes)--four expressed genes (human, bovine, rat, and mouse) and five pseudogenes (human, chimpanzee, orangutan, squirrel monkey, and bovine)--and constructed the sequence of the ancestral mammalian COX4 gene. By analyzing these sequences to determine the pattern and rate of nucleotide substitution in each branch of the evolutionary tree, we deduced that the human gene has evolved rapidly sinc...

  6. Role of the NFkB subunit p50 in particle induced, aseptic lung inflammation

    OpenAIRE

    Yin, Renfu

    2013-01-01

    We tested the hypothesis that pulmonary exposure to sterile carbon nanoparticles (CNP), a main constituent of urban air pollution, generates an acute inflammatory response by involving the p50 subunit of NFkB1, a pathway generally crucial for inflammatory gene expression in sepsis. Exposing wt and p50-deficinet mice to CNP caused an acute, aseptic neutrophilic alveolitis. While the storm of acute phase cytokines was much more pronounced in p50 mice, no genotype related difference in the numbe...

  7. A human RNA polymerase II subunit is encoded by a recently generated multigene family

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2001-11-01

    Full Text Available Abstract Background The sequences encoding the yeast RNA polymerase II (RPB subunits are single copy genes. Results While those characterized so far for the human (h RPB are also unique, we show that hRPB subunit 11 (hRPB11 is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR. Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G, and encodes a single polypeptide which is identical to subunit hRPB11a. Conclusions The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

  8. Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels

    Science.gov (United States)

    Yu, Haibo; Lin, Zhihong; Mattmann, Margrith E.; Zou, Beiyan; Terrenoire, Cecile; Zhang, Hongkang; Wu, Meng; McManus, Owen B.; Kass, Robert S.; Lindsley, Craig W.; Hopkins, Corey R.; Li, Min

    2013-01-01

    Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1–KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1–KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1–KCNE1 channels. PMID:23650380

  9. Three Homologous Subunits Form a High Affinity Peptide-gated Ion Channel in Hydra*

    Science.gov (United States)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D.; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J. P.; Holstein, Thomas W.; Gründer, Stefan

    2010-01-01

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na+ channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na+ channels (HyNaCs) 2–4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na+ channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na+ selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission. PMID:20159980

  10. Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits.

    Science.gov (United States)

    Yang, S H; Nussenzweig, A; Li, L; Kim, D; Ouyang, H; Burgman, P; Li, G C

    1996-07-01

    Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF). To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides. To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells. We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression. Conversely, expression of human Ku-80 alone does not have this effect. Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected. These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1. The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression. PMID:8668197

  11. Increased expression of nuclear factor kappa-B p65 subunit in adenomyosis

    OpenAIRE

    Park, Hana; Kim, Sung-Hoon; Cho, Yoo Mi; Ihm, Hyo Jin; Oh, Young Sang; Hong, Seung Hwa; Chae, Hee Dong; Kim, Chung-Hoon; Kang, Byung Moon

    2016-01-01

    Objective Nuclear factor kappa-B (NF-κB) is a critical proinflammatory regulator that has been suggested to play a pivotal role in the pathogenesis and pathophysiology of endometriosis. In the present study, we aimed to evaluate whether the expression of NF-κB p65 subunit is increased in the eutopic endometrium and/or in the adenomyosis nodule of women with adenomyosis. Methods Thirty-three women with histologically confirmed adenomyosis after laparoscopic or transabdominal hysterectomy were ...

  12. Effects of mutations on enzyme activity and immunoreactivity of the S1 subunit of pertussis toxin.

    OpenAIRE

    Lobet, Y; Cieplak, W; Smith, S. G.; Keith, J M

    1989-01-01

    By introducing a series of six different substitutions at and around position 9, we investigated the structural requirements of the amino-terminal region of the S1 subunit of pertussis toxin for both enzyme activity and immunoreactivity. All mutant S1 analogs with a substitution at this location exhibited severely decreased ADP-ribosyltransferase activity (range, 400- to 2,500-fold). In contrast, alteration of arginine 58 had considerably less effect. The reactivity of the mutant molecules wi...

  13. Immunogenicity and Protection Efficacy of Subunit-based Smallpox Vaccines Using Variola Major Antigens

    OpenAIRE

    Sakhatskyy, Pavlo; Wang, Shixia; Zhang, Chuanyou; Chou, Te-Hui; Kishko, Michael; Lu, Shan

    2007-01-01

    The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the ide...

  14. Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host

    OpenAIRE

    Tooley, Aaron J.; Cai, Yuping A.; Glazer, Alexander N.

    2001-01-01

    The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phyc...

  15. Fragmentations of the large-subunit rRNA in the family Rhizobiaceae.

    OpenAIRE

    Selenska-Pobell, S; Evguenieva-Hackenberg, E

    1995-01-01

    A 130-nucleotide-long rRNA species corresponding to the 5' end of the 23S rRNA gene was found in 96 strains belonging to different Rhizobium, Bradyrhizobium, and Agrobacterium species. Additional fragmentation in the central region of the large-subunit rRNA occurred in all agrobacteria, except Agrobacterium vitis, and in most Rhizobium leguminosarum and Rhizobium etli strains but did not occur in any of the other rhizobia and bradyrhizobia studied.

  16. Molecular evolution of the cytochrome c oxidase subunit 5A gene in primates

    OpenAIRE

    Wildman Derek E; Opazo Juan C; Uddin Monica; Sherwood Chet C; Hof Patrick R; Goodman Morris; Grossman Lawrence I

    2008-01-01

    Abstract Background Many electron transport chain (ETC) genes show accelerated rates of nonsynonymous nucleotide substitutions in anthropoid primate lineages, yet in non-anthropoid lineages the ETC proteins are typically highly conserved. Here, we test the hypothesis that COX5A, the ETC gene that encodes cytochrome c oxidase subunit 5A, shows a pattern of anthropoid-specific adaptive evolution, and investigate the distribution of this protein in catarrhine brains. Results In a dataset compris...

  17. Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX

    OpenAIRE

    Stinson, Benjamin M.; Baytshtok, Vladimir; Schmitz, Karl R.; Baker, Tania A.; Sauer, Robert T.

    2015-01-01

    The hexameric AAA+ ring of Escherichia. coli ClpX, an ATP-dependent protein unfolding and translocation machine, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+ ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring w...

  18. Human locus coeruleus neurons express the GABAA receptor γ2 subunit gene and produce benzodiazepine binding

    OpenAIRE

    Hellsten, Kati S.; Sinkkonen, Saku T.; Hyde, Thomas M.; Kleinman, Joel E.; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R.

    2010-01-01

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for γ-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABAA receptors are composed of a variable array of subunits forming heteropentam...

  19. Activated radixin is essential for GABAA receptor α5 subunit anchoring at the actin cytoskeleton

    OpenAIRE

    Loebrich, Sven; Bähring, Robert; Katsuno, Tatsuya; Tsukita, Sachiko; Kneussel, Matthias

    2006-01-01

    Neurotransmitter receptor clustering is thought to represent a critical parameter for neuronal transmission. Little is known about the mechanisms that anchor and concentrate inhibitory neurotransmitter receptors in neurons. GABAA receptor (GABAAR) α5 subunits mainly locate at extrasynaptic sites and are thought to mediate tonic inhibition. Notably, similar as synaptic GABAARs, these receptor subtypes also appear in cluster formations at neuronal surface membranes and are of particular interes...

  20. Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

    OpenAIRE

    Harpur, A G; Layton, M. J.; Das, P; Bottomley, M J; Panayotou, G.; Driscoll, P. C.; Waterfield, M D

    1999-01-01

    The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain a...

  1. The structure of the TFIIH p34 subunit reveals a von Willebrand factor A like fold.

    Directory of Open Access Journals (Sweden)

    Dominik R Schmitt

    Full Text Available RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH. A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.

  2. Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury.

    Science.gov (United States)

    Urich, Daniela; Eisenberg, Jessica L; Hamill, Kevin J; Takawira, Desire; Chiarella, Sergio E; Soberanes, Saul; Gonzalez, Angel; Koentgen, Frank; Manghi, Tomas; Hopkinson, Susan B; Misharin, Alexander V; Perlman, Harris; Mutlu, Gokhan M; Budinger, G R Scott; Jones, Jonathan C R

    2011-09-01

    Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3(fl/fl)), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3(fl/fl) mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury. PMID:21878500

  3. The structural basis for an essential subunit interaction in influenza virus RNA polymerase

    International Nuclear Information System (INIS)

    Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2, and PA are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The C terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the N-terminal residues of PB1 can fit by forming a 310 helix. (author)

  4. Tumor cells with low proteasome subunit expression predict overall survival in head and neck cancer patients

    International Nuclear Information System (INIS)

    Experimental and clinical data suggest that solid cancers contain treatment-resistant cancer stem cells that will impair treatment efficacy. The objective of this study was to investigate if head and neck squamous cell carcinoma (HNSCC) also contain cancer stem cells that can be identified by low 26S proteasome activity and if their presence correlates to clinical outcome. Human HNSCC cells, engineered to report lack of proteasome activity based on accumulation of a fluorescent fusion protein, were separated based on high (ZsGreen-cODCneg) or low (ZsGreen-cODCpos) proteasome activity. Self-renewal capacity, tumorigenicity and radioresistance were assessed. Proteasome subunit expression was analyzed in tissue microarrays and correlated to survival and locoregional cancer control of 174 patients with HNSCC. HNSCC cells with low proteasome activity showed a significantly higher self-renewal capacity and increased tumorigenicity. Irradiation enriched for ZsGreen-cODCpos cells. The survival probability of 82 patients treated with definitive radio- or chemo-radiotherapy exhibiting weak, intermediate, or strong proteasome subunit expression were 21.2, 28.8 and 43.8 months (p = 0.05), respectively. Locoregional cancer control was comparably affected. Subpopulations of HNSCC display stem cell features that affect patients’ tumor control and survival. Evaluating cancer tissue for expression of the proteasome subunit PSMD1 may help identify patients at risk for relapse

  5. Hco-LGC-38 is novel nematode cys-loop GABA receptor subunit.

    Science.gov (United States)

    Siddiqui, Salma Z; Brown, David D R; Accardi, Michael V; Forrester, Sean G

    2012-10-01

    We have identified and characterized a novel cys-loop GABA receptor subunit (Hco-LGC-38) from the parasitic nematode Haemonchus contortus. This subunit is present in parasitic and free-living nematodes and shares similarity to both the UNC-49 group of GABA receptor subunits from nematodes and the resistant to dieldrin (RDL) receptors of insects. Expression of the Hco-lgc-38 gene in Xenopus oocytes and subsequent electrophysiological analysis has revealed that the gene encodes a homomeric channel sensitive to GABA (EC(50) 19 μM) and the GABA analogue muscimol. The sensitivity of the Hco-LGC-38 channel to GABA is similar to reported values for the Drosophila RDL receptor whereas its lower sensitivity to muscimol is similar to nematode GABA receptors. Hco-LGC-38 is also highly sensitive to the channel blocker picrotoxin and moderately sensitive to fipronil and dieldrin. Homology modeling of Hco-LGC-38 and subsequent docking of GABA and muscimol into the binding site has uncovered several types of potential interactions with binding-site residues and overall appears to share similarity with models of other invertebrate GABA receptors. PMID:22940478

  6. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

    Science.gov (United States)

    Tu, Weihong; Xu, Xin; Peng, Lisheng; Zhong, Xiaofen; Zhang, Wenfeng; Soundarapandian, Mangala M.; Balel, Cherine; Wang, Manqi; Jia, Nali; Zhang, Wen; Lew, Frank; Chan, Sic Lung; Chen, Yanfang; Lu, Youming

    2010-01-01

    SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. PMID:20141836

  7. Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance

    Science.gov (United States)

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S.; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L.; Elpek, Kutlu G.; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W.; Makishima, Hideki; Turley, Shannon J.; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P.; Jaiswal, Siddhartha; Ebert, Benjamin L.; Rodig, Scott J.; Tyner, Jeffrey W.; Marto, Jarrod A.; Weinstock, David M.; Lane, Andrew A.

    2014-01-01

    Activating mutations of G protein alpha subunits (Gα) occur in 4–5% of all human cancers1 but oncogenic alterations in beta subunits (Gβ) have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors, and disrupt Gα-Gβγ interactions. Different mutations in Gβ proteins clustered to some extent based on lineage; for example, all eleven GNB1 K57 mutations were in myeloid neoplasms while 7 of 8 GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 alleles in Cdkn2a-deficient bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 mutations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  8. Proteasome β5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

    Science.gov (United States)

    Kirschner, Felicia; Reppe, Katrin; Andresen, Nadine; Witzenrath, Martin; Ebstein, Frédéric; Kloetzel, Peter-Michael

    2016-01-01

    Immunoproteasomes, harboring the active site subunits β5i/LMP7, β1i/LMP2, and β2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in β5i/LMP7-/- mice. Our data show that β5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in β5i/LMP7-/- mice. The impaired bacterial elimination in β5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections. PMID:27100179

  9. COX7AR is a Stress-inducible Mitochondrial COX Subunit that Promotes Breast Cancer Malignancy

    Science.gov (United States)

    Zhang, Kezhong; Wang, Guohui; Zhang, Xuebao; Hüttemann, Philipp P.; Qiu, Yining; Liu, Jenney; Mitchell, Allison; Lee, Icksoo; Zhang, Chao; Lee, Jin-sook; Pecina, Petr; Wu, Guojun; Yang, Zeng-quan; Hüttemann, Maik; Grossman, Lawrence I.

    2016-01-01

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in regulating mitochondrial energy production and cell survival. COX subunit VIIa polypeptide 2-like protein (COX7AR) is a novel COX subunit that was recently found to be involved in mitochondrial supercomplex assembly and mitochondrial respiration activity. Here, we report that COX7AR is expressed in high energy-demanding tissues, such as brain, heart, liver, and aggressive forms of human breast cancer cells. Under cellular stress that stimulates energy metabolism, COX7AR is induced and incorporated into the mitochondrial COX complex. Functionally, COX7AR promotes cellular energy production in human mammary epithelial cells. Gain- and loss-of-function analysis demonstrates that COX7AR is required for human breast cancer cells to maintain higher rates of proliferation, clone formation, and invasion. In summary, our study revealed that COX7AR is a stress-inducible mitochondrial COX subunit that facilitates human breast cancer malignancy. These findings have important implications in the understanding and treatment of human breast cancer and the diseases associated with mitochondrial energy metabolism. PMID:27550821

  10. Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.

    Science.gov (United States)

    Peréz Aguirreburualde, María Sol; Gómez, María Cristina; Ostachuk, Agustín; Wolman, Federico; Albanesi, Guillermo; Pecora, Andrea; Odeon, Anselmo; Ardila, Fernando; Escribano, José M; Dus Santos, María José; Wigdorovitz, Andrés

    2013-02-15

    Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 μg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 μg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection. PMID:23291101

  11. Identification of the mitochondrially encoded subunit 6 of F1FO ATPase in Trypanosoma brucei.

    Science.gov (United States)

    Škodová-Sveráková, Ingrid; Horváth, Anton; Maslov, Dmitri A

    2015-06-01

    Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the (35)S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence. PMID:26276057

  12. Evaluation of Pakistani wheat germplasm for bread quality based on allelic variation in HMW glutenin subunits

    International Nuclear Information System (INIS)

    Seventy six Pakistani wheat genotypes including land races were investigated for Bread quality (BQ) based on allelic variation in HMW glutenin subunits at the Glu-1 loci through SDS- polyacrylamide gel electropherosis. Twenty five different allelic combinations were detected with a total of 14 Glu-1 loci. Highest polymorphism was revealed by Glu-B locus and some single/ rare sub units were also screened out. The frequencies of dominant subunits were 50% for 2*, 42.11% for subunit pair 17+18 and 48.68% for 5+10 and 2+12 respectively. The quality scores displayed a range from 4 to 10, however generally good quality score of eight was more frequent (39. 47%). The highest quality scores of 10 and 9 were observed in 22.36% and 19.74% of genotypes respectively. The UPGMA analysis grouped genotypes into three major with two additional sub clusters for each. The cluster 'a' 'b' and 'C' were separated at 73% genetic distance which was further differentiated at a genetic distance of 50% into their sub clusters. Pakistani wheat varieties/land races exhibited large variation in term of HMW-GS. The generated information will lead to the pyrimiding of sub units for high BQ through mission oriented marker assisted breeding programmes for quality improvement of wheat. (author)

  13. Xanthan Gum as an Adjuvant in a Subunit Vaccine Preparation against Leptospirosis

    Directory of Open Access Journals (Sweden)

    Katia L. Bacelo

    2014-01-01

    Full Text Available Leptospiral immunoglobulin-like (Lig proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106, aluminium hydroxide (alhydrogel, and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, P<0.05 was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens.

  14. Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding.

    Science.gov (United States)

    Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Plückthun, Andreas; Wagner, Gerhard; Hagn, Franz

    2016-06-28

    Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. PMID:27298341

  15. Properties of human brain sodium channel α-subunits expressed in HEK293 cells and their modulation by carbamazepine, phenytoin and lamotrigine

    Science.gov (United States)

    Qiao, Xin; Sun, Guangchun; Clare, Jeffrey J; Werkman, Taco R; Wadman, Wytse J

    2014-01-01

    Background and purpose Voltage-activated Na+ channels contain one distinct α-subunit. In the brain NaV1.1, NaV1.2, NaV1.3 and NaV1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na+ channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine. Experimental approach Na+ currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits. Key results NaV1.2 and NaV1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and NaV1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to NaV1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin. Conclusions and implications The four Na+ channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na+ channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development. PMID:24283699

  16. Structural and functional influences of coagulation factor XIII subunit B heterozygous missense mutants.

    Science.gov (United States)

    Thomas, Anne; Biswas, Arijit; Ivaskevicius, Vytautas; Oldenburg, Johannes

    2015-07-01

    The coagulation factor XIII(FXIII) is a plasma circulating heterotetrameric protransglutaminase that acts at the end of the coagulation cascade by covalently cross-linking preformed fibrin clots (to themselves and to fibrinolytic inhibitors) in order to stabilize them against fibrinolysis. It circulates in the plasma as a heterotetramer composed of two homomeric catalytic Factor XIIIA2 (FXIIIA2) and two homomeric protective/carrier Factor XIIIB2 subunit (FXIIIB2). Congenital deficiency of FXIII is of two types: severe homozygous/compound heterozygous FXIII deficiency which results in severe bleeding symptoms and mild heterozygous FXIII deficiency which is associated with mild bleeding (only upon trauma) or an asymptomatic phenotype. Defects in the F13B gene (Factor XIIIB subunit) occur more frequently in mild FXIII deficiency patients than in severe FXIII deficiency. We had recently reported secretion-related defects for seven previously reported F13B missense mutations. In the present study we further analyze the underlying molecular pathological mechanisms as well as the heterozygous expression phenotype for these mutations using a combination of in vitro heterologous expression (in HEK293T cells) and confocal microscopy. In combination with the in vitro work we have also performed an in silico solvated molecular dynamic simulation study on previously reported FXIIIB subunit sushi domain homology models in order to predict the putative structure-functional impact of these mutations. We were able to categorize the mutations into the following functional groups that: (1) affect antigenic stability as well as binding to FXIIIA subunit, that is, Cys5Arg, Cys316Phe, and Pro428Ser (2) affect binding to FXIIIA subunit with little or no influence on antigenic stability, that is, Ile81Asn and Val401Gln c) influence neither aspects and are most likely causality linked polymorphisms or functional polymorphisms, that is, Leu116Phe and Val217Ile. The Cys5Arg mutation was the

  17. Homodimerization of the p51 Subunit of HIV-1 Reverse Transcriptase

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, X.; Mueller, G; Cuneo, M; DeRose, E; London, R

    2010-01-01

    The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-{sup 13}C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The {sup 1}H-{sup 13}C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a 'p66-like' conformation. SAXS data obtained for p51 samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, 'p51C' conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, 'p51E' conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51{sub L289K} has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of

  18. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    Science.gov (United States)

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M; Fox, B A; Inselburg, J

    1989-12-11

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension. PMID:2690004

  19. Influence of subunit structure on the oligomerization state of light harvesting complexes: a free energy calculation study

    CERN Document Server

    Janosi, L; Kosztin, I; Ritz, T; Janosi, Lorant; Keer, Harindar; Kosztin, Ioan; Ritz, Thorsten

    2005-01-01

    Light harvesting complexes 2 (LH2) from Rhodospirillum (Rs.) molischianum and Rhodopseudomonas (Rps.) acidophila form ring complexes out of eight or nine identical subunits, respectively. Here, we investigate computationally what factors govern the different ring sizes. Starting from the crystal structure geometries, we embed two subunits of each species into their native lipid-bilayer/water environment. Using molecular dynamics simulations with umbrella sampling and steered molecular dynamics, we probe the free energy profiles along two reaction coordinates, the angle and the distance between two subunits. We find that two subunits prefer to arrange at distinctly different angles, depending on the species, at about 42.5 deg for Rs. molischianum and at about 38.5 deg for Rps. acidophila, which is likely to be an important factor contributing to the assembly into different ring sizes. Our calculations suggest a key role of surface contacts within the transmembrane domain in constraining these angles, whereas t...

  20. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units

    DEFF Research Database (Denmark)

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-01-01

    -unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to......We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub...... structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions...

  1. A Formalism for Scattering of Complex Composite Structures: I Applications to Branched Structures of Asymmetric Sub-Units

    DEFF Research Database (Denmark)

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-01-01

    -unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to......We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub...... structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions...

  2. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

    Directory of Open Access Journals (Sweden)

    Sayyed Alireza Talaei

    2015-06-01

    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  3. Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits

    DEFF Research Database (Denmark)

    Blumenthal, T; Young, R A; Brown, S

    1976-01-01

    Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes for...... of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP as...... measured by sensitivity of EF-Ts activity to N-ethylmaleimide....

  4. Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation.

    Science.gov (United States)

    Buller, Andrew R; Brinkmann-Chen, Sabine; Romney, David K; Herger, Michael; Murciano-Calles, Javier; Arnold, Frances H

    2015-11-24

    Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope. PMID:26553994

  5. PETG_PATWE - Cytochrome b6-f complex subunit 5 {ECO:0000255|HAMAP-Rule:MF_00432} [UniProt

    Lifescience Database Archive (English)

    Full Text Available lex subunit V {ECO:0000255|HAMAP-Rule:MF_00432} petG {ECO0000255|HAMAP-RuleMF_00432} Patellifolia webbiana (Patellaria web...biana) (Beta webbiana) 35923 F:electron transporter, transferring e

  6. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia

    Directory of Open Access Journals (Sweden)

    Aaron eSathyanesan

    2013-06-01

    Full Text Available Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE and the vomeronasal organ (VNO. Our reverse transcriptase PCR (RT-PCR and realtime qPCR analyses of all known Gβ(β1,2,3,4,5 and Gγ(γ1,2,2t,3,4,5,7,8,10,11,12,13 subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs, while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβsubunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3 and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7 and 14 and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system.

  7. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

    Science.gov (United States)

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-01-01

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system. PMID:27618897

  8. Combination of Two Candidate Subunit Vaccine Antigens Elicits Protective Immunity to Ricin and Anthrax Toxin in Mice

    OpenAIRE

    David J Vance; Rong, Yinghui; Brey, Robert N.; Mantis, Nicholas J.

    2014-01-01

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.

  9. Combination of two candidate subunit vaccine antigens elicits protective immunity to ricin and anthrax toxin in mice.

    Science.gov (United States)

    Vance, David J; Rong, Yinghui; Brey, Robert N; Mantis, Nicholas J

    2015-01-01

    In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population. PMID:25475957

  10. Comparative genomic analysis of the proteasome β5t subunit gene : implications for the origin and evolution of thymoproteasomes

    OpenAIRE

    Sutoh, Yoichi; Kondo, Mizuho; Ohta, Yuko; Ota, Tatsuya; Tomaru, Utano; Flajnik, Martin F.; Kasahara, Masanori

    2012-01-01

    The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t...

  11. Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes

    OpenAIRE

    Sutoh, Yoichi; Kondo, Mizuho; Ohta, Yuko; Ota, Tatsuya; Tomaru, Utano; Flajnik, Martin F.; Kasahara, Masanori

    2011-01-01

    The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t...

  12. Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I.

    OpenAIRE

    Davis, Edward S.; Wille, Lucia; Chestnut, Barry A.; Sadler, Penny L.; Shakes, Diane C; Golden, Andy

    2002-01-01

    Two genes, originally identified in genetic screens for Caenorhabditis elegans mutants that arrest in metaphase of meiosis I, prove to encode subunits of the anaphase-promoting complex or cyclosome (APC/C). RNA interference studies reveal that these and other APC/C subunits are essential for the segregation of chromosomal homologs during meiosis I. Further, chromosome segregation during meiosis I requires APC/C functions in addition to the release of sister chromatid cohesion.

  13. Intellectual capital configurations and organizational capability: An empirical examination of human resource subunits in the multinational enterprise

    OpenAIRE

    Morris, Shad S.; Scott A Snell

    2011-01-01

    Building on the organizational capabilities literature and theories of the multinational enterprise, this paper develops and tests a framework that examines the relationship between intellectual capital configurations and organizational capabilities in human resource (HR) subunits. Looking at 187 subunits from 20 MNEs, findings show that intellectual capital dimensions vary in their usefulness for generating, sharing, and implementing HR management practices. In particular, while certain reso...

  14. Do nuclear-encoded core subunits of mitochondrial complex I confer genetic susceptibility to schizophrenia in Han Chinese populations?

    OpenAIRE

    Xiao Li; Wen Zhang; Jinsong Tang; Liwen Tan; Xiong-jian Luo; Xiaogang Chen; Yong-Gang Yao

    2015-01-01

    Schizophrenia is one of the most prevalent psychiatric disorders with complex genetic etiology. Accumulating evidence suggests that energy metabolism and oxidative stress play important roles in the pathophysiology of schizophrenia. Dysfunction of mitochondrial respiratory chain and altered expression of complex I subunits were frequently reported in schizophrenia. To investigate whether nuclear-encoded core subunit genes of mitochondrial complex I are associated with schizophrenia, we perfor...

  15. Nuclear Respiratory Factor 1 Regulates All Ten Nuclear-encoded Subunits of Cytochrome c Oxidase in Neurons*

    OpenAIRE

    Dhar, Shilpa S.; Ongwijitwat, Sakkapol; Wong-Riley, Margaret T. T.

    2007-01-01

    Cytochrome c oxidase (COX) is one of only four bigenomic proteins in mammalian cells, having ten subunits encoded in the nuclear genome and three in the mitochondrial DNA. The mechanism of its bigenomic control is not well understood. The ten nuclear subunits are on different chromosomes, and the possibility of their coordinate regulation by the same transcription factor(s) deserves serious consideration. The present study tested our hypothesis that nuclear respiratory factor 1 (NRF-1) serves...

  16. Conserved Expression of the Glutamate NMDA Receptor 1 Subunit Splice Variants during the Development of the Siberian Hamster Suprachiasmatic Nucleus

    OpenAIRE

    Duffield, Giles E.; Jens D Mikkelsen; Ebling, Francis J. P.

    2012-01-01

    Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional propertie...

  17. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    Directory of Open Access Journals (Sweden)

    Janssens Veerle

    2008-08-01

    Full Text Available Abstract Background Protein phosphatase 2A (PP2A is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

  18. Targeting of Shiga Toxin B-Subunit to Retrograde Transport Route in Association with Detergent-resistant Membranes

    OpenAIRE

    Falguières, Thomas; Mallard, Frédéric; Baron, Carole; Hanau, Daniel; Lingwood, Clifford; Goud, Bruno; Salamero, Jean; Johannes, Ludger

    2001-01-01

    In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein wa...

  19. Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

    OpenAIRE

    Rossi, Francesco Mattia; Pizzorusso, Tommaso; Porciatti, Vittorio; Marubio, Lisa M.; Maffei, Lamberto; Changeux, Jean-Pierre

    2001-01-01

    In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. ...

  20. Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE)

    OpenAIRE

    Wu, Jinzi; Luo, Xiaoting; Yan, Liang-Jun

    2015-01-01

    The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue n...

  1. The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically

    OpenAIRE

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB g...

  2. Early evolution of ionotropic GABA receptors and selective regimes acting on the mammalian-specific theta and epsilon subunits.

    Directory of Open Access Journals (Sweden)

    Christopher J Martyniuk

    Full Text Available BACKGROUND: The amino acid neurotransmitter GABA is abundant in the central nervous system (CNS of both invertebrates and vertebrates. Receptors of this neurotransmitter play a key role in important processes such as learning and memory. Yet, little is known about the mode and tempo of evolution of the receptors of this neurotransmitter. Here, we investigate the phylogenetic relationships of GABA receptor subunits across the chordates and detail their mode of evolution among mammals. PRINCIPAL FINDINGS: Our analyses support two major monophyletic clades: one clade containing GABA(A receptor alpha, gamma, and epsilon subunits, and another one containing GABA(A receptor rho, beta, delta, theta, and pi subunits. The presence of GABA receptor subunits from each of the major clades in the Ciona intestinalis genome suggests that these ancestral duplication events occurred before the divergence of urochordates. However, while gene divergence proceeded at similar rates on most receptor subunits, we show that the mammalian-specific subunits theta and epsilon experienced an episode of positive selection and of relaxed constraints, respectively, after the duplication event. Sites putatively under positive selection are placed on a three-dimensional model obtained by homology-modeling. CONCLUSIONS: Our results suggest an early divergence of the GABA receptor subunits, before the split from urochordates. We show that functional changes occurred in the lineages leading to the mammalian-specific subunit theta, and we identify the amino acid sites putatively responsible for the functional divergence. We discuss potential consequences for the evolution of mammals and of their CNS.

  3. Pantothenate is required in Neurospora crassa for assembly of subunit peptides of cytochrome c oxidase and ATPase/ATP synthase.

    OpenAIRE

    Brambl, R; Plesofsky-Vig, N

    1986-01-01

    One polypeptide subunit of cytochrome c oxidase (EC 1.9.3.1) and two subunits of the ATPase/ATP synthase (EC 3.6.1.34) in mitochondria of Neurospora crassa are covalently modified with a derivative of pantothenic acid. In asexual spores of a pantothenate auxotroph of Neurospora, deprivation of pantothenic acid blocked the increase of the specific activities of cytochrome c oxidase and the ATPase above the basal activities in the dormant spores. Under cellular panthothenate deprivation, all th...

  4. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato[OPEN

    Science.gov (United States)

    Subramaniam, Gayathery; Trusov, Yuri; Hayashi, Satomi; Batley, Jacqueline

    2016-01-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato. PMID:26668332

  5. Complex Control of GABA(A) Receptor Subunit mRNA Expression: Variation, Covariation, and Genetic Regulation

    OpenAIRE

    Mulligan, Megan K.; Wang, Xusheng; Adler, Adrienne L.; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W.

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly...

  6. Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

    OpenAIRE

    Lem, J.; Flannery, J. G.; Li, T; Applebury, M L; Farber, D B; Simon, M. I.

    1992-01-01

    The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photorecept...

  7. Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase ß subunit

    OpenAIRE

    Lem, Janis; Flannery, John G.; Li, Tiansen; Applebury, Meredithe L.; Farber, Debora B.; Simon, Melvin I.

    1992-01-01

    The ß subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE ß subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in...

  8. Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli.

    OpenAIRE

    Båga, M; Normark, S; Hardy, J.; O'Hanley, P; Lark, D; O Olsson; Schoolnik, G; Falkow, S

    1984-01-01

    The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.

  9. STUDY ABOUT THE INTERDEPENDENCE BETWEEN THE FINANCIAL POSITION AND PERFORMANCE WITHIN THE NATIONAL FOREST ADMINISTRATION – ROMSILVA SUBUNITS

    OpenAIRE

    Beletu Elena Cristina

    2013-01-01

    In order to elaborate the financial accounting diagnosis, one must evaluate the performance and the financial position, as well as the interdependence between them. In order to reflect the interdependence between financial position and performance an analysis was made using the multiple linear regression model for which 24 subunits of RNPROMSILVA were selected using as criteria three types of subunits: with large surfaces of forests, with deficit, medium. In this article I studied the correla...

  10. Assembly of the yeast mitochondrial H/sup +/ATPase: regulation by the overproduction and availability of the nuclear encoded (F/sub 1/) subunits

    Energy Technology Data Exchange (ETDEWEB)

    Burns, D.; Lewin, A.

    1986-05-01

    The assembly of the mitochondrial ATPase was studied in vitro by incubating isolated yeast mitochondria with radiolabeled mitochondrial precursors and in vivo by pulse-labeling of intact yeast cells and spheroplasts. Newly assembled F/sub 1/ ATPase (radiolabeled) was assayed by immunoprecipitation using subunit-specific antisera directed to the ..cap alpha.. subunit of the complex. Using two different experimental approaches, the authors have provided evidence suggesting that isolated mitochondria possess pools of unassembled F/sub 1/..cap alpha.. subunits and possibly F/sub 1/..beta.. subunits. In addition, the kinetics of import suggest that the F/sub 1/..beta.. subunit was imported and assembled at a slower rate than either the F/sub 1/..cap alpha.. and F/sub 1/..gamma.. subunits. Thus, the appearance of the new ATPase could be limited by the availability of the ..beta.. subunits.

  11. Assembly of the yeast mitochondrial H+ATPase: regulation by the overproduction and availability of the nuclear encoded (F1) subunits

    International Nuclear Information System (INIS)

    The assembly of the mitochondrial ATPase was studied in vitro by incubating isolated yeast mitochondria with radiolabeled mitochondrial precursors and in vivo by pulse-labeling of intact yeast cells and spheroplasts. Newly assembled F1 ATPase (radiolabeled) was assayed by immunoprecipitation using subunit-specific antisera directed to the α subunit of the complex. Using two different experimental approaches, the authors have provided evidence suggesting that isolated mitochondria possess pools of unassembled F1α subunits and possibly F1β subunits. In addition, the kinetics of import suggest that the F1β subunit was imported and assembled at a slower rate than either the F1α and F1γ subunits. Thus, the appearance of the new ATPase could be limited by the availability of the β subunits

  12. An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin.

    Science.gov (United States)

    Karpowich, Nathan K; Song, Jinmei; Wang, Da-Neng

    2016-07-31

    ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters. PMID:27312125

  13. V-Y Advancement Flaps Based on Yotsuyanagi Aesthetic Subunit Principles for Small Nasal Defects in Asian Patients.

    Science.gov (United States)

    Gu, Zi-Chun; Li, Hua; Hamann, Dathan; Xu, Fawei

    2016-06-01

    Cosmesis is paramount in the reconstruction of small nasal defects. Yotsuyanagi et al have previously described nasal aesthetic subunits in patients of Asian descent and their implications for reconstruction of large nasal defects, including forehead flap and Z-plasty. The impact of Asian skin types and aesthetic subunits on reconstruction planning of small nasal defects has not been rigorously explored. The aim of this article is to present a novel method for repairing small nose defects in patients of Asian descent using V-Y advancement flap designed using Yotsuyanagi nasal subunit aesthetic principles. A total of 21 defects ranging from 7 to 22 mm in size in 21 patients of Asian descent were repaired with either 1 or 2 V-Y advancement flaps designed along Yotsuyanagi nasal subunit borders. All reconstructions were completed successfully in one stage. Scars were inconspicuous and nasal subunits were neither displaced nor twisted. All patients were pleased with the cosmetic outcomes. The use of V-Y advancement flaps based on Asian aesthetic nasal subunit principles for the reconstruction of small nasal defects is a novel, cosmetically sensitive alternative. PMID:27248031

  14. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge.

    Science.gov (United States)

    Mallajosyula, Jyothi K; Hiatt, Ernie; Hume, Steve; Johnson, Ashley; Jeevan, Trushar; Chikwamba, Rachel; Pogue, Gregory P; Bratcher, Barry; Haydon, Hugh; Webby, Richard J; McCormick, Alison A

    2014-01-01

    Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low potency, requiring high dose or boosting to generate a sustained immune response. We have improved the immunogenicity of a plant-made HA vaccine by chemical conjugation to the surface of the Tobacco mosaic virus (TMV) which is non infectious in mammals. We have previously shown that TMV is taken up by mammalian dendritic cells and is a highly effective antigen carrier. In this work, we tested several TMV-HA conjugation chemistries, and compared immunogenicity in mice as measured by anti-HA IgG titers and hemagglutination inhibition (HAI). Importantly, pre-existing immunity to TMV did not reduce initial or boosted titers. Further optimization included dosing with and without alum or oil-in water adjuvants. Surprisingly, we were able to stimulate potent immunogenicity and HAI titers with a single 15 µg dose of HA as a TMV conjugate. We then evaluated the efficacy of the TMV-HA vaccine in a lethal virus challenge in mice. Our results show that a single dose of the TMV-HA conjugate vaccine is sufficient to generate 50% survival, or 100% survival with adjuvant, compared with 10% survival after vaccination with a commercially available H1N1 vaccine. TMV-HA is an effective dose-sparing influenza vaccine, using a single-step process to rapidly generate large quantities of highly effective flu vaccine from an otherwise low potency HA subunit protein. PMID:24378714

  15. Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

    Energy Technology Data Exchange (ETDEWEB)

    Mazur, Olga; Zimmer, Jochen (UV)

    2012-10-25

    Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear {beta}-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-{beta}-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an ({alpha}/{alpha}){sub 6}-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades {beta}-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.

  16. Arabidopsis thaliana GYRB3 does not encode a DNA gyrase subunit.

    Directory of Open Access Journals (Sweden)

    Katherine M Evans-Roberts

    Full Text Available DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3.We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer.These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.

  17. Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

    Science.gov (United States)

    Evans-Roberts, Katherine M.; Breuer, Christian; Wall, Melisa K.; Sugimoto-Shirasu, Keiko; Maxwell, Anthony

    2010-01-01

    Background DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. Methodology/Principal Findings We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer. Conclusions/Significance These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen. PMID:20360860

  18. Computational studies on the substrate interactions of influenza A virus PB2 subunit.

    Directory of Open Access Journals (Sweden)

    Ya-Jun Wang

    Full Text Available Influenza virus, which spreads around the world in seasonal epidemics and leads to large numbers of deaths every year, has several ribonucleoproteins in the central core of the viral particle. These viral ribonucleoproteins can specifically bind the conserved 3' and 5' caps of the viral RNAs with responsibility for replication and transcription of the viral RNA in the nucleus of infected cells. A fundamental question of most importance is that how the cap-binding proteins in the influenza virus discriminates between capped RNAs and non-capped ones. To get an answer, we performed molecular dynamics simulations and free energy calculations on the influenza A virus PB2 subunit, an important component of the RNP complexes, with a cap analog m7GTP. Our calculations showed that some key residues in the active site, such as Arg355, His357, Glu361 as well as Gln406, could offer significant hydrogen bonding and hydrophobic interactions with the guanine ring of the cap analog m7GTP to form an aromatic sandwich mechanism for the cap recognition and positioning in the active site. Subsequently, we applied this idea to a virtual screening procedure and identified 5 potential candidates that might be inhibitors against the PB2 subunit. Interestingly, 2 candidates Cpd1 and Cpd2 have been already reported to have inhibitory activities to the influenza virus cap-binding proteins. Further calculation also showed that they had comparatively higher binding affinities to the PB2 subunit than that of m7GTP. We believed that our findings could give an atomic insight into the deeper understanding of the cap recognition and binding mechanism, providing useful information for searching or designing novel drugs against influenza viruses.

  19. Mutations in G protein β subunits promote transformation and kinase inhibitor resistance.

    Science.gov (United States)

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L; Elpek, Kutlu G; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W; Makishima, Hideki; Turley, Shannon J; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P; Jaiswal, Siddhartha; Ebert, Benjamin L; Rodig, Scott J; Tyner, Jeffrey W; Marto, Jarrod A; Weinstock, David M; Lane, Andrew A

    2015-01-01

    Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  20. ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer

    Directory of Open Access Journals (Sweden)

    Pan Jian

    2011-12-01

    Full Text Available Abstract Background Treatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients. Methods Differential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS. Results Up regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6% compared to normal (21.2% and atypical hyperplasia (23% breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P Conclusions Over-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.

  1. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  2. KATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

    Directory of Open Access Journals (Sweden)

    Gemes Geza

    2010-01-01

    Full Text Available Abstract Background ATP-sensitive potassium (KATP channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy. Results PCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane. In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites. DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence. Conclusions We

  3. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  4. Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

    OpenAIRE

    Evans-Roberts, Katherine M.; Christian Breuer; Wall, Melisa K.; Keiko Sugimoto-Shirasu; Anthony Maxwell

    2010-01-01

    Background DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. Methodology/Principal Fi...

  5. Seasonal and Spatial Variability in Lake Michigan Sediment Small-Subunit rRNA Concentrations

    OpenAIRE

    MacGregor, Barbara J.; Moser, Duane P.; Baker, Brett J.; Alm, Elizabeth W.; Maurer, Max; Nealson, Kenneth H.; Stahl, David A.

    2001-01-01

    We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4°C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequen...

  6. Prevalence of the Prefoldin Subunit 5 Gene Deletion in Canine Mammary Tumors

    OpenAIRE

    Silvia Hennecke; Julia Beck; Kirsten Bornemann-Kolatzki; Stephan Neumann; Hugo Murua Escobar; Ingo Nolte; Susanne Conradine Hammer; Marion Hewicker-Trautwein; Johannes Junginger; Franz-Josef Kaup; Bertram Brenig; Ekkehard Schütz

    2015-01-01

    Background A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors. Me...

  7. Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury

    OpenAIRE

    Urich, Daniela; Eisenberg, Jessica L.; Hamill, Kevin J.; Takawira, Desire; Chiarella, Sergio E.; Soberanes, Saul; Gonzalez, Angel; Koentgen, Frank; Manghi, Tomas; Hopkinson, Susan B.; Misharin, Alexander V.; Perlman, Harris; Mutlu, Gokhan M; Budinger, G. R. Scott; Jonathan C.R. Jones

    2011-01-01

    Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3fl/fl), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3fl/fl mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 lami...

  8. Congenital Central Hypothyroidism due to a Homozygous Mutation in the TSHβ Subunit Gene

    OpenAIRE

    Sarah Catharina Grünert; Miriam Schmidts; Joachim Pohlenz; Matthias Volkmar Kopp; Markus Uhl; Karl Otfried Schwab

    2011-01-01

    Congenital central hypothyroidism (CCH) is a rare condition occurring in 1 : 20000 to 1 : 50000 newborns. As TSH plasma levels are low, CCH is usually not detected by TSH-based neonatal screening for hypothyroidism, and, as a result, diagnosis is often delayed putting affected children at risk for developmental delay and growth failure. We report on a girl with isolated central hypothyroidism due to a homozygous one-base pair deletion (T313del) in exon 3 of the TSHβ subunit gene. The molecula...

  9. Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase.

    OpenAIRE

    Doctor, B. P.; Camp, S; Gentry, M. K.; Taylor, S S; Taylor, P

    1983-01-01

    A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. ...

  10. Recombinant toxin-coregulated pilus A (TcpA) as a candidate subunit cholera vaccine.

    OpenAIRE

    Somayeh Kiaie; Hamid Abtahi; Ghasem Mosayebi; MohammadYosef Alikhani; Iraj Pakzad

    2014-01-01

    Background and Objectives The toxin co-regulated pilus A (TcpA) has been described as a critical pathogenicity factor of Vibrio cholerae. TcpA is a candidate for making subunit vaccine against cholera. The aim of this study was to produce a candidate vaccine by expressing recombinant TcpA in E. coli. Materials and Methods In this study, the toxin co-regulated pilus A gene from EL-Tor, V. cholerae subspecies, was amplified by PCR and sub-cloned into prokaryotic expression vector pGEX4T1. E. co...

  11. A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

    OpenAIRE

    Lagrain, Bert; Rombouts, Ine; Wieser, Herbert; Delcour, Jan; Koehler, Peter

    2012-01-01

    The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In t...

  12. Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli.

    OpenAIRE

    Muffler, A; Traulsen, D D; Lange, R. de; Hengge-Aronis, R

    1996-01-01

    The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be t...

  13. Age-Related Differences in NMDA Receptor Subunits of Prenatally Methamphetamine-Exposed Male Rats

    Czech Academy of Sciences Publication Activity Database

    Vrajová, M.; Schutová, B.; Klaschka, Jan; Štěpánková, H.; Řípová, D.; Šlamberová, R.

    2014-01-01

    Roč. 39, č. 11 (2014), s. 2040-2046. ISSN 0364-3190 Grant ostatní: GA ČR(CZ) GAP303/10/0580; Ministerstvo školství(CZ) CSM 7/CRP/2014; Univerzita Karlova(CZ) Prvouk P34; Univerzita Karlova(CZ) 260045/SVV/2014; Prague Psychiatric Center(CZ) MH CZ–DRO: 00023752 Institutional support: RVO:67985807 Keywords : methamphetamine * in-utero * NMDA receptor subunits * hippocampus Subject RIV: FH - Neurology Impact factor: 2.593, year: 2014

  14. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

    OpenAIRE

    Run Lei; Ke Zhang; Yanxia Wei; Min Chen; Weinstein, Lee S.; Yang Hong; Minyan Zhu; Hongchang Li; Huashun Li

    2016-01-01

    The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and cranio...

  15. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  16. Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit

    OpenAIRE

    Suzuki, Toshiharu; Wakabayashi, Chiaki; TANAKA, Kazumi; Feniouk, Boris A.; Yoshida, Masasuke

    2011-01-01

    The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achi...

  17. Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

    OpenAIRE

    Johnson, G.,; Widner, W; Xin, W N; Feiss, M

    1991-01-01

    Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to...

  18. A randomized controlled trial comparing split and subunit influenza vaccines in adults in Colombia

    OpenAIRE

    Morales, A.; J. Arias Salazar; Y. Salazar; Garcia, A; Arnoux, S.; A. Arancibia; C. Deroche; Rey, E

    2003-01-01

    In a two-center, comparative trial, 344 adults were randomly assigned to receive a single dose of inactivated split-virion (Imovax Gripeâ) or sub-unit (Agrippal S1â) influenza vaccine (1999-2000 formulations). For analysis, study groups were subdivided into adult (18-60 years old) and elderly (over 60 years) subjects. Blood was drawn immediately before and one month after vaccination, safety was evaluated using a blind-observer design based on reporting of solicited and unsolicited adverse ev...

  19. Phylogenetic position of the genus Perkinsus (Protista, Apicomplexa) based on small subunit ribosomal RNA.

    Science.gov (United States)

    Goggin, C L; Barker, S C

    1993-07-01

    Parasites of the genus Perkinsus destroy marine molluscs worldwide. Their phylogenetic position within the kingdom Protista is controversial. Nucleotide sequence data (1792 bp) from the small subunit rRNA gene of Perkinsus sp. from Anadara trapezia (Mollusca: Bivalvia) from Moreton Bay, Queensland, was used to examine the phylogenetic affinities of this enigmatic genus. These data were aligned with nucleotide sequences from 6 apicomplexans, 3 ciliates, 3 flagellates, a dinoflagellate, 3 fungi, maize and human. Phylogenetic trees were constructed after analysis with maximum parsimony and distance matrix methods. Our analyses indicate that Perkinsus is phylogenetically closer to dinoflagellates and to coccidean and piroplasm apicomplexans than to fungi or flagellates. PMID:8366895

  20. Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis

    OpenAIRE

    Yang, Qingzhu; Yu, Kun; Yan, Liming; Li, Yuanyuan; Chen, Cheng; Li, Xuemei

    2011-01-01

    The aspartate kinase (AK) from Mycobacterium tuberculosis (Mtb) catalyzes the biosynthesis of aspartate family amino acids, including lysine, threonine, isoleucine and methionine. We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone (referred to as MtbAKβ) and in complex with threonine (referred to as MtbAKβ-Thr) at resolutions of 2.6 Å and 2.0 Å, respectively. MtbAKβ is composed of two perpendicular non-equivalent ACT domains [aspartate kinase, ch...

  1. Characterization of the gene encoding the hemocyanin subunit e from the tarantula Eurypelma californicum.

    OpenAIRE

    Voll, W; Voit, R

    1990-01-01

    The gene for the hemocyanin subunit e of the tarantula Eurypelma californicum has been isolated from a genomic phage library by using a corresponding cDNA clone as a probe. The transcriptional unit spans a chromosomal region of about 55 kilobase pairs (kbp). The gene consists of nine exons that are separated by large introns. The intron/exon boundaries were determined by direct comparison of genomic and cDNA sequences. A putative promoter region ("TATA" box, reversed "CAAT" box) 100 bp 5' to ...

  2. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    OpenAIRE

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein se...

  3. Cloning and Expression of Beta Subunit Gene of Phycocyanin From Spirulina platensis in Escherichia coli

    OpenAIRE

    Shoja, Zahra; Rajabi Memari, Hamid; Roayaei Ardakani, Mohammd

    2015-01-01

    Background: C-Phycocyanin (C-PC) from blue-green algae such as Spirulina has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-tumor activities. Recombinant β-subunit of C-PC (C-PC/β) is an inhibitor of cell proliferation and an inducer of cancer cell apoptosis. Objectives: Since C-PC/β has a big potential to be used as a promising cancer prevention or therapy agent, the purpose of this study was to clone and express Spirulina platensis cpcB ge...

  4. An immune stimulating complex (iscom) subunit rabies vaccine protects dogs and mice against street rabies challenge.

    OpenAIRE

    Fekadu, M.; Schaddock, J.H.; Ekström, J.; Osterhaus, Ab; Sanderlin, D W; Sundquist, B; Morein, Bror

    1992-01-01

    textabstractDogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two intraperitoneal (i.p.) doses of 360 ng ISCOM or 0.5 ml HDCV protected 95% (38/40) and 90% (36/40) of mice, respectively, against a lethal intracerebral (i.c.) dose with challenge virus strain (CVS). On...

  5. Absence of prostaglandin E2-induced hyperalgesia in NMDA receptor ε subunit knockout mice

    OpenAIRE

    Minami, Toshiaki; Sugatani, Junko; Sakimura, Kenji; Abe, Manabu; Mishina, Masayoshi; Ito, Seiji

    1997-01-01

    We have previously found that intrathecal administration of prostaglandins E2 (PGE2) and D2 (PGD2) into conscious mice induced hyperalgesia by the hot plate test. The present study investigated the involvement of N-methyl-D-aspartate (NMDA) receptor in the prostaglandin-induced hyperalgesia by use of mice lacking NMDA receptor ε1, ε4, or ε1/ε4 subunits.PGE2 induced hyperalgesia over a wide range of doses from 50 pg to 500 ng kg−1 in wild-type mice. But PGE2 could not induce hyperalgesia in ε1...

  6. Multiple Roles of the τ131 Subunit of Yeast Transcription Factor IIIC (TFIIIC) in TFIIIB Assembly

    OpenAIRE

    Dumay-Odelot, Hélène; Acker, Joël; Arrebola, Rosalia; Sentenac, André; Marck, Christian

    2002-01-01

    Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, τ131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (τ131-ΔTPR2) harboring a deletion in τ131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in v...

  7. The casein kinase II beta subunit binds to Mos and inhibits Mos activity.

    OpenAIRE

    Chen, M.; D. Li; Krebs, E G; Cooper, J. A.

    1997-01-01

    Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation. Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK). We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos. The Mos-interacting region of CKII beta was mapped to the C terminus. Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta a...

  8. Thermodynamic analysis reveals that GTP binding affects the interaction between the α- and γ-subunits of translation initiation factor 2

    International Nuclear Information System (INIS)

    Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔCp) values. The ΔH and ΔCp values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA

  9. Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

    Science.gov (United States)

    Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

  10. Effects of Exposure to Aluminum on Long-term Potentiation and AMPA Receptor Subunits in Ratsin vivo

    Institute of Scientific and Technical Information of China (English)

    SONG Jing; LIU Ying; ZHANG Hui Fang; ZHANG Qin Li; NIU Qiao

    2014-01-01

    ObjectiveTo explore the effects of exposure to aluminum(Al) on long-term potentiation(LTP) and AMPA receptor subunits in rats in vivo. MethodsDifferent dosages of aluminum-maltolate complex[Al(mal)3] were given to rats via acute intracerebroventricular (i.c.v.)injection and subchronic intraperitoneal (i.p.) injection. Following Al exposure, the hippocampal LTP were recorded by field potentiation techniquein vivo and the expression of AMPAR subunit proteins (GluR1 and GluR2) in both total and membrane-enriched extracts from the CA1 area of rat hippocampus were detected by Western blot assay. ResultsAcute Al treatment produced dose-dependent suppression of LTP in the rat hippocampus and dose-dependent decreases of GluR1and GluR2in membrane extracts; however, no similar changes were found in the total cell extracts, which suggests decreased trafficking of AMPA receptor subunits from intracellular pools to synaptic sites in the hippocampus. Thedose-dependent suppressive effects on LTP and the expression of AMPA receptor subunits both in the membrane and in total extracts were found after subchronic Al treatment, indicating a decrease in AMPA receptor subunit trafficking from intracellular poolsto synaptic sites and an additional reduction in the expression of the subunits. ConclusionAl(mal)3obviously and dose-dependently suppressed LTP in the rat hippocampal CA1 region in vivo, and this suppression may be related to both trafficking and decreases in the expression of AMPA receptor subunit proteins. However, the mechanisms underlying these observations need further investigation.

  11. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa.

    Directory of Open Access Journals (Sweden)

    Christen M Klinger

    Full Text Available Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs, factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during

  12. Targeting the γ-Aminobutyric Acid A Receptor α4 Subunit in Airway Smooth Muscle to Alleviate Bronchoconstriction.

    Science.gov (United States)

    Yocum, Gene T; Gallos, George; Zhang, Yi; Jahan, Rajwana; Stephen, Michael Rajesh; Varagic, Zdravko; Puthenkalam, Roshan; Ernst, Margot; Cook, James M; Emala, Charles W

    2016-04-01

    We previously demonstrated that airway smooth muscle (ASM) cells express γ-aminobutyric acid A receptors (GABAARs), and that GABAAR agonists acutely relax ASM. Among the GABAAR α subunits, human ASM cells express only α4 and α5, providing the opportunity for selective pharmacologic targeting. Novel GABAAR-positive allosteric modulators designed for enhanced α4/α6 subunit selectivity were synthesized using iterative computational analyses (CMD-45 and XHe-III-74). Studies using oocyte heterologous expression systems confirmed that CMD-45 and XHe-III-74 led to significantly greater augmentation of currents induced by a 3% maximal effective concentration (EC3) of GABA [EC3]-induced currents in oocytes expressing α4 or α6 subunits (along with β3 and γ2) compared with other α subunits. CMD-45 and XHe-III-74 also led to greater ex vivo relaxation of contracted wild-type mouse tracheal rings compared with tracheal rings from GABAAR α4 subunit (Gabra4) knockout mice. Furthermore, CMD-45 and XHe-III-74 significantly relaxed precontracted human ASM ex vivo, and, at a low concentration, both ligands led to a significant leftward shift in albuterol-mediated ASM relaxation. In vivo, inhaled XHe-III-74 reduced respiratory system resistance in an asthmatic mouse model. Pretreatment of human ASM cells with CMD-45 and XHe-III-74 inhibited histamine-induced increases in intracellular calcium concentrations in vitro, an effect that was lost when calcium was omitted from the extracellular buffer, suggesting that inhibition of calcium influx due to alterations in plasma membrane potential may play a role in the mechanism of ASM relaxation. Selective targeting of the GABAAR α4 subunit with inhaled ligands may be a novel therapeutic pathway to treat bronchoconstriction, while avoiding sedative central nervous system effects, which are largely mediated by α1-3 subunit-containing GABAARs in the brain. PMID:26405827

  13. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  14. High-Molecular-Weight Glutenin Subunit Composition of Chinese Wheat Germplasm

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ling-li; LI Xiu-quan; YANG Xin-ming; LI Hong-jie; WANG Hui; LI Li-hui

    2007-01-01

    The objective of the present study was to characterize the high molecular glutenin subunits (HMW-GS) composition and the presence of 1B/1R translocation in newly developed wheat (Triticum aestivum L.) germplasm, which have one or more traits that are useful in wheat improvement. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE)and acid polyacrylamide-gel electrophoresis (A-PAGE) were used to detect HMW-GS composition and the presence of 1B/1R wheat-rye (Secale cereale L.) chromosome translocation in the wheat germplasm. Bread-making quality scores of these lines were determined. A high level of variations in HMW-GS encoded by Glu-1 locus was observed. Sixteen major HMW-GS, with 30 combinations, were detected. The percentage of cultivars with more than two desirable subunits was 38.7%. Thirteen cultivars had bread-making quality scores of 10 in combination with one or two desirable agronomical traits, such as high-yield potential, dwarfing stem, resistance to diseases, and/or tolerance to abiotic stress. Sixty-eight (36.6%) cultivars possessed 1B/1R translocation. The newly developed germplasm with HMW-GS for good quality can be promising resources for improving bread-making quality of wheat.

  15. NDH-1L interacts with ferredoxin via the subunit NdhS in Thermosynechococcus elongatus.

    Science.gov (United States)

    He, Zhihui; Zheng, Fangfang; Wu, Yaozong; Li, Qinghua; Lv, Jing; Fu, Pengcheng; Mi, Hualing

    2015-12-01

    The large size complex of cyanobacterial NAD(P)H dehydrogenase (NDH-1) complex (NDH-1L) plays crucial role in a variety of bioenergetic reactions such as respiration and cyclic electron flow around photosystem I. Although the complex has been isolated and identified, its biochemical function still remains to be clarified. Here, we highly purified the NDH-1L complex from the cells of Thermosynechococcus elongatus by Ni(2+) affinity chromatography and size-exclusion chromatography. The purified NDH-1L complex has an apparent total molecular mass of approximately 500 kDa. 14 known subunits were identified by mass spectrometry and immunoblotting, including the NdhS subunit containing ferredoxin (Fd)-docking site domain. Surface plasmon resonance measurement demonstrates that the NDH-1L complex could bind to Fd with the binding constant (K D) of 59 µM. Yeast two-hybrid system assay further confirmed the interaction of Fd with NdhS and indicated that NdhH is involved in the interaction. Our results provide direct biochemical evidence that the cyanobacterial NDH-1 complex catalyzes the electron transport from reduced Fd to plastoquinone via NdhS and NdhH. PMID:25630976

  16. Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

    Science.gov (United States)

    Kleyman, T R; Zuckerman, J B; Middleton, P; McNulty, K A; Hu, B; Su, X; An, B; Eaton, D C; Smith, P R

    2001-08-01

    The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability. PMID:11457713

  17. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  18. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    Science.gov (United States)

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra

    2008-10-15

    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  19. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Science.gov (United States)

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  20. Role of calmodulin (δ-subunit) in activation of phosphorylase kinase from rabbit skeletal muscles

    International Nuclear Information System (INIS)

    The structure of the inactivated and activated forms of phospholyase kinase was compared. The enzyme was activated by incubation in an alkaline medium (pH 8.5), phosphorylation of the catalytic subunit of cAMP-dependent protein kinase, and limited proteolysis. Hydrophobic chromatography on phenyl-Sepharose and electrophoresis in a polyacrylamide gel density gradient were employed for a comparison of these forms of the enzyme. Activation of the enzyme was accompanied by the separation of a low-molecular-weight component (M/sub r/ about 17,000). The low-molecular-weight protein was obtained in a homogeneous state by chromatography on phenyl-Sepharose. It was established that its properties are similar to those of calmodulin. The presence of calmodulin in preparations of phosphorylase kinase was judged by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in much the same way as bovine brain calmodulin. The results obtained suggest that the δ-subunit is a protein inhibitor of the enzyme

  1. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  2. Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Zhengui Zheng

    Full Text Available Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18 affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC and AGAMOUS (AG. A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

  3. Sucrose regulation of ADP-glucose pyrophosphorylase subunit genes transcript levels in leaves and fruits

    Science.gov (United States)

    Li, Xiangyang; Xing, Jinpeng; Gianfagna, Thomas J.; Janes, Harry W.

    2002-01-01

    ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) is a key regulatory enzyme in starch biosynthesis. The enzyme is a heterotetramer with two S and two B subunits. In tomato, there are three multiple forms of the S subunit gene. Agp S1, S2 and B are highly expressed in fruit from 10 to 25 days after anthesis. Agp S3 is only weakly expressed in fruit. Sucrose significantly elevates expression of Agp S1, S2 and B in both leaves and fruits. Agp S1 exhibits the highest degree of regulation by sucrose. In fact, sucrose may be required for Agp S1 expression. For excised leaves incubated in water, no transcripts for Agp S1 could be detected in the absence of sucrose, whereas it took up to 16 h in water before transcripts were no longer detectable for Agp S2 and B. Neither Agp S3 nor the tubulin gene is affected by sucrose, demonstrating that this response is specifically regulated by a carbohydrate metabolic signal, and is not due to a general increase in metabolism caused by sucrose treatment. Truncated versions of the promoter for Agp S1 indicate that a specific region 1.3-3.0 kb upstream from the transcription site is responsible for sucrose sensitivity. This region of the S1 promoter contains several cis-acting elements present in the promoters of other genes that are also regulated by sucrose. c2002 Elsevier Science Ireland Ltd. All rights reserved.

  4. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

    Science.gov (United States)

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

    2002-01-01

    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  5. A link between the cytoplasmic engulfment protein Elmo1 and the Mediator complex subunit Med31.

    Science.gov (United States)

    Mauldin, Joshua P; Lu, Mingjian; Das, Soumita; Park, Daeho; Ernst, Peter B; Ravichandran, Kodi S

    2013-01-21

    The cytoplasmic Elmo1:Dock180 complex acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac and functions downstream of the phagocytic receptor BAI1 during apoptotic cell clearance, and in the entry of Salmonella and Shigella into cells. We discovered an unexpected binding between Elmo1 and the Mediator complex subunit Med31. The Mediator complex is a regulatory hub for nearly all gene transcription via RNA polymerase II, bridging the general transcription machinery with gene-specific regulatory proteins. Med31 is the smallest and the most evolutionarily conserved Mediator subunit, and knockout of Med31 results in embryonic lethality in mice; however, Med31 function in specific biological contexts is poorly understood. We observed that in primary macrophages, during Salmonella infection, Elmo1 and Med31 specifically affected expression of the cytokine genes Il10 and Il33 among the >25 genes monitored. Although endogenous Med31 is predominantly nuclear localized, Elmo1 increased the cytoplasmic localization of Med31. We identify ubiquitination as a novel posttranslational modification of Med31, with the cytoplasmic monoubiquitinated form of Med31 being enhanced by Elmo1. These data identify Elmo1 as a novel regulator of Med31, revealing a previously unrecognized link between cytoplasmic signaling proteins and the Mediator complex. PMID:23273896

  6. Evolutionary paths of the cAMP-dependent protein kinase (PKA catalytic subunits.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available 3',5'-cyclic adenosine monophosphate (cAMP dependent protein kinase or protein kinase A (PKA has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

  7. Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

    Science.gov (United States)

    Hocky, Glen M; Baker, Joseph L; Bradley, Michael J; Sinitskiy, Anton V; De La Cruz, Enrique M; Voth, Gregory A

    2016-05-26

    Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

  8. Mutagenesis of Ser24 of cytochrome b559 α subunit affects PSⅡ activities in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    MA JingJing; LI LiangBi; JING YuXiang; KUANG TingYun

    2007-01-01

    In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSⅡ) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in c subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSⅡ activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSll (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore,SDS-PAGE and Western-blotting analysis indicated that the expression levels of c subunit of Cyt b559,LHCⅡ and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSⅡ complex activity of oxygen evolution although it is not directly bound to heme group.

  9. A randomized controlled trial comparing split and subunit influenza vaccines in adults in Colombia.

    Science.gov (United States)

    Morales, Alvaro; Arias Salazar, Javier; Salazar, Yolanda; García, Albert; Arnoux, Sabine; Arancibia, Andrea; Deroche, Christèle; Rey, Elena

    2003-01-01

    In a two-center, comparative trial, 344 adults were randomly assigned to receive a single dose of inactivated split-virion (Imovax Gripe) or sub-unit (Agrippal S1) influenza vaccine (1999-2000 formulations). For analysis, study groups were subdivided into adult (18-60 years old) and elderly (over 60 years) subjects. Blood was drawn immediately before and one month after vaccination, safety was evaluated using a blind-observer design based on reporting of solicited and unsolicited adverse events. Both vaccines were very well tolerated, had similar reactogenicity profiles, and elicited fewer reports of reactions in elderly individuals. Post-vaccination Imovax Gripe induced seroprotective antibody titers against the three vaccine strains in 94-99% of adults and 88-97% of elderly subjects, compared with 88-100% and 88-98%, respectively, of those given Agrippal S1. In conclusion, the split-virion and sub-unit influenza vaccines had similar safety and reactogenicity profiles, and elicited satisfactory immunity in adult and elderly subjects. However, higher post-vaccination geometric mean titer (GMT) values in response to the B strain were seen with the split vaccine Imovax Gripe, giving it a better overall immunogenicity. PMID:12876902

  10. Structural view of the regulatory subunit of aspartate kinase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Yang, Qingzhu; Yu, Kun; Yan, Liming; Li, Yuanyuan; Chen, Cheng; Li, Xuemei

    2011-09-01

    The aspartate kinase (AK) from Mycobacterium tuberculosis (Mtb) catalyzes the biosynthesis of aspartate family amino acids, including lysine, threonine, isoleucine and methionine. We determined the crystal structures of the regulatory subunit of aspartate kinase from Mtb alone (referred to as MtbAKβ) and in complex with threonine (referred to as MtbAKβ-Thr) at resolutions of 2.6 Å and 2.0 Å, respectively. MtbAKβ is composed of two perpendicular non-equivalent ACT domains [aspartate kinase, chorismate mutase, and TyrA (prephenate dehydrogenase)] per monomer. Each ACT domain contains two α helices and four antiparallel β strands. The structure of MtbAKβ shares high similarity with the regulatory subunit of the aspartate kinase from Corynebacterium glutamicum (referred to as CgAKβ), suggesting similar regulatory mechanisms. Biochemical assays in our study showed that MtbAK is inhibited by threonine. Based on crystal structure analysis, we discuss the regulatory mechanism of MtbAK. PMID:21976064

  11. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Juan [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Microbiology and Immunology, Nanjing Medical University (China); Wang, Shixia [China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States); Gan, Weihua [Department of Pediatrics, The Second Affiliated Hospital, Nanjing Medical University (China); Zhang, Wenhong [Department of Infectious Diseases, Huashan Hospital, Fudan University (China); Ju, Liwen [School of Public Health, Fudan University (China); Huang, Zuhu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Lu, Shan, E-mail: shan.lu@umassmed.edu [Department of Infectious Diseases, The First Affiliated Hospital, Nanjing Medical University (China); China-US Vaccine Research Center, The First Affiliated Hospital, Nanjing Medical University (China); Department of Medicine, University of Massachusetts Medical School (United States)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer EV71 is a major emerging infectious disease in many Asian countries. Black-Right-Pointing-Pointer Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. Black-Right-Pointing-Pointer Developing subunit based EV71 vaccines is significant and novel antigen design is needed. Black-Right-Pointing-Pointer DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. Black-Right-Pointing-Pointer Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  12. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

    Energy Technology Data Exchange (ETDEWEB)

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J.G.; Sunahara, Roger K. (Michigan)

    2012-03-15

    No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

  13. Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD

    Energy Technology Data Exchange (ETDEWEB)

    Barbieri, J.T.; Mende-Mueller, L.M.; Rappuoli, R.; Collier, R.J. (Medical College of Wisconsin, Milwaukee (USA))

    1989-11-01

    UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from (3H-nicotinamide)NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from (32P-adenylate)NAD (0.2 mol/mol of protein). Label from (3H-nicotinamide)NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with (3H-nicotinamide)NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.

  14. Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens

    International Nuclear Information System (INIS)

    Highlights: ► EV71 is a major emerging infectious disease in many Asian countries. ► Inactivated EV71 vaccines are in clinical studies but their safety and efficacy are unknown. ► Developing subunit based EV71 vaccines is significant and novel antigen design is needed. ► DNA immunization is an efficient tool to test the immunogenicity of VP1 based EV71 vaccines. ► Multiple VP1 antigens are developed showing immunogenic potential. -- Abstract: Hand, foot, and mouth disease (HFMD) is a common viral illness in young children. HFMD is caused by viruses belonging to the enterovirus genus of the picornavirus family. Recently, enterovirus 71 (EV71) has emerged as a virulent agent for HFMD with severe clinical outcomes. In the current report, we conducted a pilot antigen engineering study to optimize the expression and immunogenicity of subunit VP1 antigen for the design of EV71 vaccines. DNA immunization was adopted as a simple technical approach to test different designs of VP1 antigens without the need to express VP1 protein in vitro first. Our studies indicated that the expression and immunogenicity of VP1 protein can be improved with alternated VP1 antigen designs. Data presented in the current report revealed novel pathways to optimize the design of VP1 antigen-based EV71 vaccines.

  15. Quality-control method for the determination of biological activity of engineered calcineurin subunit B.

    Science.gov (United States)

    Shi, Xinchang; Yang, Huan; Xu, Li; Li, Xiang; Huang, Zongwen; Han, Yudong; Wei, Qun; Rao, Chunming

    2016-06-01

    The aim of this study was to establish a quality-control method for calcineurin subunit B (CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate (pNPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant (CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding pNPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05-50 μg mL(-1), with sensitivity of 50 μg mL(-1). Tested cytokines did not induce CNAΔ316 dephosphorylation of pNPP. The chosen CNAΔ316 concentration range did not affect activity determinations. PMID:27053126

  16. Tie2-dependent deletion of α6 integrin subunit in mice reduces tumor growth and angiogenesis.

    Science.gov (United States)

    Bouvard, Claire; Segaoula, Zacharie; De Arcangelis, Adèle; Galy-Fauroux, Isabelle; Mauge, Laetitia; Fischer, Anne-Marie; Georges-Labouesse, Elisabeth; Helley, Dominique

    2014-11-01

    The α6 integrin subunit (α6) has been implicated in cancer cell migration and in the progression of several malignancies, but its role in tumor angiogenesis is unclear. In mice, anti-α6 blocking antibodies reduce tumor angiogenesis, whereas Tie1-dependent α6 gene deletion enhances neovessel formation in melanoma and lung carcinoma. To clarify the discrepancy in these results we used the cre-lox system to generate a mouse line, α6fl/fl‑Tie2Cre(+), with α6 gene deletion specifically in Tie2-lineage cells: endothelial cells, pericytes, subsets of hematopoietic stem cells, and Tie2-expressing monocytes/macrophages (TEMs), known for their proangiogenic properties. Loss of α6 expression in α6fl/fl‑Tie2Cre(+) mice reduced tumor growth in a murine B16F10 melanoma model. Immunohistological analysis of the tumors showed that Tie2-dependent α6 gene deletion was associated with reduced tumor vascularization and with reduced infiltration of proangiogenic Tie2-expressing macrophages. These findings demonstrate that α6 integrin subunit plays a major role in tumor angiogenesis and TEM infiltration. Targeting α6 could be used as a strategy to reduce tumor growth. PMID:25176420

  17. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    Full Text Available BACKGROUND: The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  18. Evaluation of quality and identity of wheat genotypes on basis of high molecular weight glutenin subunit composition and morphological description

    International Nuclear Information System (INIS)

    Ten spring wheat genotypes were analyzed for high molecular weight glutenin subunit (HMW-GS) composition. The glutenin subunits as revealed by sodium de decyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were numbered according to Payne's numbering system and genotypes were assigned theoretical quality scores accordingly. All genotypes were found to possess either subunit 1 or 2 coded by the A genome. Subunit 7+9 of the B genome and 5+10 of the D genome were found predominantly in this set of genotypes. The composition of HMG-GS were of good quality since all except open genotype had quality scores of 9 or 10 with a high (9.2) average score. The results in this study indicated that wheat genotypes bred in Pakistan had a narrow genetic base in terms of HMW glutenin subunits. On the basis of HMW-GS, two genotypes were completely discriminated whereas, remaining separated into 3 groups. However, all genotypes were distinguishable from each other when HMW-GS composition was used in combination with morphological and grain characters. (author)

  19. Two Chlamydomonas OPR proteins stabilize chloroplast mRNAs encoding small subunits of photosystem II and cytochrome b6 f.

    Science.gov (United States)

    Wang, Fei; Johnson, Xenie; Cavaiuolo, Marina; Bohne, Alexandra-Viola; Nickelsen, Joerg; Vallon, Olivier

    2015-06-01

    In plants and algae, chloroplast gene expression is controlled by nucleus-encoded proteins that bind to mRNAs in a specific manner, stabilizing mRNAs or promoting their splicing, editing, or translation. Here, we present the characterization of two mRNA stabilization factors of the green alga Chlamydomonas reinhardtii, which both belong to the OctotricoPeptide Repeat (OPR) family. MCG1 is necessary to stabilize the petG mRNA, encoding a small subunit of the cytochrome b6 f complex, while MBI1 stabilizes the psbI mRNA, coding for a small subunit of photosystem II. In the mcg1 mutant, the small RNA footprint corresponding to the 5'-end of the petG transcript is reduced in abundance. In both cases, the absence of the small subunit perturbs assembly of the cognate complex. Whereas PetG is essential for formation of a functional cytochrome b6 f dimer, PsbI appears partly dispensable as a low level of PSII activity can still be measured in its absence. Thus, nuclear control of chloroplast gene expression is not only exerted on the major core subunits of the complexes, but also on small subunits with a single transmembrane helix. While OPR proteins have thus far been involved in translation or trans-splicing of plastid mRNAs, our results expand the potential roles of this repeat family to their stabilization. PMID:25898982

  20. Do the A subunits contribute to the differences in the toxicity of Shiga toxin 1 and Shiga toxin 2?

    Science.gov (United States)

    Basu, Debaleena; Tumer, Nilgun E

    2015-05-01

    Shiga toxin producing Escherichia coli O157:H7 (STEC) is one of the leading causes of food-poisoning around the world. Some STEC strains produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or variants of either toxin, which are critical for the development of hemorrhagic colitis (HC) or hemolytic uremic syndrome (HUS). Currently, there are no therapeutic treatments for HC or HUS. E. coli O157:H7 strains carrying Stx2 are more virulent and are more frequently associated with HUS, which is the most common cause of renal failure in children in the US. The basis for the increased potency of Stx2 is not fully understood. Shiga toxins belong to the AB5 family of protein toxins with an A subunit, which depurinates a universally conserved adenine residue in the α-sarcin/ricin loop (SRL) of the 28S rRNA and five copies of the B subunit responsible for binding to cellular receptors. Recent studies showed differences in the structure, receptor binding, dependence on ribosomal proteins and pathogenicity of Stx1 and Stx2 and supported a role for the B subunit in differential toxicity. However, the current data do not rule out a potential role for the A1 subunits in the differential toxicity of Stx1 and Stx2. This review highlights the recent progress in understanding the differences in the A1 subunits of Stx1 and Stx2 and their role in defining toxicity. PMID:25938272