Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

PubMed

Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

2018-06-08

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Peptide Regulation of Cells Renewal Processes in Kidney Tissue Cultures from Young and Old Animals.

PubMed

Chalisova, N I; Lin'kova, N S; Nichik, T E; Ryzhak, A P; Dudkov, A V; Ryzhak, G A

2015-05-01

Polypeptide complex isolated from calf kidneys stimulates the processes of cell renewal in organotypic kidney tissue cultures from young and old rats. The polypeptide complex enhances expression of proliferation marker Ki-67 and reduces expression of proapoptotic peptide p53 in kidney explants obtained from young and old animals. Short peptides T-31 (AED) and T-35 (EDL) also stimulate proliferation and reduce apoptosis of the kidney cells, but to a lesser degree than the polypeptide complex. The results provide the basis for further investigation of the polypeptide complex as a preparation for the therapy of kidney diseases, including age-related pathologies.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

PubMed

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils.

PubMed

Sikirzhytski, Vitali; Topilina, Natalya I; Higashiya, Seiichiro; Welch, John T; Lednev, Igor K

2008-05-07

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.

Enhanced processive cellulases

DOEpatents

Adney, William S.; Beckham, Gregg T.; Jarvis, Eric; Himmel, Michael E.; Decker, Stephen R.; Linger, Jeffrey G.; Podkaminer, Kara; Baker, John O.; Taylor, II, Larry; Xu, Qi; Singh, Arjun

2017-06-20

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not in the trigeminal nucleus caudalis, and no significant differences were found in the expression of the VPAC1 and VPAC2 receptors. Conclusions This study demonstrated the chronic alteration of pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in the rat, which suggests the crucial involvement of pituitary adenylate cyclase-activating polypeptide in the development of migraine. The selective increase in pituitary adenylate cyclase-activating polypeptide-related receptors suggests that the PAC1 receptor pathway is a novel target for the treatment of migraine.

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

PubMed Central

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

1972-01-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

NASA Astrophysics Data System (ADS)

Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

2014-09-01

A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03058c

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, E.

1998-12-15

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed. 5 figs.

Use of linalool synthase in genetic engineering of scent production

DOEpatents

Pichersky, Eran

1998-01-01

A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha; Schultz, Peter G.

2013-01-22

The invention related to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural acids at genetically-programmed positions.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2011-06-14

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Systems for the expression of orthogonal translation components eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2012-06-12

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

PubMed Central

Sebastian, Katrin; Detro-Dassen, Silvia; Rinis, Natalie; Fahrenkamp, Dirk; Müller-Newen, Gerhard; Merk, Hans F.; Schmalzing, Günther

2013-01-01

Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration. PMID:24376674

Compositions and methods for making selenocysteine containing polypeptides

DOEpatents

Soll, Dieter; Aldag, Caroline; Hohn, Michael

2016-10-11

Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

A single aromatic core mutation converts a designed “primitive” protein from halophile to mesophile folding

PubMed Central

Longo, Liam M; Tenorio, Connie A; Kumru, Ozan S; Middaugh, C Russell; Blaber, Michael

2015-01-01

The halophile environment has a number of compelling aspects with regard to the origin of structured polypeptides (i.e., proteogenesis) and, instead of a curious niche that living systems adapted into, the halophile environment is emerging as a candidate “cradle” for proteogenesis. In this viewpoint, a subsequent halophile-to-mesophile transition was a key step in early evolution. Several lines of evidence indicate that aromatic amino acids were a late addition to the codon table and not part of the original “prebiotic” set comprising the earliest polypeptides. We test the hypothesis that the availability of aromatic amino acids could facilitate a halophile-to-mesophile transition by hydrophobic core-packing enhancement. The effects of aromatic amino acid substitutions were evaluated in the core of a “primitive” designed protein enriched for the 10 prebiotic amino acids (A,D,E,G,I,L,P,S,T,V)—having an exclusively prebiotic core and requiring halophilic conditions for folding. The results indicate that a single aromatic amino acid substitution is capable of eliminating the requirement of halophile conditions for folding of a “primitive” polypeptide. Thus, the availability of aromatic amino acids could have facilitated a critical halophile-to-mesophile protein folding adaptation—identifying a selective advantage for the incorporation of aromatic amino acids into the codon table. PMID:25297559

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili.

PubMed

Dougan, G; Dowd, G; Kehoe, M

1983-01-01

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.

Facile preparation of well-defined AB2 Y-shaped miktoarm star polypeptide copolymer via the combination of ring-opening polymerization and click chemistry.

PubMed

Rao, Jingyi; Zhang, Yanfeng; Zhang, Jingyan; Liu, Shiyong

2008-10-01

Well-defined AB2 Y-shaped miktoarm star polypeptide copolymer, PZLL-b-(PBLG)2, was synthesized via a combination of ring-opening polymerization (ROP) of alpha-amino acid N-carboxyanhydride (NCA) and click chemistry, where PZLL is poly(epsilon-benzyloxycarbonyl-L-lysine) and PBLG is poly(gamma-benzyl-L-glutamate). First, two types of primary-amine-containing initiators, N-aminoethyl 3,5-bis(propargyloxyl)-benzamide and 3-azidopropylamine, were synthesized and employed for the ROP of NCA, leading to the formation of dialkynyl-terminated PZLL and azide-terminated PBLG, dialkynyl-PZLL and PBLG-N3, respectively. The subsequent copper(I)-catalyzed cycloaddition reaction between dialkynyl-PZLL and slightly excess PBLG-N3 led to facile preparation of PZLL-b-(PBLG)2 Y-shaped miktoarm star polypeptide copolymer. The excess PBLG-N3 was scavenged off by reacting with alkynyl-functionalized Wang resin. The obtained Y-shaped miktoarm star polypeptide copolymer was characterized by gel permeation chromatograph (GPC), Fourier transform-infrared spectroscopy (FT-IR), and (1)H NMR. Moreover, after the hydrolysis of protecting benzyl and benzyloxycarbonyl groups of PZLL-b-(PBLG)2, water-soluble pH-responsive Y-shaped miktoarm star polypeptide copolymer, PLL-b-(PLGA)2, was obtained, where PLL is poly(L-lysine) and PLGA is poly(L-glutamic acid). It can self-assemble into PLGA-core micelles at acidic pH and PLL-core micelles at alkaline pH, accompanied with the coil-to-helix transition of PLGA and PLL sequences, respectively. The spontaneous pH-responsive supramolecular assembly of PLL-b-(PLGA)2 miktoarm star polypeptide copolymer has been investigated via a combination of (1)H NMR, laser light scattering (LLS), transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

1998-10-13

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

1998-01-01

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous gene

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2007-03-20

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Recombinant cells that highly express chromosomally-integrated heterologous genes

DOEpatents

Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

2000-08-22

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

Signal transduction by the Wnt family of ligands.

PubMed Central

Dale, T C

1998-01-01

The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in diverse developmental processes. The loss or inappropriate activation of Wnt expression has been shown to alter cell fate, morphogenesis and mitogenesis. Recent progress has identified Wnt receptors and components of an intracellular signalling pathway that mediate Wnt-dependent transcription. This review will highlight this 'core' Wnt signal-transduction pathway, but also aims to reveal the potential diversity of Wnt signalling targets. Particular attention will be paid to the overlap between developmental biology and oncogenesis, since recent progress shows Wnt signalling forms a paradigm for an interdisciplinary approach. PMID:9425102

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

Facilitating protein solubility by use of peptide extensions

DOEpatents

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Dock 'n roll: folding of a silk-inspired polypeptide into an amyloid-like beta solenoid.

PubMed

Zhao, Binwu; Cohen Stuart, Martien A; Hall, Carol K

2016-04-20

Polypeptides containing the motif ((GA)mGX)n occur in silk and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two hydrophobic shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II' β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for molecules to reach the folded state.

Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

PubMed

Pechmann, Sebastian; Frydman, Judith

2013-02-01

The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs1[w

PubMed Central

Mergaert, Peter; Nikovics, Krisztina; Kelemen, Zsolt; Maunoury, Nicolas; Vaubert, Danièle; Kondorosi, Adam; Kondorosi, Eva

2003-01-01

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60–90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed. PMID:12746522

Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli.

PubMed Central

Neidhardt, F C; VanBogelen, R A; Lau, E T

1983-01-01

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon. Images PMID:6337122

Inhibition of vaccinia virus maturation by zinc chloride.

PubMed Central

Katz, E; Margalith, E

1981-01-01

Zinc chloride (0.1 mM) inhibited by 96.4% the growth of vaccinia virus in HeLa cells. Approximately 50% inhibition in formation of particles that sedimented in sucrose gradients similarly to vaccinia virions occurred in the presence of zinc ions. Whereas the synthesis of the viral deoxyribonucleic acid was not affected by zinc chloride, a decrease in the overall synthesis of viral polypeptides and inhibition of the cleavage of precursors to the core polypeptides were observed. Images PMID:7347557

Design and preparation of beta-sheet forming repetitive and block-copolymerized polypeptides.

PubMed

Higashiya, Seiichiro; Topilina, Natalya I; Ngo, Silvana C; Zagorevskii, Dmitri; Welch, John T

2007-05-01

The design and rapid construction of libraries of genes coding beta-sheet forming repetitive and block-copolymerized polypeptides bearing various C- and N-terminal sequences are described. The design was based on the assembly of DNA cassettes coding for the (GA)3GX amino acid sequence where the (GAGAGA) sequences would constitute the beta-strand units of a larger beta-sheet assembly. The edges of this beta-sheet would be functionalized by the turn-inducing amino acids (GX). The polypeptides were expressed in Escherichia coli using conventional vectors and were purified by Ni-nitriloacetic acid (NTA) chromatography. The correlation of polymer structure with molecular weight was investigated by gel electrophoresis and mass spectrometry. The monomer sequences and post-translational chemical modifications were found to influence the mobility of the polypeptides over the full range of polypeptide molecular weights while the electrophoretic mobility of lower molecular weight polypeptides was more susceptible to C- and N-termini polypeptide modifications.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Plants with elevated levels of glucan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pauly, Markus; Kraemer, Florian J.; Hake, Sarah

The present disclosure relates to mutations in licheninase genes encoding polypeptides with decreased licheninase activity, which when expressed in plants results in elevated levels of glucan in the plants. In particular, the disclosure relates to licheninase nucleic acids and polypeptides related to glucan accumulation in plants, plants with reduced expression of a licheninase nucleic acid, and methods related to the generation of plants with increased glucan content in the cell walls of leaf tissue.

Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

DOEpatents

Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

2010-04-20

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J.; Dahlbacka, Glen; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser; Ellanskaya, deceased, Irina

2007-12-11

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J.; Dahlbacka, Glen; Elleskaya, Irina; Ellanskaya, legal representative; Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

2010-08-10

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J [Waukee, IA; Dahlbacka, Glen [Oakland, CA; Elleskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, IA; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

2011-04-12

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Antifungal polypeptides

DOEpatents

Altier, Daniel J [Granger, IA; Dahlbacka, Glen [Oakland, CA; Ellanskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, TX; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

2012-04-03

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

Improvement of Learning and Memory Induced by Cordyceps Polypeptide Treatment and the Underlying Mechanism

PubMed Central

2018-01-01

Our previous research revealed that Cordyceps militaris can improve the learning and memory, and although the main active ingredient should be its polypeptide complexes, the underlying mechanism of its activity remains poorly understood. In this study, we explored the mechanisms by which Cordyceps militaris improves learning and memory in a mouse model. Mice were given scopolamine hydrobromide intraperitoneally to establish a mouse model of learning and memory impairment. The effects of Cordyceps polypeptide in this model were tested using the Morris water maze test; serum superoxide dismutase activity; serum malondialdehyde levels; activities of acetyl cholinesterase, Na+-k+-ATPase, and nitric oxide synthase; and gamma aminobutyric acid and glutamate contents in brain tissue. Moreover, differentially expressed genes and the related cellular signaling pathways were screened using an mRNA expression profile chip. The results showed that the genes Pik3r5, Il-1β, and Slc18a2 were involved in the effects of Cordyceps polypeptide on the nervous system of these mice. Our findings suggest that Cordyceps polypeptide may improve learning and memory in the scopolamine-induced mouse model of learning and memory impairment by scavenging oxygen free radicals, preventing oxidative damage, and protecting the nervous system. PMID:29736181

Enzymes that cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kravit, Nancy G.; Schmidt, Katherine A.

The patent application relates to isolated polypeptides that specifically cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides, and to cDNAs encoding the polypeptides. The patent application also relates to nucleic acid constructs, expression vectors and host cells comprising the cDNAs, as well as methods of producing and using the isolated polypeptides for treating pulp and biomass to increase soluble saccharide yield and enrich lignin fractions.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, David B.; Lao, Guifang

1998-01-01

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

The two subunits of the human asialoglycoprotein receptor have different fates when expressed alone in fibroblasts

PubMed Central

Shia, Michael A.; Lodish, Harvey F.

1989-01-01

Two related polypeptides, H1 and H2, comprise the human asialoglycoprotein receptor (ASGP-R). Stable lines of murine NIH 3T3 fibroblasts expressing H1 alone or H2 alone do not bind or internalize the ligand asialoorosomucoid (ASOR), which contains triantennary oligosaccharides. In contrast, cells expressing H1 and H2 together bind and degrade ASOR with properties indistinguishable from those of the ASPG-R in human hepatoma HepG2 cells. Whether or not H2 is coexpressed, H1 is synthesized as a 40-kDa precursor bearing high-mannose oligosaccharides, processed to its mature 46-kDa form, and transported to the cell surface. In cells expressing only H1, homodimers and -trimers of H1 are formed. In contrast, when expressed in 3T3 cells without H1, H2 is synthesized as its 43-kDa precursor, bearing high-mannose oligosaccharides, but is rapidly degraded. When H1 and H2 are coexpressed in the same cell, the H1 polypeptide “rescues” the H2 polypeptide; H2 is processed to its characteristic 50-kDa mature form and is transported to the surface. We conclude that the human ASGP-R is a multichain heterooligomer, probably a trimer of H1 molecules in noncovalent association with one, two, or three H2 molecules, and that the two polypeptides normally interact early in biosynthesis. Images PMID:2919187

In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

DOEpatents

Young, Travis; Schultz, Peter G.

2017-08-15

The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

DOEpatents

Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

2014-02-11

The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

Analysis of urine composition in type Ⅱ diabetic mice after intervention therapy using holothurian polypeptides

NASA Astrophysics Data System (ADS)

Li, Yanyan; Xu, Jiajie; Su, Xiurong

2017-07-01

Hydrolysates and peptide fractions (PF) obtained from sea cucumber with commercial enzyme were studied on the hpyerglycemic and renal protective effects on db/db rats using urine metabolomics. Compared with the control group the polypeptides from the two species could significantly reduce the urine glucose and urea. We also tried to address the compositions of highly expressed urinary proteins using a proteomics approach. They were serum albumins, AMBP proteins, negative trypsin, elastase and urinary protein, GAPDH, a receptor of urokinase-type plasminogen activator (uPAR), and Ig kappa chain C region. We used the electronic nose to quickly detect changes in the volatile substances in mice urine after holothurian polypeptides fed, and the results show it can identify the difference between treatment groups with the control group without overlapping. The protein express mechanism of holothurian polypeptides treating diabetes was discussed, and we suggested these two peptides with the hypoglycemic and renal protective activity might be utilized as nutraceuticals.

Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

DOEpatents

Freimuth, Paul I.

2010-04-06

The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

Restriction/modification polypeptides, polynucleotides, and methods

DOEpatents

Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

2015-02-24

The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

Dock ’n Roll: Folding of a Silk-Inspired Polypeptide into an Amyloid-like Beta Solenoid

PubMed Central

Zhao, Binwu; Cohen Stuart, Martien A.; Hall, Carol K.

2016-01-01

Polypeptides containing the motif ((GA)mGX)n occur in silk (we refer to them as ‘silk-like’) and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II’ β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for molecules to reach the folded state. PMID:26947809

Voluntary scheduled exercise alters diurnal rhythms of behaviour, physiology and gene expression in wild-type and vasoactive intestinal peptide-deficient mice

PubMed Central

Schroeder, Analyne M; Truong, Danny; Loh, Dawn H; Jordan, Maria C; Roos, Kenneth P; Colwell, Christopher S

2012-01-01

The circadian system co-ordinates the temporal patterning of behaviour and many underlying biological processes. In some cases, the regulated outputs of the circadian system, such as activity, may be able to feed back to alter core clock processes. In our studies, we used four wheel-access conditions (no access; free access; early night; and late night) to manipulate the duration and timing of activity while under the influence of a light–dark cycle. In wild-type mice, scheduled wheel access was able to increase ambulatory activity, inducing a level of exercise driven at various phases of the light–dark cycle. Scheduled exercise also manipulated the magnitude and phasing of the circadian-regulated outputs of heart rate and body temperature. At a molecular level, the phasing and amplitude of PER2::LUCIFERASE (PER2::LUC) expression rhythms in the SCN and peripheral tissues of Per2::Luc knockin mice were altered by scheduled exercise. We then tested whether scheduled wheel access could improve deficits observed in vasointestinal polypeptide-deficient mice under the influence of a light–dark cycle. We found that scheduled wheel access during the late night improved many of the behavioural, physiological and molecular deficits previously described in vasointestinal polypeptide-deficient mice. Our results raise the possibility that scheduled exercise could be used as a tool to modulate daily rhythms and, when applied, may counteract some of the negative impacts of ageing and disease on the circadian system. PMID:22988135

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex 1

PubMed Central

Leto, Kenneth J.; Keresztes, Aron; Arntzen, Charles J.

1982-01-01

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [35S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. Images PMID:16662421

Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins.

PubMed

Rintamäki, E; Salonen, M; Suoranta, U M; Carlberg, I; Andersson, B; Aro, E M

1997-11-28

An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the light-harvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, D.B.; Lao, G.

1998-01-06

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

PubMed Central

Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

1996-01-01

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

Ectopic expression of the gastric inhibitory polypeptide receptor gene is a sufficient genetic event to induce benign adrenocortical tumor in a xenotransplantation model.

PubMed

Mazzuco, Tania L; Chabre, Olivier; Sturm, Nathalie; Feige, Jean-Jacques; Thomas, Michaël

2006-02-01

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

Nucleic acids encoding antifungal polypeptides and uses thereof

DOEpatents

Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

2010-11-02

Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.

PubMed Central

Oliver, N A; Wallace, D C

1982-01-01

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria. Images PMID:6955589

Methods for transforming and expression screening of filamentous fungal cells with a DNA library

DOEpatents

Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

2015-06-02

The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Mike; Novick, Scott

2013-04-16

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Michael R; Novick, Scott J

2013-08-20

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Polypeptide composition of fraction 1 protein of the somatic hybrid between Petunia parodii and Petunia parviflora.

PubMed

Kumar, A; Wilson, D; Cocking, E C

1981-04-01

The analysis of the subunit polypeptide composition of Fraction 1 protein provides information on the expression of both chloroplast and nuclear genomes. Fraction 1 protein, isolated from leaves of the somatic hybrid plants derived form the fusion of protoplasts of Petunia parodii and P. parviflora, was analyzed for its subunit polypeptide composition by isoelectric focusing in 8 M urea. The fraction 1 protein enzyme oligomer in the somatic hybrid plants contained small subunits resulting from the expression of both parental nuclear genomes, but probably only one of the parental large subunits, namely that of P. parodii. The relevance of such somatic hybrid material for the study of nucleocytoplasmic interrelationship is discussed, as well as the use of these fraction 1 protein isoelectric focusing patterns for the analysis of taxonomic relationships in Petunia.

Promoters and proteins from Clostridium thermocellum and uses thereof

DOEpatents

Wu, J. H. David; Newcomb, Michael

2012-11-13

The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

PubMed Central

Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

2015-01-01

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

Two novel genes, fanA and fanB, involved in the biogenesis of K99 fimbriae.

PubMed

Roosendaal, E; Boots, M; de Graaf, F K

1987-08-11

The nucleotide sequence of the region located transcriptionally upstream of the K99 fimbrial subunit gene (fanC) was determined. Several putative transcription signals and two open reading frames, designated fanA and fanB, became apparent. Frameshift mutations in fanA and fanB reduced K99 fimbriae expression 8-fold and 16-fold, respectively. Complementation of the mutants in trans restored the K99 expression to about 75% of the wild type level, indicating that fanA and fanB code for transacting polypeptides involved in the biogenesis of K99 fimbriae. The fanA and fanB gene products FanA and FanB were not detectable in minicell preparations, indicating that both polypeptides are synthesized in very small amounts. However, in an in vitro DNA directed translation system FanA and FanB could be identified. The deduced amino acid sequences of FanA and FanB showed that both polypeptides contain no signal peptides, indicating a cytoplasmic location. Furthermore, the polypeptides are very hydrophilic, mainly basic, and exhibit remarkable homology to each other and to a regulatory protein (papB) encoded by the pap-operon (1). Some of these features are characteristics of nucleic acid binding proteins, which suggests that FanA and FanB have a regulatory function in the synthesis of FanC and the auxiliary polypeptides FanD-H.

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs.

PubMed

O'Leary, Brendan; Park, Joonho; Plaxton, William C

2011-05-15

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus.

PubMed

Smith, A R; Boursnell, M E; Binns, M M; Brown, T D; Inglis, S C

1990-01-01

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides

PubMed Central

Kostova, Kamena K.; Hickey, Kelsey L.; Osuna, Beatriz A.; Hussmann, Jeffrey A.; Frost, Adam; Weinberg, David E.; Weissman, Jonathan S.

2017-01-01

Ribosome stalling leads to recruitment of the Ribosome Quality control Complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a Carboxy-terminal Alanine and Threonine (CAT) tail through a non-canonical elongation reaction. Here we explore the role of CATtailing in nascent-chain degradation in budding yeast. We show that Ltn1p can efficiently access only nascent chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enables degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates. PMID:28751611

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

PubMed

Macnaughton, T B; Gowans, E J; Reinboth, B; Jilbert, A R; Burrell, C J

1990-06-01

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

PubMed Central

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

2013-01-01

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

Oncoprotein protein kinase

DOEpatents

Karin, M.; Hibi, M.; Lin, A.

1997-02-25

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

PubMed

Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

2007-07-01

A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

PubMed

Keeley, Fred W; Bellingham, Catherine M; Woodhouse, Kimberley A

2002-02-28

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells.

PubMed

Gerster, Anja; Wodarczyk, Claas; Reichenbächer, Britta; Köhler, Janet; Schulze, Andreas; Krause, Felix; Müller, Dethardt

2016-12-01

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development. Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios. Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide 1

PubMed Central

Lin, Chentao; Thomashow, Michael F.

1992-01-01

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed. ImagesFigure 1Figure 4Figure 5Figure 6Figure 7 PMID:16668917

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

PubMed Central

Farza, H; Hadchouel, M; Scotto, J; Tiollais, P; Babinet, C; Pourcel, C

1988-01-01

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication. Images PMID:2845128

Purification and biophysical characterization of the core protease domain of anthrax lethal factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkazonis, Petros V.; Dalkas, Georgios A.; Chasapis, Christos T.

2010-06-04

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn{sup 2+} binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site aremore » essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF{sub 672-776}) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ({sup 1}H, {sup 13}C, {sup 15}N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn{sup 2+}, suitable for high resolution structural analysis by NMR.« less

Changes in the Polypeptide Patterns of Barley Seedlings Exposed to Jasmonic Acid and Salinity 1

PubMed Central

Maslenkova, Liliana Todorova; Miteva, Tania Simeonova; Popova, Losanka P.

1992-01-01

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins. ImagesFigure 1Figure 4Figure 5 PMID:16668698

Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: Synthesis, characterization and implications for anticancer drug delivery

PubMed Central

Kim, Jong Oh; Oberoi, Hardeep S.; Desale, Swapnil; Kabanov, Alexander V.; Bronich, Tatiana K.

2014-01-01

Polymer nanogels have gained considerable attention as a potential platform for drug delivery applications. Here we describe the design and synthesis of novel polypeptide-based nanogels with hydrophobic moieties in the cross-linked ionic cores. Diblock copolymer, poly(ethylene glycol)-b-poly(L-glutamic acid), hydrophobically modified with L-phenylalanine methyl ester moieties was used for controlled template synthesis of nanogels with small size (ca. 70 nm in diameter) and narrow particle size distribution. Steady-state and time-resolved fluorescence studies using coumarin C153 indicated the existence of hydrophobic domains in the ionic cores of the nanogels. Stable doxorubicin-loaded nanogels were prepared at high drug capacity (30 w/w%). We show that nanogels are enzymatically-degradable leading to accelerated drug release under simulated lysosomal acidic pH. Furthermore, we demonstrate that the nanogel-based formulation of doxorubicin is well tolerated and exhibit an improved antitumor activity compared to a free doxorubicin in an ovarian tumor xenograft mouse model. Our results signify the point to a potential of these biodegradable nanogels as attractive carriers for delivery of chemotherapeutics. PMID:23998716

Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways.

PubMed Central

Müller, M; Laxton, C; Briscoe, J; Schindler, C; Improta, T; Darnell, J E; Stark, G R; Kerr, I M

1993-01-01

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon-stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN-alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-alpha and -gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN-alpha response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-alpha and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN. Images PMID:7693454

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

PubMed

Davis, J Q; Bennett, V

1993-01-01

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Compositions and Methods for the Treatment of Pierce's Disease

DOEpatents

Gupta, Goutam

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

PubMed Central

Rabert, Claudia; Gutiérrez-Moraga, Ana; Navarrete-Gallegos, Alejandro; Navarrete-Campos, Darío; Bravo, León A.; Gidekel, Manuel

2014-01-01

The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed. PMID:24514564

The universality of β-hairpin misfolding indicated by molecular dynamics simulations.

PubMed

Shao, Qiang; Wang, Jinan; Shi, Jiye; Zhu, Weiliang

2013-10-28

Previous molecular dynamics simulations showed that besides the experimentally measured folded structures, several β-structured polypeptides could also have misfolded "out-of-register" structures. Through the enhanced sampling molecular dynamics simulations on a series of polypeptides using either implicit or explicit solvent model, the present study systematically investigated the universality of β-hairpin misfolding and its determinants. It was observed that the misfolding could take place for almost all polypeptides under study, especially in the presence of weak side chain hydrophobicity. Moreover, the observed misfolded structures for various polypeptides share the following common features: (1) the turn length in misfolded structure is one-residue shorter than that in folded structure; (2) the hydrophobic side chains on the two strands are pointed to the opposite directions instead of packing in the same direction to form hydrophobic core cluster in the folded structure; and (3) the misfolded structure is one-residue-shifted asymmetric β-hairpin structure. The detailed analysis suggested that the misfolding of β-hairpin is the result of the competition between the formation of the alterable turn configurations and the inter-strand hydrophobic interactions. These predictions are desired to be tested by experiments.

The universality of β-hairpin misfolding indicated by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Shao, Qiang; Wang, Jinan; Shi, Jiye; Zhu, Weiliang

2013-10-01

Previous molecular dynamics simulations showed that besides the experimentally measured folded structures, several β-structured polypeptides could also have misfolded "out-of-register" structures. Through the enhanced sampling molecular dynamics simulations on a series of polypeptides using either implicit or explicit solvent model, the present study systematically investigated the universality of β-hairpin misfolding and its determinants. It was observed that the misfolding could take place for almost all polypeptides under study, especially in the presence of weak side chain hydrophobicity. Moreover, the observed misfolded structures for various polypeptides share the following common features: (1) the turn length in misfolded structure is one-residue shorter than that in folded structure; (2) the hydrophobic side chains on the two strands are pointed to the opposite directions instead of packing in the same direction to form hydrophobic core cluster in the folded structure; and (3) the misfolded structure is one-residue-shifted asymmetric β-hairpin structure. The detailed analysis suggested that the misfolding of β-hairpin is the result of the competition between the formation of the alterable turn configurations and the inter-strand hydrophobic interactions. These predictions are desired to be tested by experiments.

Expression and role of the genes involved in the transport of bile acids in the liver and kidneys in mice.

PubMed

Attakpa, Eugène S; Djibril, Naguibou M; Baba-Moussa, Farid; Yessoufou, Ganiou; Sezan, Alphonse

2013-01-01

Bile acids are synthesized in the liver from cholesterol. This study investigated the impact and expression of different carriers of bile acid in the liver and kidneys. Eight-week-old male mice were used, which were fed for 15 days and divided into two groups: 15 mice fed with standard diet (control group) and another 15 mice fed with a rich diet of 5% cholesterol (second group). Bile acid dosage was based on their oxidation by 7α hydroxyl-steroid dehydrogenize. The mRNA expression was quantitatively analyzed by the real time of polymerase chain reaction (RT-PCR), and the expression of the renal carrier bile acid protein was analyzed by Western blot. The expression of bile salt export pump involved in the uptake of bile acids in the basolateral membrane of hepatocytes revealed no differences between the two groups of mice. However, the expression of multidrug resistance-associated protein 2 was reduced in mice of the second group. Moreover, the expressions of organic anion transporting polypeptide 4, organic anion transporting polypeptide 1, and sodium taurocholate co-transporting polypeptide (Ntcp) involved in the uptake of bile acids in the apical pole of hepatocytes are suppressed in mice of the second group. The expression of multidrug resistance-associated protein 3 involved in the secretion of bile acids in the apical membrane of hepatocytes revealed no significant differences between the two groups. In mice of the second group, blood concentration of bile acids on the last day was increased. In those mice, the expression of intestinal bile acid transporter was reduced in the kidneys compared with the control mice.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

PubMed Central

Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

2015-01-01

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation. PMID:26404319

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph)

PubMed Central

Liu, Hongbin; Al Nasr, Ibrahim; Liu, Xianyong; Suo, Xun; Barta, John

2015-01-01

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3–10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima. PMID:26641262

From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph).

PubMed

El-Ashram, Saeed; Yin, Qing; Liu, Hongbin; Al Nasr, Ibrahim; Liu, Xianyong; Suo, Xun; Barta, John

2015-01-01

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3-10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

PubMed

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

2015-06-01

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bender, M.A.; Gelinas, R.E.; Miller

1989-04-01

Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide.more » DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.« less

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

PubMed

Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

2016-08-30

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice.

PubMed

Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 -and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality.

Recombinant soluble adenovirus receptor

DOEpatents

Freimuth, Paul I.

2002-01-01

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures

PubMed Central

Ramboer, Eva; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

2015-01-01

The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures. PMID:26648816

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides.

PubMed

Kostova, Kamena K; Hickey, Kelsey L; Osuna, Beatriz A; Hussmann, Jeffrey A; Frost, Adam; Weinberg, David E; Weissman, Jonathan S

2017-07-28

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Gene and genon concept: coding versus regulation

PubMed Central

2007-01-01

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon. PMID:18087760

Memory T-Cell-Mediated Immune Responses Specific to an Alternative Core Protein in Hepatitis C Virus Infection

PubMed Central

Bain, Christine; Parroche, Peggy; Lavergne, Jean Pierre; Duverger, Blandine; Vieux, Claude; Dubois, Valérie; Komurian-Pradel, Florence; Trépo, Christian; Gebuhrer, Lucette; Paranhos-Baccala, Glaucia; Penin, François; Inchauspé, Geneviève

2004-01-01

In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen. PMID:15367612

Wall-associated kinase-like polypeptide mediates nutritional status perception and response

DOEpatents

Yang, Zhenbiao; Karr, Stephen

2014-02-11

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex.

PubMed

López Solís, Remigio O; Weis, Ulrike Kemmerling; Ceballos, Alicia Ramos; Salas, Gustavo Hoecker

2003-12-01

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP). Copyright 2003 Wiley-Liss, Inc.

Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments

PubMed Central

Kim, Walter M.; Sigalov, Alexander B.; Stern, Lawrence J.

2010-01-01

HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning. PMID:20124696

High density growth of T7 expression strains with auto-induction option

DOEpatents

Studier, F. William

2010-07-20

A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

PubMed

Walker, Candace Rae; Pushpavanam, Karthik; Nair, Divya Geetha; Potta, Thrimoorthy; Sutiyoso, Caesario; Kodibagkar, Vikram D; Sapareto, Stephen; Chang, John; Rege, Kaushal

2013-08-13

Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

Primary light-harvesting system: phycobilisomes and associated membranes. Progress report, January 1, 1981-December 31, 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, E.

1981-01-01

Phycobilisomes, serving as primary light harvesting complexes in cyanobacteria and red algae, were investigated. Structurally the phycobilisomes of both groups have the same fundamental phycobiliprotein arrangement. Allophycocyanin is in the center near the thylakoid. Stacked rods composed of phycocyanin, or phycocyanin-phycoerythrin radiate peripherally from the allophycocyanin core. Phycobilisomes of Nostoc sp. and Fremyella diplosiphon, after separation into separate allophycocyanin and phycoerythrin-phycocyanin fractions have been associated in vitro. Hybrid phycobilisomes, derived from mixtures of phycobiliprotein from these species were also obtained. The interaction is specific since reassociation was not obtained with phycobiliprotein complexes of some other algae. Phycobilisomes, whether native, ormore » associated in vitro, were similar in their sedimentation, absorption, fluorescence excitation, fluorescence emission, and by electron microscopy. Furthermore, many of the colorless polypeptides were also highly similar between Nostoc and Fremyella. The similarity formed may reflect an evolutionary relationship between the two species. The polypeptide composition of Porphyridium cruentum phycobilisomes is the most complex of any thus far examined. The phycobiliprotein containing polypeptides comprised 84% of the total stainable protein, while the remaining were colorless. Most of the colorless polypeptides occurred in a pelletable fraction, which was enriched in allophycocyanin and phycocyanin, it is probable that some are involved in the linking of these phycobiliproteins.« less

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-11

PubMed Central

Chia, Catherine P.; Duesing, John H.; Arntzen, Charles J.

1986-01-01

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EFs particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O2 evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664990

Molecular cloning, characterization and expression analysis of HSP60, HSP70 and HSP90 in the golden apple snail, Pomacea canaliculata.

PubMed

Xu, Yipeng; Zheng, Guowan; Dong, Shengzhang; Liu, Guangfu; Yu, Xiaoping

2014-12-01

The golden apple snail, Pomacea canaliculata, has strong tolerance to high temperature, facilitating its invasion in East and Southeast Asia. In the present study, three cDNAs encoding heat shock proteins (PocaHSP60, PocaHSP70, PocaHSP90) in P. canaliculata were cloned and characterized. The PocaHSP60 cDNA was 2447 bp, containing an ORF encoding a polypeptide of 574 amino acids. The PocaHSP70 cDNA was 2644 bp, containing an ORF encoding a polypeptide of 643 amino acids. The PocaHSP90 cDNA was 2546 bp, containing an ORF encoding a polypeptide of 726 amino acids. Genomic DNA analysis showed that PocaHSP60 had 11 introns in the coding region and PocaHSP90 had 7 introns but PocaHSP70 had no one. The expression changes of these three PocaHSPs in the gill, digestive gland, kidney and foot muscle of P. canaliculata exposed to high and low temperature were investigated. The results of quantitative PCR and western blotting showed that the expression level of PocaHSP90 was much higher than PocaHSP60 and PocaHSP70 at room temperature, and PocaHSP70 expression level was the lowest among them. Afterheat shock, PocaHSP70 expression increased rapidly, much more significantly than PocaHSP90 expression, and the effect of heat shock on the expression of PocaHSP70 and PocaHSP90 in the different tissues of P. canaliculata was not the same. Unlike PocaHSP70 and PocaHSP90, PocaHSP60 expression seemed not to be affected by heat shock, because its expression was moderately induced only in the foot muscle. However, cool shock had little effect on the expression change of above three PocaHSPs. These results indicated that HSPs might be related to the thermal resistance of P. canaliculata.

Changes in Gene Expression during Tomato Fruit Ripening 1

PubMed Central

Biggs, M. Scott; Harriman, Robert W.; Handa, Avtar K.

1986-01-01

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)+ and poly(A)− RNAs. Peak levels of total RNA, poly(A)+ RNA, and poly(A)+ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)+ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)+ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products. Images Fig. 1 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16664828

Heat Increases the Editing Efficiency of Human Papillomavirus E2 Gene by Inducing Upregulation of APOBEC3A and 3G.

PubMed

Yang, Yang; Wang, Hexiao; Zhang, Xinrui; Huo, Wei; Qi, Ruiqun; Gao, Yali; Zhang, Gaofeng; Song, Bing; Chen, Hongduo; Gao, Xinghua

2017-04-01

Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3 proteins have been identified as potent viral DNA mutators and have broad antiviral activity. In this study, we demonstrated that apolipoprotein B mRNA-editing catalytic polypeptide 3A (A3A) and A3G expression levels were significantly upregulated in human papillomavirus (HPV)-infected cell lines and tissues. Heat treatment resulted in elevated expression of A3A and A3G in a temperature-dependent manner in HPV-infected cells. Correspondingly, HPV-infected cells heat-treated at 44 °C showed accumulated G-to-A or C-to-T mutation in HPV E2 gene. Knockdown of A3A or A3G could promote cell viability, along with the lower frequency of A/T in HPV E2 gene. In addition, regressing genital viral warts also harbored high G-to-A or C-to-T mutation in HPV E2 gene. Taken together, we demonstrate that apolipoprotein B mRNA-editing catalytic polypeptide 3 expression and editing function was heat sensitive to a certain degree, partly explaining the mechanism of action of local hyperthermia to treat viral warts. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Expression and immunogenic analysis of recombinant polypeptides derived from capsid protein VP1 for developing subunit vaccine material against hepatitis A virus.

PubMed

Jang, Kyoung Ok; Park, Jong-Hwa; Lee, Hyun Ho; Chung, Dae Kyun; Kim, Wonyong; Chung, In Sik

2014-08-01

Three recombinant polypeptides, VP1-His, VP1-3N-His, and 3D2-His, were produced by Escherichia coli expression system. Recombinant VP1-His, VP1-3N-His, and 3D2-His were expressed as bands with molecular weights of 32, 38, and 30 kDa, respectively. These were purified by affinity chromatography using Ni-NTA Fast-flow resin and/or ion-exchange chromatography using DEAE-Sepharose Fast-flow resin. Intraperitoneal immunizations of recombinant polypeptides successfully elicited the productions of VP1-His, VP1-3N-His, and 3D2-His specific IgG antibodies (IgG subclass distribution of IgG1>IgG2a>IgG2b>IgG3) in sera and induced the secretions of cytokines IFN-γ and IL-6 in spleen cells. Sera from recombinant VP1-His-, VP1-3N-His-, and 3D2-His-immunized mice neutralized the propagation of HAV. The highest neutralizing activity was shown in sera from recombinant VP1-3N-His-immunized mice. These results suggest that recombinant VP1-3N-His can be a useful source for developing hepatitis A virus (HAV) subunit vaccine candidates. Copyright © 2014 Elsevier Inc. All rights reserved.

Transgenic rescue of adipocyte glucose-dependent insulinotropic polypeptide receptor expression restores high fat diet-induced body weight gain.

PubMed

Ugleholdt, Randi; Pedersen, Jens; Bassi, Maria Rosaria; Füchtbauer, Ernst-Martin; Jørgensen, Signe Marie; Kissow, Hanne-Louise; Nytofte, Nikolaj; Poulsen, Steen Seier; Rosenkilde, Mette Marie; Seino, Yutaka; Thams, Peter; Holst, Peter Johannes; Holst, Jens Juul

2011-12-30

The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

PubMed

Khan, Aliyya; Eikani, Carlo K; Khan, Hana; Iavarone, Anthony T; Pesavento, James J

2018-01-05

The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas' versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.

Insulin-Like Growth Factor II Targets the mTOR Pathway to Reverse Autism-Like Phenotypes in Mice.

PubMed

Steinmetz, Adam B; Stern, Sarah A; Kohtz, Amy S; Descalzi, Giannina; Alberini, Cristina M

2018-01-24

Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social interaction and repetitive behavior, and is also associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASD symptomatology; thus, identifying novel therapies is urgently needed. We used male BTBR T + Itpr3 tf /J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, to test the effects of systemic administration of insulin-like growth factor II (IGF-II), a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-II treatments reverse the typical defects in social interaction, cognitive/executive functions, and repetitive behaviors reflective of ASD-like phenotypes. In BTBR mice, IGF-II, via IGF-II receptor, but not via IGF-I receptor, reverses the abnormal levels of the AMPK-mTOR-S6K pathway and of active translation at synapses. Thus, IGF-II may represent a novel potential therapy for ASD. SIGNIFICANCE STATEMENT Currently, there is no effective treatment for autism spectrum disorder (ASD), a developmental disability affecting a high number of children. Using a mouse model that expresses most of the key core as well as associated behavioral deficits of ASD, that are, social, cognitive, and repetitive behaviors, we report that a systemic administration of the polypeptide insulin-like growth factor II (IGF-II) reverses all these deficits. The effects of IGF-II occur via IGF-II receptors, and not IGF-I receptors, and target both basal and learning-dependent molecular abnormalities found in several ASD mice models, including those of identified genetic mutations. We suggest that IGF-II represents a potential novel therapeutic target for ASD. Copyright © 2018 the authors 0270-6474/18/371015-15$15.00/0.

The NS2 polypeptide of parvovirus MVM is required for capsid assembly in murine cells.

PubMed

Cotmore, S F; D'Abramo, A M; Carbonell, L F; Bratton, J; Tattersall, P

1997-05-12

Mutants of minute virus of mice (MVM) which express truncated forms of the NS2 polypeptide are known to exhibit a host range defect, replicating productively in transformed human cells but not in cells from their normal murine host. To explore this deficiency we generated viruses with translation termination codons at various positions in the second exon of NS2. In human cells these mutants were viable, but showed a late defect in progeny virion release which put them at a selective disadvantage compared to the wildtype. In murine cells, however, duplex viral DNA amplification was reduced to 5% of wildtype levels and single-strand DNA synthesis was undetectable. These deficiencies could not be attributed to a failure to initiate infection or to a generalized defect in viral gene expression, since the viral replicator protein NS1 was expressed to normal or elevated levels early in infection. In contrast, truncated NS2 gene products failed to accumulate, so that each mutant exhibited a similar NS2-null phenotype. Expression of the capsid polypeptides VP1 and VP2 and their subsequent assembly into intact particles were examined in detail. Synchronized infected cell populations labeled under pulse-chase conditions were analyzed by differential immunoprecipitation of native or denatured extracts using antibodies which discriminated between intact particles and isolated polypeptide chains. These analyses showed that at early times in infection, capsid protein synthesis and stability were normal, but particle assembly was impaired. Unassembled VP proteins were retained in the cell for several hours, but as the unprocessed material accumulated, capsid protein synthesis progressively diminished, so that at later times relatively few VP molecules were synthesized. Thus in NS2-null infections of mouse cells there is a major primary defect in the folding or assembly processes required for effective capsid production.

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-05-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed Central

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-01-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit. PMID:8685266

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peters, J.; Peters, M.; Lottspeich, F.

1987-11-01

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

PubMed

Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis

PubMed Central

Ericson, Brad L.; Carlson, Darby J.

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses. PMID:27298753

Thermostable cellulase from a thermomonospora gene

DOEpatents

Wilson, David B.; Walker, Larry P.; Zhang, Sheng

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity.

Sequences Of Amino Acids For Human Serum Albumin

NASA Technical Reports Server (NTRS)

Carter, Daniel C.

1992-01-01

Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals.

PubMed

Biasin, Mara; Piacentini, Luca; Lo Caputo, Sergio; Kanari, Yasuyoshi; Magri, Giuliana; Trabattoni, Daria; Naddeo, Valentina; Lopalco, Lucia; Clivio, Alberto; Cesana, Eugenio; Fasano, Francesca; Bergamaschi, Cristina; Mazzotta, Francesco; Miyazawa, Masaaki; Clerici, Mario

2007-04-01

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

PubMed

Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

1993-07-15

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed

Ustav, M; Stenlund, A

1991-02-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

PubMed

Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

2017-10-01

Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

PubMed

Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

2008-09-01

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

Comprehensive analysis of CLE polypeptide signaling gene expression and over-expression activity in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis thaliana, only a few families of extracellular signaling molecules have been discovered and their biological roles are lar...

Characterization and developmental expression of genes encoding the early carotenoid biosynthetic enzymes in Citrus paradisi Macf.

PubMed

Costa, Marcio G C; Moreira, Cristina D; Melton, John R; Otoni, Wagner C; Moore, Gloria A

2012-02-01

In the present study, the full-length cDNA sequences of PSY, PDS, and ZDS, encoding the early carotenoid biosynthetic enzymes in the carotenoid pathway of grapefruit (Citrus paradisi), were isolated and characterized for the first time. CpPSY contained a 1311-bp open reading frame (ORF) encoding a polypeptide of 436 amino acids, CpPDS contained a 1659-bp ORF encoding a polypeptide of 552 amino acids, and CpZDS contained a 1713-bp ORF encoding a polypeptide of 570 amino acids. Phylogenetic analysis indicated that CpPSY shares homology with PSYs from Citrus, tomato, pepper, Arabidopsis, and the monocot PSY1 group, while CpPDS and CpZDS are most closely related to orthologs from Citrus and tomato. Expression analysis revealed fluctuations in CpPSY, CpPDS, and CpZDS transcript abundance and a non-coordinated regulation between the former and the two latter genes during fruit development in albedo and juice vesicles of white ('Duncan') and red ('Flame') grapefruits. A 3× higher upregulation of CpPSY expression in juice vesicles of red-fleshed 'Flame' as compared to white-fruited 'Duncan' was observed in the middle stages of fruit development, which correlates with the well documented accumulation pattern of lycopene in red grapefruit. Together with previous data, our results suggest that the primary mechanism controlling lycopene accumulation in red grapefruit involves the transcriptional upregulation of CpPSY, which controls the flux into the carotenoid pathway, and the downregulated expression of CpLCYB2, which controls the step of cyclization of lycopene in chromoplasts during fruit ripening. A correlation between CpPSY expression and fruit color evolution in red grapefruit is demonstrated.

Polypeptide Liquid Crystal Assisted Assembly of Cylindrically Symmetric Silica-Polypeptide Hybrid Microparticles

NASA Astrophysics Data System (ADS)

Russo, Paul; Rosu, Cornelia; Jacobeen, Shane; Park, Katherine; Yunker, Peter; Reichmanis, Elsa

Liquid crystals can organize dispersed particles into exotic structures. Matching the particle surface coating to the chemistry of the mesogenic phase permits a tight focus on factors such as extended particle shape. The colloidal particles developed for this work consist of a magnetic and fluorescent cylinder-like silica core. One end of the silica is rounded, almost hemispherical, giving the particles a bullet-like shape. These particles are functionalized with helical poly(γ-stearyl-L-glutamate) and dispersed, at different concentrations in cholesteric liquid crystals (ChLC) of the same polymer in tetrahydrofuran. Defects introduced by the particles to the director field of the bulk PSLG/THF host led to a variety of phases, including a quasi-hexagonal alignment of the particles. National Science Foundation.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps.

PubMed

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-03-07

To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R)and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone,8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). The VPCAP2-R mRNA expression level in the control group (1.09+/-0.58) was lower than that in the gallbladder polyp group (1.64+/-0.56) and the gallstone group (1.55+/-0.45) (P<0.05) while the VPCAP1-R mRNA expression level in the control group (1.15+/-0.23) was not apparently different from that in the gallbladder polyp group (1.28+/-0.56) and the gallstone group (1.27+/-0.38). The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps.

Neuropeptides in Lower Urinary Tract (LUT) Function

PubMed Central

Arms, Lauren; Vizzard, Margaret A.

2014-01-01

Numerous neuropeptide/receptor systems including vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, calcitonin gene-related peptide, substance P, neurokinin A, bradykinin, and endothelin-1 are expressed in the lower urinary tract (LUT) in both neural and non-neural (e.g., urothelium) components. LUT neuropeptide immunoreactivity is present in afferent and autonomic efferent neurons innervating the bladder and urethra and in the urothelium of the urinary bladder. Neuropeptides have tissue-specific distributions and functions in the LUT and exhibit neuroplastic changes in expression and function with LUT dysfunction following neural injury, inflammation and disease. LUT dysfunction with abnormal voiding including urinary urgency, increased voiding frequency, nocturia, urinary incontinence and pain may reflect a change in the balance of neuropeptides in bladder reflex pathways. LUT neuropeptide/receptor systems may represent potential targets for therapeutic intervention. PMID:21290237

Thermostable cellulase from a thermomonospora gene

DOEpatents

Wilson, D.B.; Walker, L.P.; Zhang, S.

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities.

PubMed

Suarez, M; Entenza, J M; Doerries, C; Meyer, E; Bourquin, L; Sutherland, J; Marison, I; Moreillon, P; Mermod, N

2003-01-05

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings. Copyright 2002 Wiley Periodicals, Inc.

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeang, K.T.; Cho, M.S.; Hayward, G.S.

1984-10-01

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably bymore » a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.« less

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed Central

Ustav, M; Stenlund, A

1991-01-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells. Images PMID:1846806

Prognostic value of alcohol dehydrogenase mRNA expression in gastric cancer.

PubMed

Guo, Erna; Wei, Haotang; Liao, Xiwen; Xu, Yang; Li, Shu; Zeng, Xiaoyun

2018-04-01

Previous studies have reported that alcohol dehydrogenase (ADH) isoenzymes possess diagnostic value in gastric cancer (GC). However, the prognostic value of ADH isoenzymes in GC remains unclear. The aim of the present study was to identify the prognostic value of ADH genes in patients with GC. The prognostic value of ADH genes was investigated in patients with GC using the Kaplan-Meier plotter tool. Kaplan-Meier plots were used to assess the difference between groups of patients with GC with different prognoses. Hazard ratios (HR) and 95% confidence intervals (CI) were used to assess the relative risk of GC survival. Overall, 593 patients with GC and 7 ADH genes were included in the survival analysis. High expression of ADH 1A (class 1), α polypeptide ( ADH1A; log-rank P=0.043; HR=0.79; 95% CI: 0.64-0.99), ADH 1B (class 1), β polypeptide ( ADH1B ; log-rank P=1.9×10 -05 ; HR=0.65; 95% CI: 0.53-0.79) and ADH 5 (class III), χ polypeptide ( ADH5 ; log-rank P=0.0011; HR=0.73; 95% CI: 0.6-0.88) resulted in a significantly decreased risk of mortality in all patients with GC compared with patients with low expression of those genes. Furthermore, protective effects may additionally be observed in patients with intestinal-type GC with high expression of ADH1B (log-rank P=0.031; HR=0.64; 95% CI: 0.43-0.96) and patients with diffuse-type GC with high expression of ADH1A (log-rank P=0.014; HR=0.51; 95% CI: 0.3-0.88), ADH1B (log-rank P=0.04; HR=0.53; 95% CI: 0.29-0.98), ADH 4 (class II), π polypeptide (log-rank P=0.033; HR=0.58; 95% CI: 0.35-0.96) and ADH 6 (class V) (log-rank P=0.037; HR=0.59; 95% CI: 0.35-0.97) resulting in a significantly decreased risk of mortality compared with patients with low expression of those genes. In contrast, patients with diffuse-type GC with high expression of ADH5 (log-rank P=0.044; HR=1.66; 95% CI: 1.01-2.74) were significantly correlated with a poor prognosis. The results of the present study suggest that ADH1A and ADH1B may be potential prognostic biomarkers of GC, whereas the prognostic value of other ADH genes requires further investigation.

Prognostic value of alcohol dehydrogenase mRNA expression in gastric cancer

PubMed Central

Guo, Erna; Wei, Haotang; Liao, Xiwen; Xu, Yang; Li, Shu; Zeng, Xiaoyun

2018-01-01

Previous studies have reported that alcohol dehydrogenase (ADH) isoenzymes possess diagnostic value in gastric cancer (GC). However, the prognostic value of ADH isoenzymes in GC remains unclear. The aim of the present study was to identify the prognostic value of ADH genes in patients with GC. The prognostic value of ADH genes was investigated in patients with GC using the Kaplan-Meier plotter tool. Kaplan-Meier plots were used to assess the difference between groups of patients with GC with different prognoses. Hazard ratios (HR) and 95% confidence intervals (CI) were used to assess the relative risk of GC survival. Overall, 593 patients with GC and 7 ADH genes were included in the survival analysis. High expression of ADH 1A (class 1), α polypeptide (ADH1A; log-rank P=0.043; HR=0.79; 95% CI: 0.64–0.99), ADH 1B (class 1), β polypeptide (ADH1B; log-rank P=1.9×10−05; HR=0.65; 95% CI: 0.53–0.79) and ADH 5 (class III), χ polypeptide (ADH5; log-rank P=0.0011; HR=0.73; 95% CI: 0.6–0.88) resulted in a significantly decreased risk of mortality in all patients with GC compared with patients with low expression of those genes. Furthermore, protective effects may additionally be observed in patients with intestinal-type GC with high expression of ADH1B (log-rank P=0.031; HR=0.64; 95% CI: 0.43–0.96) and patients with diffuse-type GC with high expression of ADH1A (log-rank P=0.014; HR=0.51; 95% CI: 0.3–0.88), ADH1B (log-rank P=0.04; HR=0.53; 95% CI: 0.29–0.98), ADH 4 (class II), π polypeptide (log-rank P=0.033; HR=0.58; 95% CI: 0.35–0.96) and ADH 6 (class V) (log-rank P=0.037; HR=0.59; 95% CI: 0.35–0.97) resulting in a significantly decreased risk of mortality compared with patients with low expression of those genes. In contrast, patients with diffuse-type GC with high expression of ADH5 (log-rank P=0.044; HR=1.66; 95% CI: 1.01–2.74) were significantly correlated with a poor prognosis. The results of the present study suggest that ADH1A and ADH1B may be potential prognostic biomarkers of GC, whereas the prognostic value of other ADH genes requires further investigation. PMID:29552190

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice

PubMed Central

Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 –and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality. PMID:29155851

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus.

PubMed

Antle, Michael C; LeSauter, Joseph; Silver, Rae

2005-06-09

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

PubMed

Sato, T; Oeller, P W; Theologis, A

1991-02-25

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Evidence for a hepatocellular lineage in a combined hepatocellular-cholangiocarcinoma of transitional type.

PubMed

Fisher, H P; Doppl, W; Osborn, M; Altmannsberger, M

1988-01-01

A combined hepatocellular-cholangiocarcinoma (CHC) of transitional subtype and the surrounding cirrhotic liver tissue were investigated immunocytochemically by monoclonal antibodies specific for each of the keratin polypeptides 7, 8, 18 and 19. Different keratin subsets were found in different parts of the tumour. The hepatocellular component reveals keratins 8 and 18, with the bordering cells of trabecular formations additionally expressing keratins 7 and 19. The same keratins i.e. 7, 8, 18, 19 were found in normal bile duct epithelium as well as in cholangiocarcinomatous and transitional areas of hepatocellular and cholangiocellular differentiation. Normal hepatocytes express only keratin 8 and 18. In cirrhotic liver some modified hepatocytes additionally express keratin 7. When ductal transformation is observed in the marginal parts of portal tracts and fibrous septa the keratin polypeptide pattern mimics that of bile duct epithelium. The cholangiocellular metaplasia of hepatocytes observed here correlates well with findings in hepato-organogenesis and hepatocarcinogenesis and suggests that the transitional subtype of combined hepatocellular-cholangiocarcinoma is a variant of hepatocellular carcinoma.

Polypeptide N-acetylgalactosaminyltransferase-6 expression in gastric cancer

PubMed Central

Guo, Yan; Shi, Jingjing; Zhang, Jun; Li, Haixin; Liu, Ben; Guo, Hua

2017-01-01

Gastric cancer (GC) is one of the leading causes of cancer-related deaths, with limited improvement in its clinical outcome worldwide. Aberrant mucin-type O-glycosylation is a critical event widespread in the development of GC. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) regulate the initial step and determine the sites of mucin-type O-glycoprotein bio-synthesis. GALNT6 has considerable potential as a biomarker in various cancers. The roles of GALNT6 in GC were analyzed, and the results showed that GALNT6 expression markedly increased in GC tissues compared with those in adjacent gastric tissues. High intratumoral GALNT6 density was associated with the clinicopathological parameters of TNM stage and distant metastasis. GALNT6 was identified as an independent prognosticator for the poor prognosis of GC patients. Moreover, the high expression level of GALNT6 was significantly associated with the low expression levels of E-cadherin and β-catenin and the high expression levels of MMP9. These findings indicated that GALNT6 could provide new insights into the characterization of GC as well as contribute to the development of an efficient prognostic indicator and novel therapeutic modalities for GC. PMID:28744137

Differential accumulation of dehydrins in response to water stress for hybrid and common bermudagrass genotypes differing in drought tolerance.

PubMed

Hu, Longxing; Wang, Zhaolong; Du, Hongmei; Huang, Bingru

2010-01-15

Expression of dehydrin proteins may be induced or enhanced by environmental stresses that lead to cell dehydration. The objective of the this study was to investigate genetic variation in dehydrin protein accumulation in response to drought stress of whole-plants or dehydration of detached leaves and to identify dehydrins differentially expressed in bermudagrass (Cynodon spp.) genotypes differing in drought tolerance. Plants of four hybrid bermudagrass (Cynodondactylon L. xCynodontransvaalensis L.) ('Tifway', 'Tifdwarf', 'Tifeagle', 'Kan1') and four common bermudagrass (Cynodon dactylon) ('C299', 'Sportbermuda', 'H10', and 'H19') genotypes were subjected to 14d of drought stress and detached leaves of two genotypes were exposed to dehydration in growth chambers. Turf quality and leaf relative water content (RWC) decreased while electrolyte leakage (EL) increased during whole-plant drought stress for all genotypes, with more pronounced changes in each parameter for 'C299' and 'Tifeagle' than those for other genotypes ('Tifway', 'Kan 1', 'Sportbermuda', 'H10', and H19'), suggesting that the former two genotypes were more sensitive to drought stress than the other genotypes. During dehydration of detached leaves, relative water loss rate (RWL) was significantly lower in drought-tolerant 'Tifway' than in drought-sensitive 'C299'. Immunoblotting analysis indicated that no dehydrin polypeptides were detected in all genotypes under well-watered conditions. A 24-kDa polypeptide was detected in 'C299' at 6 d of drought, but not in the other genotypes. The dehydrin polypeptides of about 14-74kDa accumulated at 10d of drought stress and in a range of RWL for detached leaves, and two dehydrins (31 and 40kDa) exhibited differential accumulation in the drought-sensitive 'C299' and tolerant 'Tifway', as demonstrated by the whole-plant drought responses. The 31-kDa dehydrin polypeptide was present only in 'Tifway' and 'H19' at 10d of drought stress, and accumulated with the increasing RWL in detached leaves of 'Tifway'. The expression level of 40-kDa dehydrin polypeptides was greater in 'Tifway'' than in 'C299' at the same level of water deficit (from 10% to 65% RWL). These results indicated that the accumulation of 31- and 40-kDa dehydrins may contribute to drought or dehydration tolerance in warm-season bermudagrass.

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina1

PubMed Central

Pick, Uri; Gounaris, Kleoniki; Barber, James

1987-01-01

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using 32P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions. Images Fig. 4 PMID:16665656

Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid.

PubMed

Koike, M; Takezawa, D; Arakawa, K; Yoshida, S

1997-06-01

Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 microM ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE. N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-1) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pI value (10.2). Expression of WPM-1 mRNA was dramatically induced by 50 microM ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.

The bark of Robinia pseudoacacia contains a complex mixture of lectins.Characterization of the proteins and the cDNA clones.

PubMed Central

Van Damme, E J; Barre, A; Smeets, K; Torrekens, S; Van Leuven, F; Rougé, P; Peumans, W J

1995-01-01

Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes. PMID:7716244

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin structure (ARC) are identical to portions of the deduced open reading frame of TIG-1 mRNA.

Expression of pituitary adenylate cyclase-activating polypeptide 1 and 2 receptor mRNA in gallbladder tissue of patients with gallstone or gallbladder polyps

PubMed Central

Zhang, Zhen-Hai; Wu, Shuo-Dong; Gao, Hong; Shi, Gang; Jin, Jun-Zhe; Kong, Jing; Tian, Zhong; Su, Yang

2006-01-01

AIM: To detect the expression of pituitary adenylate cyclase-activating polypeptide receptor 1 (VPCAP1-R) and VPCAP2-R mRNA in gallbladder tissues of patients with gallstone or gallbladder polyps. METHODS: The expression of VPCAP1-R and VPCAP2-R mRNA in gallbladder tissues was detected in 25 patients with gallstone, 8 patients with gallbladder polyps and 7 donors of liver transplantation by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The VPCAP2-R mRNA expression level in the control group (1.09±0.58) was lower than that in the gallbladder polyp group (1.64 ± 0.56) and the gallstone group (1.55±0.45) (P < 0.05) while the VPCAP1-R mRNA expression level in the control group (1.15 ± 0.23) was not apparently different from that in the gallbladder polyp group (1.28±0.56) and the gallstone group (1.27 ± 0.38). CONCLUSION: The abnormal expression of VPCAP2-R mRNA in gallbladder tissue may play a role in the formation of gallbladder stone and gallbladder polyps. PMID:16552823

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean.

PubMed

Voelker, T A; Staswick, P; Chrispeels, M J

1986-12-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained.

Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides

Treesearch

James M. Slavicek

1991-01-01

Genomic expression of the Lymantriu dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was studied. Viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through Northern analysis. Sixty-five distinct transcripts were identified in this...

In vivo disruption of T cell development by expression of a dominant-negative polypeptide designed to abolish the SLP-76/Gads interaction.

PubMed

Jordan, Martha S; Maltzman, Jonathan S; Kliche, Stefanie; Shabason, Jacob; Smith, Jennifer E; Obstfeld, Amrom; Schraven, Burkhart; Koretzky, Gary A

2007-10-01

Multi-molecular complexes nucleated by adaptor proteins play a central role in signal transduction. In T cells, one central axis consists of the assembly of several signaling proteins linked together by the adaptors linker of activated T cells (LAT), Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads). Each of these adaptors has been shown to be important for normal T cell development, and their proper sub-cellular localization is critical for optimal function in cell lines. We previously demonstrated in Jurkat T cells and a rat basophilic leukemic cell line that expression of a 50-amino acid polypeptide identical to the site on SLP-76 that binds to Gads blocks proper localization of SLP-76 and SLP-76-dependent signaling events. Here we extend these studies to investigate the ability of this polypeptide to inhibit TCR-induced integrin activity in Jurkat cells and to inhibit in vivo thymocyte development and primary T cell function. These data provide evidence for the in vivo function of a dominant-negative peptide based upon the biology of SLP-76 action and suggest the possibility of therapeutic potential of targeting the SLP-76/Gads interaction.

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

New Class of Cargo Protein in Tetrahymena thermophila Dense Core Secretory Granules

PubMed Central

Haddad, Alex; Bowman, Grant R.; Turkewitz, Aaron P.

2002-01-01

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules. PMID:12456006

Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

PubMed Central

2014-01-01

Background Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose. Methods Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay. Results Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed. Conclusions This concludes that TTV N22 region may be expressed and safely used as antigen for blot assay to detect anti-TTV antibodies in sera. PMID:24884576

Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing.

PubMed

Wang, Ziqiang; Liu, Qing; Lu, Jinhua; Fan, Ping; Xie, Weidong; Qiu, Wei; Wang, Fan; Hu, Guangnan; Zhang, Yaou

2016-12-16

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Biology of Proteins Acting in Immune Response Mechanisms

DTIC Science & Technology

1988-06-01

Studies of the bigsynthesis and processing of LAMP-i and LAMP-2 by pulse -labeling with [ S]methionine showed that the proteins were synthesized as...about Mr 90,000, and post-translationally processed by the addition of a heterogeneous mixture of complex-type oligosaccharides to form mature...centrifugation, and analysis of oligosaccharide processing (D’Souza et -l., 1986). Synthesis and glycosylation of the core polypeptide in the rough

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

PubMed

Wei, Haiyan; Yang, Lijiang; Gao, Yi Qin

2010-09-16

Following the studies of urea denaturation of β-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of β-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Polypeptide-Based Gold Nanoshells for Photothermal Therapy.

PubMed

Mayle, Kristine M; Dern, Kathryn R; Wong, Vincent K; Sung, Shijun; Ding, Ke; Rodriguez, April R; Taylor, Zachary; Zhou, Z Hong; Grundfest, Warren S; Deming, Timothy J; Kamei, Daniel T

2017-02-01

Targeted killing of cancer cells by engineered nanoparticles holds great promise for noninvasive photothermal therapy applications. We present the design and generation of a novel class of gold nanoshells with cores composed of self-assembled block copolypeptide vesicles with photothermal properties. Specifically, poly(L-lysine) 60 - block-poly(L-leucine) 20 (K 60 L 20 ) block copolypeptide vesicles coated with a thin layer of gold demonstrate enhanced absorption of light due to surface plasmon resonance (SPR) in the near-infrared range. We show that the polypeptide-based K 60 L 20 gold nanoshells have low toxicity in the absence of laser exposure, significant heat generation upon exposure to near-infrared light, and, as a result, localized cytotoxicity within the region of laser irradiation in vitro. To gain a better understanding of our gold nanoshells in the context of photothermal therapy, we developed a comprehensive mathematical model for heat transfer and experimentally validated this model by predicting the temperature as a function of time and position in our experimental setup. This model can be used to predict which parameters of our gold nanoshells can be manipulated to improve heat generation for tumor destruction. To our knowledge, our results represent the first ever use of block copolypeptide vesicles as the core material of gold nanoshells.

Assembly Properties of Divergent Tubulin Isotypes and Altered Tubulin Polypeptides in Vivo

NASA Astrophysics Data System (ADS)

Gu, Wei

1990-01-01

Mbeta1 is one of the closely related (though distinct) gene products termed isotypes encoded by the mouse beta-tubulin multigene family. These isotypes typically share 95%-98% homology at the amino acid level. However, Mbeta 1 is unusual in its relatively high degree of divergence compared to other beta-tubulin isotypes; furthermore, its tissue-restricted pattern of expression (Mbeta1 is only expressed in hematopoietic tissue) led to speculation that this isotype might be specialized for assembly into unique microtubule structures (such as the marginal band in some erythropoietic cell types). To test if this isotype is capable of coassembly into microtubules in cell types other than those in which it is normally expressed, a method was developed for the generation of an anti-Mbeta1 specific antibody. The Mbeta1 tubulin isotype was introduced into tissue culture cells by transfection and its expression and assembly properties were studied in both transiently transfected cells and stable cell lines using the anti -Mbeta1 specific antibody. The successful expression and coassembly of a 'foreign' tubulin isotype into microtubules in tissue culture cells and the generation of an antibody that can specifically recognize this isotype provided an approach to study the properties of altered beta-tubulin polypeptides in vivo. beta-tubulin synthesis in eukaryotic cells is autoregulated by a posttranscriptional mechanism in which the first four amino acids are responsible for determining the stability of beta -tubulin mRNA. To test if the beta -tubulin amino-terminal regulatory domain also contributes to the capacity of the tubulin monomer to polymerize into microtubules, altered sequences encoding Mbeta 1 but containing deletions encompassing amino acids 2-5 were expressed in HeLa cells. Stable cell lines expressing the altered Mbeta1 isotype were also generated. The assembly properties and stability of these altered Mbeta1 tubulin polypeptides were tested using the anti-Mbeta1 specific antibody. The data suggest that the first four amino acids of beta-tubulin play a regulatory rather than a structural role.

Fibrillar dimer formation of islet amyloid polypeptides

DOE PAGES

Chiu, Chi -cheng; de Pablo, Juan J.

2015-05-08

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

Fibrillar dimer formation of islet amyloid polypeptides

NASA Astrophysics Data System (ADS)

Chiu, Chi-cheng; de Pablo, Juan J.

2015-09-01

Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

Transplantations and Cloning of an Immortal Cell Line from Rat SCN

DTIC Science & Technology

1994-05-31

of SCN peptides in colonies was examined using rabbit polyclonal antisera against arginine vasopressin (AVP; Arnel Products), gastrin releasing ...examined for expression of arginine vasopressin (AVP), gastrin releasing peptide (GRP), somatostatin (SMT) and vasoactive intestinal polypeptide (VIP...neuronal markers and peptides found within SCN neurons in situ. Concordant with immunostaining data, content, release and mRNA expression of SCN

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

PubMed

Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

2003-07-01

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Absolute measurement of species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) and its modulation in cultured hepatocytes.

PubMed

Qiu, Xi; Bi, Yi-An; Balogh, Larissa M; Lai, Yurong

2013-09-01

Species differences among membrane transporters can be remarkable and difficult to properly assess by conventional methods. Herein, we employed the first use of stable isotope labeling in mammals or stable isotope-labeled peptides combined with mass spectrometry to identify species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) protein expression in liver tissue and to characterize the modulation of protein expression in sandwich-cultured human (SCHH) and rat hepatocytes (SCRH). The lower limit of quantification was established to be 5 fmol on column with a standard curve that was linear up to 2000 fmol. The accuracy and precision were evaluated with three quality control samples and known amounts of synthetic proteotypic peptides that were spiked into the membrane protein extracts. The overall relative error and coefficient of variation were less than 10%. The expression of Ntcp in mouse and rat was significant higher than that in human (five-fold) and monkey (two-fold) and ranked as mouse > rat > monkey > human. In the cultured hepatocytes, although significant downregulation of Ntcp expression in SCRH at day 5 after the culture was detected, NTCP expression in SCHH was comparable to the suspension hepatocytes. The results suggested that NTCP/Ntcp modulation in cultured hepatocytes is species specific. Copyright © 2013 Wiley Periodicals, Inc.

Structural properties of hydration shell around various conformations of simple polypeptides.

PubMed

Czapiewski, Dariusz; Zielkiewicz, Jan

2010-04-08

In this paper we investigate structural properties of water within the solvation shell around the peptide core created by a well-defined conformation of polypeptide chain. The following secondary structures are investigated: linear (straight chain), and three helices PII (polyproline-like), 3(10), and alpha. We propose using the two-particle contribution to entropy as a rational measure of the water structural ordering within the solvation layer. This contribution divides into two terms, depending on the peptide-water and water-water interactions, respectively, and in this paper both terms are investigated. The structure of "solvation" water is described by the second term, and therefore it mainly attracts our attention. Determination of this term, however, is not an easy task, requiring some controversial approximations. Therefore, we have transformed this term to the form of some rational parameter which measures the local structural ordering of water within the solvation shell. Moreover, the results of several independent investigations are reported: we adopt the harmonic approximation for an independent estimation of the water entropy within the solvation shell, and we also study structure of the water-water hydrogen bond network, mean geometry of a single hydrogen bond, the self-diffusion coefficients (both translational and rotational) of water, and the mean lifetimes of water-water and water-peptide hydrogen bonds. All the obtained results lead to the conclusion that the local structure of water within the solvation shell changes only slightly in comparison to the bulk one. If so, the measure of local water ordering proposed by us is exploited with the aim to gain the deeper insight on the structural properties of "solvation" water. It has been shown that this parameter can be factored into three terms, which measure translational, configurational, and orientational ordering, respectively. Using this factoring, the ordering map for a precise description of the water local ordering has been built. An interesting correlation is observed: the points on this map lie approximately on the straight line, while the linear conformations clearly deviate from the general tendency. Further analysis of the obtained results allows us to express the supposition that an increasing local ordering of water around given secondary structure corresponds to an increasing relative stability of this structure in aqueous solution. Analyzing the geometry of the water-water hydrogen bond network within the solvation layer, we find some systematic deviations of this geometry from the bulk water properties. We also observe that the alanine peptides (excluding the linear form) disturb the hydrogen bond network in the less range, and in another way than the various conformations of polyglycine, while the linear form of polyalanine behaves very similarly to the glycine ones. Next, investigating the dynamic properties, we also conclude that water near the peptide surface creates a pseudorigid structure, a "halo" around the peptide core. This "halo" is stabilized by slightly higher energy of the hydrogen bonds network: we have found that within this region the hydrogen bonds network is slightly less distorted, the water-water hydrogen bonds are a little more stable and their mean lifetime is clearly longer that that of bulk water. Significant differences between the alanine- and glycine-based polypeptides are also visible. It has also been found that this solvation layer interacts with the polyalanine in another way than with polyglycine. Although in the case of the glycine-based polypeptide this layer slides relatively freely over the peptide surface, for the alanine-based polypeptide this sliding is strongly hindered by the presence of the methyl groups, and this effect is additionally enhanced by a rise in the solvation layer rigidity. Thus, the survey of various dynamic properties allows us to perceive and to explain distinct differences in behavior of water within the solvation shell around both glycine and alanine peptides.

Expression of Molecular Markers in Brain, Serum, and Lung Tissues Following Hypobaric Hypoxia

DTIC Science & Technology

2018-01-01

8 4.2 HIF-1α ELISA Results...9 4.3 Prolyl-4-hydroxylase Alpha Polypeptide I (P4Ha1) ELISA Results...10 4.4 Vascular Endothelial Growth Factor ELISA Results .......................................................12

Method to determine transcriptional regulation pathways in organisms

DOEpatents

Gardner, Timothy S.; Collins, James J.; Hayete, Boris; Faith, Jeremiah

2012-11-06

The invention relates to computer-implemented methods and systems for identifying regulatory relationships between expressed regulating polypeptides and targets of the regulatory activities of such regulating polypeptides. More specifically, the invention provides a new method for identifying regulatory dependencies between biochemical species in a cell. In particular embodiments, provided are computer-implemented methods for identifying a regulatory interaction between a transcription factor and a gene target of the transcription factor, or between a transcription factor and a set of gene targets of the transcription factor. Further provided are genome-scale methods for predicting regulatory interactions between a set of transcription factors and a corresponding set of transcriptional target substrates thereof.

The Regulation of Steroid Action by Sulfation and Desulfation

PubMed Central

Mueller, Jonathan W.; Gilligan, Lorna C.; Idkowiak, Jan; Arlt, Wiebke

2015-01-01

Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined. PMID:26213785

Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids †

PubMed Central

Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

1980-01-01

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex. Images PMID:16789190

Development and Application of a Saccharomyces cerevisiae-Expressed Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Infectious Bronchitis Virus

PubMed Central

Gibertoni, Aliandra M.; Montassier, Maria de Fátima S.; Sena, Janete A. D.; Givisiez, Patrícia E. N.; Furuyama, Cibele R. A. G.; Montassier, Hélio J.

2005-01-01

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. PMID:15815038

Transgenic rabbit that expresses a functional human lipoprotein (a)

DOEpatents

Rouy, Didier; Duverger, Nicolas; Emmanuel, Florence; Denefle, Patrice; Houdebine, Louis-Marie; Viglietta, Celine; Rubin, Edward M.; Hughes, Steven D.

2003-01-01

A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

DOEpatents

Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

2015-12-08

This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis1[C][W

PubMed Central

Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo

2011-01-01

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis. PMID:22021419

Molecular Characterization of Tomato 3-Dehydroquinate Dehydratase-Shikimate:NADP Oxidoreductase1

PubMed Central

Bischoff, Markus; Schaller, Andreas; Bieri, Fabian; Kessler, Felix; Amrhein, Nikolaus; Schmid, Jürg

2001-01-01

Analysis of cDNAs encoding the bifunctional 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase (DHQase-SORase) from tomato (Lycopersicon esculentum) revealed two classes of cDNAs that differed by 57 bp within the coding regions, but were otherwise identical. Comparison of these cDNA sequences with the sequence of the corresponding single gene unequivocally proved that the primary transcript is differentially spliced, potentially giving rise to two polypeptides that differ by 19 amino acids. Quantitative real-time polymerase chain reaction revealed that the longer transcript constitutes at most 1% to 2% of DHQase-SORase transcripts. Expression of the respective polypeptides in Escherichia coli mutants lacking the DHQase or the SORase activity gave functional complementation only in case of the shorter polypeptide, indicating that skipping of a potential exon is a prerequisite for the production of an enzymatically active protein. The deduced amino acid sequence revealed that the DHQase-SORase is most likely synthesized as a precursor with a very short (13-amino acid) plastid-specific transit peptide. Like other genes encoding enzymes of the prechorismate pathway in tomato, this gene is elicitor-inducible. Tissue-specific expression resembles the patterns obtained for 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 2 and dehydroquinate synthase genes. This work completes our studies of the prechorismate pathway in that cDNAs for all seven enzymes (including isozymes) of the prechorismate pathway from tomato have now been characterized. PMID:11299368

Vectorial transport of bile salts across MDCK cells expressing both rat Na+-taurocholate cotransporting polypeptide and rat bile salt export pump.

PubMed

Mita, Sachiko; Suzuki, Hiroshi; Akita, Hidetaka; Stieger, Bruno; Meier, Peter J; Hofmann, Alan F; Sugiyama, Yuichi

2005-01-01

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.

The plant vacuolar Na+/H+ antiport.

PubMed

Barkla, B J; Apse, M P; Manolson, M F; Blumwald, E

1994-01-01

Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species. One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium. The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast. Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles. In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium. This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide. In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride. Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity. Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport. Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library. A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus. This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

Ethylene insensitive plants

DOEpatents

Ecker, Joseph R [Carlsbad, CA; Nehring, Ramlah [La Jolla, CA; McGrath, Robert B [Philadelphia, PA

2007-05-22

Nucleic acid and polypeptide sequences are described which relate to an EIN6 gene, a gene involved in the plant ethylene response. Plant transformation vectors and transgenic plants are described which display an altered ethylene-dependent phenotype due to altered expression of EIN6 in transformed plants.

Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

PubMed Central

Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S.; DiSpirito, Alan A.

2014-01-01

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. PMID:25416758

Gastric De Novo Muc13 Expression and Spasmolytic Polypeptide-Expressing Metaplasia during Helicobacter heilmannii Infection

PubMed Central

Liu, Cheng; Blaecher, Caroline; Flahou, Bram; Ducatelle, Richard; Linden, Sara; Haesebrouck, Freddy

2014-01-01

Helicobacter heilmannii is a zoonotic bacterium that has been associated with gastric disease in humans. In this study, the mRNA expression of mucins in the stomach of BALB/c mice was analyzed at several time points during a 1-year infection with this bacterium, during which gastric disease progressed in severity. Markers for acid production by parietal cells and mucous metaplasia were also examined. In the first 9 weeks postinfection, the mRNA expression of Muc6 was clearly upregulated in both the antrum and fundus of the stomach of H. heilmannii-infected mice. Interestingly, Muc13 was upregulated already at 1 day postinfection in the fundus of the stomach. Its expression level remained high in the stomach over the course of the infection. This mucin is, however, not expressed in a healthy stomach, and high expression of this mucin has so far only been described in gastric cancer. In the later stages of infection, mRNA expression of H+/K+-ATPase α/β and KCNQ1 decreased, whereas the expression of Muc4, Tff2, Dmbt1, and polymeric immunoglobulin receptor (pIgR) increased starting at 16 weeks postinfection onwards, suggesting the existence of spasmolytic polypeptide-expressing metaplasia in the fundus of the stomach. Mucous metaplasia present in the mucosa surrounding low-grade mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions was also histologically confirmed. Our findings indicate that H. heilmannii infection causes severe gastric pathologies and alterations in the expression pattern of gastric mucins, such as Muc6 and Muc13, as well as disrupting gastric homeostasis by inducing the loss of parietal cells, resulting in the development of mucous metaplasia. PMID:24866791

Competition between metals for binding to methanobactin enables expression of soluble methane monooxygenase in the presence of copper.

PubMed

Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

2015-02-01

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Expansin polynucleotides, related polypeptides and methods of use

DOEpatents

Cosgrove, Daniel J.; Wu, Yajun

2006-02-21

The present invention relates to beta expansin polypeptides, nucleotide sequences encoding the same and regulatory elements and their use in altering cell wall structure in plants. Nucleic acid constructs comprising a beta expansin sequence operably linked to a promoter, or other regulatory sequence are disclosed as well as vectors, plant cells, plants, and transformed seeds containing such constructs are provided. Methods for the use of such constructs in repressing or inducing expression of a beta expansin sequences in a plant are also provided as well as methods for harvesting transgenic expansin proteins. In addition, methods are provided for inhibiting or improving cell wall structure in plants by repression or induction of expansin sequences in plants.

Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

PubMed Central

Plesiat, P; Grandguillot, M; Harayama, S; Vragar, S; Michel-Briand, Y

1991-01-01

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp SalI fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp SalI fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed delta 1-dehydrogenase in Escherichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacIq plasmid pRG1 and pTEK21, the expression of delta 1-dehydrogenase was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Delta 1-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction. The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases. Images FIG. 4 PMID:1657885

Molecular analysis of two phytohemagglutinin genes and their expression in Phaseolus vulgaris cv. Pinto, a lectin-deficient cultivar of the bean

PubMed Central

Voelker, Toni A.; Staswick, Paul; Chrispeels, Maarten J.

1986-01-01

Phytohemagglutinin (PHA), the seed lectin of the common bean, Phaseolus vulgaris, is encoded by two highly homologous, tandemly linked genes, dlec1 and dlec2, which are coordinately expressed at high levels in developing cotyledons. Their respective transcripts translate into closely related polypeptides, PHA-E and PHA-L, constituents of the tetrameric lectin which accumulates at high levels in developing seeds. In the bean cultivar Pinto UI111, PHA-E is not detectable, and PHA-L accumulates at very reduced levels. To investigate the cause of the Pinto phenotype, we cloned and sequenced the two PHA genes of Pinto, called Pdlec1 and Pdlec2, and determined the abundance of their respective mRNAs in developing cotyledons. Both genes are more than 90% homologous to the normal PHA genes found in other cultivars. Pdlec1 carries a 1-bp frameshift mutation close to the 5' end of its coding sequence. Only very truncated polypeptides could be made from its mRNA. The gene Pdlec2 encodes a polypeptide, which resembles PHA-L and its predicted amino acid sequence agrees with the available Pinto PHA amino acid sequence data. Analysis of the mRNA of developing cotyledons revealed that the Pdlec1 message is reduced 600-fold, and Pdlec2 mRNA is reduced 20-fold with respect to mRNA levels in normal cultivars. A comparison of the sequences which are upstream from the coding sequence shows that Pdlec2 has a 100-bp deletion compared to the other genes (dlec1, dlec2 and Pdlec1). This deletion which contains a large tandem repeat may be responsible for the low level of expression of Pdlec2. The very low expression of Pdlec1 is as yet unexplained. ImagesFig. 5. PMID:16453730

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

An anti-HIV-1 compound that increases steady-state expression of apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G.

PubMed

Ejima, Tomohiko; Hirota, Mayuko; Mizukami, Tamio; Otsuka, Masami; Fujita, Mikako

2011-10-01

Human apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) 3G (A3G) is an antiviral protein that blocks HIV-1 replication. However, the antiviral activity of A3G is overcome by the HIV-1 protein Vif. This inhibitory function of Vif is related to its ability to degrade A3G in the proteasome. This finding prompted us to examine the activities of 4-(dimethylamino)-2,6-bis[(N-(2-[(2-nitrophenyl)dithio]ethyl)amino)methyl]pyridine (SN-2) and SN-3. We found that 5 µM SN-2 increases the expression of A3G to a level much higher than that observed in the absence of Vif, without affecting the level of Vif expression. The proteasome inhibitor MG-132 increased the level of both A3G and Vif expression. These results demonstrate that A3G is ubiquitinated and degraded in the proteasome by a factor other than Vif, and that SN-2 selectively inhibits these processes. Furthermore, 5 µM SN-2 significantly inhibited the MAGI cell infectivity of wild-type HIV-1. These findings may contribute to the development of a novel anti-HIV-1 drug.

Isolation of pheromone precursor genes of Magnaporthe grisea.

PubMed

Shen, W C; Bobrowicz, P; Ebbole, D J

1999-01-01

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia. Copyright 1999 Academic Press.

Two-color Dye-swap DNA Microarray approach toward confident gene expression profiling in PMCAO mouse model for ischemia-related and PACAP38-influenced genes

PubMed Central

Hori, Motohide; Shibato, Junko; Nakamachi, Tomoya; Rakwal, Randeep; Ogawa, Tetsuo; Shioda, Seiji; Numazawa, Satoshi

2015-01-01

Toward twin goals of identifying molecular factors in brain injured by ischemic stroke, and the effects of neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain, we have established the permanent middle cerebral artery occlusion (PMCAO) mouse model and utilized the Agilent mouse whole genome 4 × 44 K DNA chip. PACAP38 (1 pmol) injection was given intracerebroventrically in comparison to a control saline (0.9% NaCl) injection, to screen genes responsive to PACAP38. Two sets of tissues were prepared, whole hemispheres (ischemic and non-ischemic) and infract core and penumbra regions at 6 and 24 h. In this study, we have detailed the experimental design and protocol used therein and explained the quality controls for the use of total RNA in the downstream DNA microarray experiment utilizing a two-color dye-swap approach for stringent and confident gene identification published in a series of papers by Hori and coworkers (Hori et al., 2012–2015). PMID:26484166

Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

PubMed

Park, Hongsuk; Cho, Sungpil; Janat-Amsbury, Margit M; Bae, You Han

2015-12-01

Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Technical Abstract: Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered...

Uses of Ku70

DOEpatents

Li, Gloria C.; Cordon-Cardo, Carlos; Ouyang, Honghai

2004-10-26

This invention provides a method of diagnosing a predisposition to cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and; (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and (b) determining whether one or more of the subject's Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject's expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: determining the subcellular localization of Ku70 in the subject, wherein an abnormal subcellular localization of Ku70 indicates a predisposition to cancer.

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus

PubMed Central

1984-01-01

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament- desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2- aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein. PMID:6491602

In Search of Functional Advantages of Knots in Proteins.

PubMed

Dabrowski-Tumanski, Pawel; Stasiak, Andrzej; Sulkowska, Joanna I

2016-01-01

We analysed the structure of deeply knotted proteins representing three unrelated families of knotted proteins. We looked at the correlation between positions of knotted cores in these proteins and such local structural characteristics as the number of intra-chain contacts, structural stability and solvent accessibility. We observed that the knotted cores and especially their borders showed strong enrichment in the number of contacts. These regions showed also increased thermal stability, whereas their solvent accessibility was decreased. Interestingly, the active sites within these knotted proteins preferentially located in the regions with increased number of contacts that also have increased thermal stability and decreased solvent accessibility. Our results suggest that knotting of polypeptide chains provides a favourable environment for the active sites observed in knotted proteins. Some knotted proteins have homologues without a knot. Interestingly, these unknotted homologues form local entanglements that retain structural characteristics of the knotted cores.

Glucagon-related peptides in the mouse retina and the effects of deprivation of form vision.

PubMed

Mathis, Ute; Schaeffel, Frank

2007-02-01

In chickens, retinal glucagon amacrine cells play an important role in emmetropization, since they express the transcription factor ZENK (also known as NGFI-A, zif268, tis8, cef5, Krox24) in correlation with the sign of imposed image defocus. Pharmacological studies have shown that glucagon can act as a stop signal for axial eye growth, making it a promising target for pharmacological intervention of myopia. Unfortunately, in mammalian retina, glucagon itself has not yet been detected by immunohistochemical staining. To learn more about its possible role in emmetropization in mammals, we studied the expression of different members of the glucagon hormone family in mouse retina, and whether their abundance is regulated by visual experience. Black wildtype C57BL/6 mice, raised under a 12/12 h light/dark cycle, were studied at postnatal ages between P29 and P40. Frosted hemispherical thin plastic shells (diffusers) were placed in front of the right eyes to impose visual conditions that are known to induce myopia. The left eyes remained uncovered and served as controls. Transversal retinal cryostat sections were single- or double-labeled by indirect immunofluorescence for early growth response protein 1 (Egr-1, the mammalian ortholog of ZENK), glucagon, glucagon-like peptide-2 (GLP-2), glucose-dependent insulinotropic polypeptide (GIP), peptide histidine isoleucine (PHI), growth hormone-releasing hormone (GHRH), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, and vasoactive intestinal polypeptide (VIP). In total, retinas of 45 mice were studied, 28 treated with diffusers, and 17 serving as controls. Glucagon itself was not detected in mouse retina. VIP, PHI, PACAP and GIP were localized. VIP was co-localized with PHI and Egr-1, which itself was strongly regulated by retinal illumination. Diffusers, applied for various durations (1, 2, 6, and 24 h) had no effect on the expression of VIP, PHI, PACAP, and GIP, at least at the protein level. Similarly, even if the analysis was confined to cells that also expressed Egr-1, no difference was found between VIP expression in eyes with diffusers and in eyes with normal vision. Several members of the glucagon super family are expressed in mouse retina (although not glucagon itself), but their expression pattern does not seem to be regulated by visual experience.

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Developmental and cell-specific expression of thyroid hormone transporters in the mouse cochlea.

PubMed

Sharlin, David S; Visser, Theo J; Forrest, Douglas

2011-12-01

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr(-/-) mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development.

Pancreatic polypeptide controls energy homeostasis via Npy6r signaling in the suprachiasmatic nucleus in mice.

PubMed

Yulyaningsih, Ernie; Loh, Kim; Lin, Shu; Lau, Jackie; Zhang, Lei; Shi, Yanchuan; Berning, Britt A; Enriquez, Ronaldo; Driessler, Frank; Macia, Laurence; Khor, Ee Cheng; Qi, Yue; Baldock, Paul; Sainsbury, Amanda; Herzog, Herbert

2014-01-07

Y-receptors control energy homeostasis, but the role of Npy6 receptors (Npy6r) is largely unknown. Young Npy6r-deficient (Npy6r(-/-)) mice have reduced body weight, lean mass, and adiposity, while older and high-fat-fed Npy6r(-/-) mice have low lean mass with increased adiposity. Npy6r(-/-) mice showed reduced hypothalamic growth hormone releasing hormone (Ghrh) expression and serum insulin-like growth factor-1 (IGF-1) levels relative to WT. This is likely due to impaired vasoactive intestinal peptide (VIP) signaling in the suprachiasmatic nucleus (SCN), where we found Npy6r coexpressed in VIP neurons. Peripheral administration of pancreatic polypeptide (PP) increased Fos expression in the SCN, increased energy expenditure, and reduced food intake in WT, but not Npy6r(-/-), mice. Moreover, intraperitoneal (i.p.) PP injection increased hypothalamic Ghrh mRNA expression and serum IGF-1 levels in WT, but not Npy6r(-/-), mice, an effect blocked by intracerebroventricular (i.c.v.) Vasoactive Intestinal Peptide (VPAC) receptors antagonism. Thus, PP-initiated signaling through Npy6r in VIP neurons regulates the growth hormone axis and body composition. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

PubMed

Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

2014-06-01

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

PubMed Central

Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-01-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

PubMed

Juhász, Tamás; Szentléleky, Eszter; Somogyi, Csilla Szűcs; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-07-29

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

[Cloning, expressing of exendin-4 analogue and bioactivity analysis in vivo].

PubMed

Li, Taiming; Gu, Chunjiao; Ge, Xiaoyu; Li, Zhezhe; Wang, Dan; Ma, Yanhong; Liu, Tao; Zhang, Meiyou; Li, Li; Liu, Jingjing

2012-07-01

To construct, express and purify Exendin-4 analogue and detect its biological activity in vivo. Insert gene sequence into fusion partner ofpED plasmid which is helped to purification, entitled the new recombinant plasmid 5 Exendin-4 analogue polypeptide gene and fusion partner gene was linked by acid hydrolysisgene, transformed to E. coli BL21 and the fusion protein was induced by lactose. After acid hydrolysis, the Exendin-4 analogue polypeptide separated from fusion chaperon. Anion charge chromatography were used to further purification. 6 to 8 week-old ICR mice were injected (s.c) with Exendin-4 analogue, blood glucose and plasma insulin level was detected in different period after oral glucose tolerance test. The results show that high expression of inclusion body was induced by lactose, which accounted for 40% of germ proteins, the Exendin-4 analogue was obtained with the purity of 91.8% after being purified by anion charge chromatography. Bioactivity assay showed that the level of blood glucose of mouse which treated with exendin-4 analogue was obviously decreased to normal (P < 0.01), and the level of plasma insulin was increased obviously (P < 0.01).

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

Formation of hybrid phycobilisomes by association of phycobiliproteins from Nostoc and Fremyella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canaani, O.; Gantt, E.

1982-09-01

Formation of phycobilisomes has been accomplished in vitro from isolated phycobiliprotein fraction obtained from the same blue-green alga (intrageneric) and from different blue-green algae (intergeneric). Phycobilisomes, which are supramolecular complexes of phycobiliproteins, serve as major light-harvesting antennae for photosynthesis in blue-green and red algae. Intrageneric association into energetically functional phycobilisomes, previously reported to occur with Nostoc sp. allophycocyanin and phycoerythrin-phycocyanin complexes has been obtained with Fremyella diplosiphon. By their spectral propeties (absorption, fluorescence excitation, and emission) and electron microscopic images, the native and in vitro-associated phycobilisomes were virtually indistinguishable. Intergeneic phycobilisomes have been produced from allophycocyanin of Nostoc sp. strainmore » Mac, and phycoerythrin-phycocyanin of F. diplosiphon, as well as from the reverse mixtures. Phycobilisomes of Nostoc and Fremyella, analyzed by NaDodSO/sub 4//polyacrylamide gel electrophoresis, possessed a number of polypeptides having similar molecular weights: the usual ..cap alpha..- and ..beta..-phycobilin-containing polypeptides of M/sub r/ 15,000-22,000, a faint band at M/sub r/ ca. 95,000, and a prominent band at M/sub r/ ca. 31,000. The M/sub r/ 31,000 polypeptide is assumed to provide the recognition site for attachment of the phycoerythrin-phycocyanin complexes with the allophycocyanin core. In vitro association was not obtained between allophycocyanin from Nostoc and phycoerythrin-phycocyanin complexes from Phormidium persicinum or Porphyridium sordidum.« less

Primary light-harvesting system: phycobilisomes and associated membranes. Progress report, December 1979-January 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, E.

1983-01-01

The structure of phycobilisomes, which serve as primary light-harvesting complexes for photosynthesis, were investigated in red and blue-green algae (cyanobacteria). Structurally the phycobilisomes have the same fundamental arrangement in the various species, with allophycocyanin being in the core from which peripheral rods composed of phycocyanin or phycocyanin-phycoerythyrin radiate. Such studies were extended to show that energetically functional phycobilisomes could be formed in vitro from separated fractions of allophycocyanin and phycoerythrin-phycocyanin within the same species (Nostoc) and between separate species (Fremyella and Nostoc). A large (95 kD) blue-colored polypeptide is being regarded as an anchor component between phycobilisomes and thylakoids. Themore » polypeptide, isolated from phycobilisomes, had two long wavelength-emitting types of chromophores, one similar to allophycocyanin and another to chlorophyll. Such a large (95 kD) polypeptide occurred in phycobilisomes of red and blue-green algae examined, with the exception of Anacystis where it was somewhat smaller (75 kD). The effect of light quality on phycobilisome structure varied among the blue-green species studied. The relationship of the photosystems and phycobilisomes was investigated in Anacystis wild type cells, and in mutants deficient in phycocyanin. Although the phycocyanin and chlorophyll content per cell changed with light quality the phycobilisome per Reaction Center 2 ratio remained at ca. 0.9 +- 0.3 suggesting that only the size of the antennae varied.« less

Serum peptide expression and treatment responses in patients with advanced non-small-cell lung cancer

PubMed Central

An, Juan; Tang, Chuan-Hao; Wang, Na; Liu, Yi; Lv, Jin; Xu, Bin; Li, Xiao-Yan; Guo, Wan-Feng; Gao, Hong-Jun; He, Kun; Liu, Xiao-Qing

2018-01-01

Epidermal growth factor receptor (EGFR) mutation is an important predictor for response to personalized treatments of patients with advanced non-small-cell lung cancer (NSCLC). However its usage is limited due to the difficult of obtaining tissue specimens. A novel prediction system using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a perspective tool in European countries to identify patients who are likely to benefit from EGFR tyrosine kinase inhibitor (TKI) treatment. In the present study, MALDI-TOF MS was used on pretreatment serum samples of patients with advanced non-small-cell lung cancer to discriminate the spectra between disease control and disease progression groups in one cohort of Chinese patients. The candidate features for classification were subsequently validated in a blinded fashion in another set of patients. The correlation between plasma EGFR mutation status and the intensities of representative spectra for classification was evaluated. A total of 103 patients that were treated with EGFR-TKIs were included. It was determined that 8 polypeptides peaks were significant different between the disease control and disease progression group. A total of 6 polypeptides were established in the classification algorithm. The sensitivity of the algorithm to predict treatment responses was 76.2% (16/21) and the specificity was 81.8% (18/22). The accuracy rate of the algorithm was 79.1% (34/43). A total of 3 polypeptides were significantly correlated with EGFR mutations (P=0.04, P=0.03 and P=0.04, respectively). The present study confirmed that MALDI-TOF MS analysis can be used to predict responses to EGFR-TKI treatment of the Asian population where the EGFR mutation status differs from the European population. Furthermore, the expression intensities of the three polypeptides in the classification model were associated with EGFR mutation. PMID:29844828

In vitro myogenesis induced by human recombinant elastin-like proteins.

PubMed

D'Andrea, Paola; Scaini, Denis; Ulloa Severino, Luisa; Borelli, Violetta; Passamonti, Sabina; Lorenzon, Paola; Bandiera, Antonella

2015-10-01

Mammalian adult skeletal muscle has a limited ability to regenerate after injury, usage or trauma. A promising strategy for successful regenerative technology is the engineering of bio interfaces that mimic the characteristics of the extracellular matrix. Human elastin-like polypeptides (HELPs) have been synthesized as biomimetic materials that maintain some peculiar properties of the native protein. We developed a novel Human Elastin Like Polypeptide obtained by fusing the elastin-like backbone to a domain present in the α2 chain of type IV collagen, containing two RGD motives. We employed this peptide as adhesion substrate for C2C12 myoblasts and compared its effects to those induced by two other polypeptides of the HELP series. Myoblast adhered to all HELPs coatings, where they assumed morphology and cytoarchitecture that depended on the polypeptide structure. Adhesion to HELPs stimulated at a different extent cell proliferation and differentiation, the expression of Myosin Heavy Chain and the fusion of aligned fibers into multinucleated myotubes. Adhesion substrates significantly altered myotubes stiffness, measured by Atomic Force Microscopy, and differently affected the cells Ca(2+) handling capacity and the maturation of excitation-contraction coupling machinery, evaluated by Ca(2+) imaging. Overall, our findings indicate that the properties of HELP biopolymers can be exploited for dissecting the molecular connections underlying myogenic differentiation and for designing novel substrates for skeletal muscle regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of cell penetrating Notch inhibitory peptide conjugated to elastin-like polypeptide on glioblastoma cells.

PubMed

Opačak-Bernardi, Teuta; Ryu, Jung Su; Raucher, Drazen

2017-07-01

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood-brain barrier penetration. The SynB1-ELP1-dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes' expression. SynB1-ELP1-dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1-ELP1-dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.

KnotProt: a database of proteins with knots and slipknots

PubMed Central

Jamroz, Michal; Niemyska, Wanda; Rawdon, Eric J.; Stasiak, Andrzej; Millett, Kenneth C.; Sułkowski, Piotr; Sulkowska, Joanna I.

2015-01-01

The protein topology database KnotProt, http://knotprot.cent.uw.edu.pl/, collects information about protein structures with open polypeptide chains forming knots or slipknots. The knotting complexity of the cataloged proteins is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. The pattern visible in the matrix gives the knotting fingerprint of a given protein and permits users to determine, for example, the minimal length of the knotted regions (knot's core size) or the depth of a knot, i.e. how many amino acids can be removed from either end of the cataloged protein structure before converting it from a knot to a different type of knot. In addition, the database presents extensive information about the biological functions, families and fold types of proteins with non-trivial knotting. As an additional feature, the KnotProt database enables users to submit protein or polymer chains and generate their knotting fingerprints. PMID:25361973

Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

PubMed Central

LoGrasso, P V; Moll, F; Cross, T A

1988-01-01

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished. PMID:2462923

Neuroendocrine carcinoma in the nasal cavity of ten dogs.

PubMed

Sako, T; Shimoyama, Y; Akihara, Y; Ohmachi, T; Yamashita, K; Kadosawa, T; Nakade, T; Uchida, E; Okamoto, M; Hirayama, K; Taniyama, H

2005-01-01

Neuroendocrine (NE) carcinoma was diagnosed in 10 dogs. In six cases examined by cephalometric radiography and computerized tomography, a large mass was seen to fill the nasal cavity. Histopathologically, sheets, nests or ribbons of neoplastic cells were separated by delicate or thick fibrovascular stroma. The neoplastic cells were round, oval, or spindle-shaped; cytoplasmic granules and hyperchromatic nuclei with prominent nucleoli were present. Neoplastic cells were invariably immunohistochemically positive for cytokeratin (CK) AE1/AE3, neuron-specific enolase, chromogranin A and vasoactive intestinal polypeptide. Eight dogs were positive for S100 protein, seven for synaptophysin, five for protein gene product 9.5, two for somatostatin, and one for Leu-7. Immunolabelling gave negative results for CK 8, CK 19, calcitonin, calcitonin gene-related polypeptide, neurofilaments, serotonin, gastrin and glial fibrillary acidic protein. Ultrastructurally, the neoplastic cells contained a large number of round, membrane-bounded, densely-cored granules corresponding to neurosecretory granules. These observations were consistent with the neuroendocrine nature of the carcinomas.

Cyclin Polynucleotides, Polypeptides And Uses Thereof.

DOEpatents

Lowe, Keith S.; Tao, Yumin; Gordon-Kamm, William J.; Gregory, Carolyn A.; Hoerster, George J.; McElver, John A.

2003-02-11

The invention provides isolated polynucleotides and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content and/or composition of plants.

Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

PubMed

Soria-Guerra, Ruth E; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; López-Revilla, Rubén; Alpuche-Solís, Angel G

2011-03-01

DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

PubMed

Pushnova, E A; Ostanin, K; Thelen, M P

2001-11-01

Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

NASA Astrophysics Data System (ADS)

Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

2015-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

Identification and expression analysis of zebrafish polypeptide α-N-acetylgalactosaminyltransferase Y-subfamily genes during embryonic development.

PubMed

Nakayama, Yoshiaki; Nakamura, Naosuke; Kawai, Tamiko; Kaneda, Eiichi; Takahashi, Yui; Miyake, Ayumi; Itoh, Nobuyuki; Kurosaka, Akira

2014-09-01

Mucin-type glycosylation is one of the most common posttranslational modifications of secretory and membrane proteins and has diverse physiological functions. The initial biosynthesis of mucin-type carbohydrates is catalyzed by UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) encoded by GALNT genes. Among these, GalNAc-T8, -T9, -T17, and -T18 form a characteristic subfamily called "Y-subfamily" and have no or very low in vitro transferase activities when assayed with typical mucin peptides as acceptor substrates. Although the Y-subfamily isozymes have been reported to be possibly involved in various diseases, their in vivo functions have not been reported. Here, we isolated zebrafish Y-subfamily galnt genes, and determined their spatial and temporal expressions during the early development of zebrafish. Our study demonstrated that all the Y-subfamily isozymes were well conserved in zebrafish with GalNAc-T18 having two orthologs, galnt18a and galnt18b, and with the other three isozymes each having a corresponding ortholog, galnt8, galnt9, and galnt17. The galnt8 was expressed in the cephalic mesoderm and hatching gland during early developmental stages, and differently expressed in the head, somatic muscles, and liver in the later stages. The other three orthologs also exhibited the characteristic expression patterns, although their expressions were generally strong in the nervous systems. In addition to the expression in the brain, galnt17 and galnt18a were expressed in the somitic muscles, and galnt18a and galnt18b in the notochord. These expression patterns may contribute to the functional analysis of the Y-subfamily, whose physiological roles still remain to be elucidated. Copyright © 2014 Elsevier B.V. All rights reserved.

Degradation of triglycerides by a pseudomonad isolated from milk: molecular analysis of a lipase-encoding gene and its expression in Escherichia coli.

PubMed Central

Johnson, L A; Beacham, I R; MacRae, I C; Free, M L

1992-01-01

Psychrotrophic lipolytic bacteria represent a significant problem in the storage of refrigerated dairy products. A lipase-encoding gene has been cloned and characterized from a highly lipolytic strain of Pseudomonas. The nucleotide sequence of the gene predicts a polypeptide of M(r) 49,905, which was identified when the gene was expressed in Escherichia coli. Images PMID:1622251

Multiple polypeptides immunologically related to beta-poly(L-malate) hydrolase (polymalatase) in the plasmodium of the slime mold Physarum polycephalum.

PubMed

Karl, M; Holler, E

1998-01-15

Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.

Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[submore » r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.« less

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

PubMed

Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-10-25

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

PubMed Central

Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-01-01

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians. PMID:7937944

Who knows not where an anemone does wear his sting? Could polypeptides released from the columnar vesicles of Bunodosoma cangicum induce apoptosis in the ZF-L cell line?

PubMed

Bastos, Claudio L Q; Varela, Antonio Sergio; Ferreira, Shana Pires; Nornberg, Bruna Felix; Boyle, Robert Tew

2016-12-15

We provide ultrastructural and cytological evidence that the tentacles of the sea anemone Bunodosoma cangicum does not contain cytotoxic venom. However, we show that the stimulated secretion of an apparent mixture of biomolecules containing polypeptides from the columnar vesicles of Bunodosoma cangicum is apparently a potent inducer of apoptosis in the zebrafish cell line, ZF-L. Microscopic fluorescence, cell morphology and flow cytometric assays confirm the apoptotic activity. Crude vesicle venom was partially purified by size exclusion chromatography. PAGE analysis shows that this venom contains low weight polypeptides but no measurable protein. The apoptotic activity is heat labile, and the observed peptides concurrent with this activity have a molecular weight of approximately 2000 Da. This manuscript is the first report of biologically active molecules and peptides associated with columnar vesicles of anemones, and the first to confirm that the tentacles of B. cangicum do not contain cytotoxic venom, and express spirocytes exclusively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

2012-02-21

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian; Kleff, Susanne; Guettler, Michael V

2013-04-30

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Functional analyses of the cell wall hydrolase from Lactobacillus paracasei NRRL B-50314 expressed in Bacillus megaterium

USDA-ARS?s Scientific Manuscript database

This study reports the production and characterization of a novel antibacterial polypeptide, designated laparaxin, which is secreted by Lactobacillus paracasei NRRL B-50314. Crude laparaxin has antibacterial activity against a range of Gram-positive bacteria including the following: lactic acid bact...

Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swartz, James

2017-01-25

Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.

Polypeptide Synthesis in Simian Virus 5-Infected Cells

PubMed Central

Peluso, Richard W.; Lamb, Robert A.; Choppin, Purnell W.

1977-01-01

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F0) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F1 and F2. No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed. Images PMID:196101

Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family.

PubMed

Schwartz, N B; Pirok, E W; Mensch, J R; Domowicz, M S

1999-01-01

Proteoglycans are complex macromolecules, consisting of a polypeptide backbone to which are covalently attached one or more glycosaminoglycan chains. Molecular cloning has allowed identification of the genes encoding the core proteins of various proteoglycans, leading to a better understanding of the diversity of proteoglycan structure and function, as well as to the evolution of a classification of proteoglycans on the basis of emerging gene families that encode the different core proteins. One such family includes several proteoglycans that have been grouped with aggrecan, the large aggregating chondroitin sulfate proteoglycan of cartilage, based on a high number of sequence similarities within the N- and C-terminal domains. Thus far these proteoglycans include versican, neurocan, and brevican. It is now apparent that these proteins, as a group, are truly a gene family with shared structural motifs on the protein and nucleotide (mRNA) levels, and with nearly identical genomic organizations. Clearly a common ancestral origin is indicated for the members of the aggrecan family of proteoglycans. However, differing patterns of amplification and divergence have also occurred within certain exons across species and family members, leading to the class-characteristic protein motifs in the central carbohydrate-rich region exclusively. Thus the overall domain organization strongly suggests that sequence conservation in the terminal globular domains underlies common functions, whereas differences in the central portions of the genes account for functional specialization among the members of this gene family.

AmgRS-mediated envelope stress-inducible expression of the mexXY multidrug efflux operon of Pseudomonas aeruginosa

PubMed Central

Lau, Calvin Ho-Fung; Krahn, Thomas; Gilmour, Christie; Mullen, Erin; Poole, Keith

2015-01-01

AmgRS is an envelope stress-responsive two-component system and aminoglycoside resistance determinant in Pseudomonas aeruginosa that is proposed to protect cells from membrane damage caused by aminoglycoside-generated mistranslated polypeptides. Consistent with this, a ΔamgR strain showed increased aminoglycoside-promoted membrane damage, damage that was largely absent in AmgRS-activated amgS-mutant strains. Intriguingly, one such mutation, V121G, while providing for enhanced resistance to aminoglycosides, rendered P. aeruginosa susceptible to several ribosome-targeting nonaminoglycoside antimicrobials that are inducers and presumed substrates of the MexXY-OprM multidrug efflux system. Surprisingly, the amgSV121G mutation increased mexXY expression threefold, suggesting that export of these nonaminoglycosides was compromised in the amgSV121G mutant. Nonetheless, a link was established between AmgRS activation and mexXY expression and this was confirmed in studies showing that aminoglycoside-promoted mexXY expression is dependent on AmgRS. While nonaminoglycosides also induced mexXY expression, this was not AmgRS-dependent, consistent with these agents not generating mistranslated polypeptides and not activating AmgRS. The aminoglycoside inducibility of mexXY was abrogated in a mutant lacking the AmgRS target genes htpX and PA5528, encoding a presumed cytoplasmic membrane-associated protease and a membrane protein of unknown function, respectively. Thus, aminoglycoside induction of mexXY is a response to membrane damage and activation of the AmgRS two-component system. PMID:25450797

Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor

PubMed Central

Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

2016-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1+ were large in size, had oval nuclei, and merged with CD34+ cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1−/− mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1−/− and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

PubMed

Mead, Emma J; Chiverton, Lesley M; Spurgeon, Sarah K; Martin, Elaine B; Montague, Gary A; Smales, C Mark; von der Haar, Tobias

2012-01-01

Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-Tmore » Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.« less

Developmental and Cell-Specific Expression of Thyroid Hormone Transporters in the Mouse Cochlea

PubMed Central

Sharlin, David S.; Visser, Theo J.

2011-01-01

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr−/− mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development. PMID:21878515

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

PubMed Central

1986-01-01

A collection of 17 monoclonal antibodies elicited against the light- harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC- II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b. PMID:3528171

Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro.

PubMed

Kolesinski, Piotr; Belusiak, Iwona; Czarnocki-Cieciura, Mariusz; Szczepaniak, Andrzej

2014-09-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi-step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea mays). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF1 from Thermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in Escherichia coli cells. Moreover, when co-expressed with RbcL alone, a stable RbcL-RAF1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size-exclusion chromatography coupled with multi-angle light scattering indicates that it consists of an RbcL dimer and two RAF1 molecules. A purified RbcL-RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL8) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL-RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process. © 2014 FEBS.

Expression of eukaryotic polypeptides in chloroplasts

DOEpatents

Mayfield, Stephen P.

2013-06-04

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Cell cycle nucleic acids, polypeptides and uses thereof

DOEpatents

Gordon-Kamm, William J [Urbandale, IA; Lowe, Keith S [Johnston, IA; Larkins, Brian A [Tucson, AZ; Dilkes, Brian R [Tucson, AZ; Sun, Yuejin [Westfield, IN

2007-08-14

The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

Cloning of the chaperonin t-complex polypeptide 1 gene from Schistosoma mansoni and studies of its expression levels under heat shock and oxidative stress.

PubMed

Campos, E G; Hamdan, F F

2000-03-01

The protein TCP-1 (t-complex polypeptide 1) is a subunit of the hetero-oligomeric complex CCT (chaperonin containing TCP- 1) present in the eukaryotic cytosol. Chaperone function may be critical for the development and survival of the different life stages of Schistosoma mansoni, a parasite that is exposed to drastic environmental changes during its development. We isolated a full-length S. mansoni TCP-1 cDNA (SmTCP-1A) encoding a protein highly homologous with TCP-1. The deduced SmTCP-1A amino-acid sequence shows up to 65% identity with other eukaryotic CCT family members. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the mRNA expression levels of SmTCP-1A in adult S. mansoni were down-regulated in worms subjected to heat shock and oxidative stress conditions. This down-regulation of SmTCP-1A mRNA may reflect a switch in CCT subunits as an adaptive response to heat shock and oxidative stress conditions.

The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshino, Tomoko; Maeda, Yasuo; Amagai, Aiko

2007-08-03

Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method.more » Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.« less

Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus

PubMed Central

Yan, Huan; Zhong, Guocai; Xu, Guangwei; He, Wenhui; Jing, Zhiyi; Gao, Zhenchao; Huang, Yi; Qi, Yonghe; Peng, Bo; Wang, Haimin; Fu, Liran; Song, Mei; Chen, Pan; Gao, Wenqing; Ren, Bijie; Sun, Yinyan; Cai, Tao; Feng, Xiaofeng; Sui, Jianhua; Li, Wenhui

2012-01-01

Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV. DOI: http://dx.doi.org/10.7554/eLife.00049.001 PMID:23150796

Expression of VGF mRNA in developing neuroendocrine and endocrine tissues.

PubMed

Snyder, S E; Peng, B; Pintar, J E; Salton, S R J

2003-11-01

Analysis of knockout mice suggests that the neurotropin-inducible secreted polypeptide VGF (non-acronymic) plays an important role in the regulation of energy balance. VGF is synthesized by neurons in the central and peripheral nervous systems (CNS, PNS), as well as in the adult pituitary, adrenal medulla, endocrine cells of the stomach and pancreatic beta cells. Thus VGF, like cholecystokinin, leptin, ghrelin and other peptide hormones that have been shown to regulate feeding and energy expenditure, is synthesized in both the gut and the brain. Although detailed developmental studies of VGF localization in the CNS and PNS have been completed, little is known about the ontogeny of VGF expression in endocrine and neuroendocrine tIssues. Here, we report that VGF mRNA is detectable as early as embryonic day 15.5 in the developing rat gastrointestinal and esophageal lumen, pancreas, adrenal, and pituitary, and we further demonstrate that VGF mRNA is synthesized in the gravid rat uterus, together supporting possible functional roles for this polypeptide outside the nervous system and in the enteric plexus.

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

PubMed

Park, Hyun-Jeong; Choi, Bum-Chae; Song, Sang-Jin; Lee, Dong-Sik; Roh, Jaesook; Chun, Sang-Young

2010-01-01

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

PubMed Central

Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

2011-01-01

Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

Proteins in Relation to Vigor and Viability of White Lupin (Lupinus albus L.) Seed Stored for 26 Years

PubMed Central

Dobiesz, Malwina; Piotrowicz-Cieślak, Agnieszka I.

2017-01-01

The aim of the study was to evaluate the vigor and viability as well as to determine and compare the contents of selected protein fractions of white lupin (Lupinus albus L.) seeds stored for 26 years at temperatures of -14°C and +20°C. The seeds stored at -14°C germinated in 86.3%, while the seeds stored at +20°C did not germinate at all. The viability evaluation was confirmed by the measuring electroconductivity of seed exudates. In seeds stored at -14°C the contents of γ, δ, and β conglutin were 14, 4 and 69 mg g-1 fresh mass, respectively, while in seed stored at +20°C they were 15.5, 3, 65 mg g-1 fresh mass, respectively. One-dimensional electrophoresis of γ and δ conglutin fractions indicated the presence of several intense polypeptide bands with molecular weights from 23.0 to 10.3 kDa. Polypeptide bands with a molecular weight of 22.4 and 19.8 kDa exhibited almost two times higher expression in the seeds stored at -14°C compared to the seeds stored at +20°C. Electrophoresis revealed 310 protein spots on the maps generated for seeds stored at -14°C, and 228 spots for seeds stored at +20°C. In seeds stored at +20°C most polypeptide subunits had a pI ranging from 4.5 to 7 and a molecular weight of 10–97 kDa. The greatest differences in the contents of polypeptides between the analyzed variants was observed within the range of 20–45 kDa (-14°C: 175, +20°C: 115 protein spots) and within the range of 65–97 kDa (-14°C: 103, +20°C: 75 protein spots). In seeds stored at +20°C, a clear decline in basic (8–10 pI) polypeptides was observed. The study demonstrated that the polypeptides identified as γ and δ conglutins are probably closely related to vigor and viability of seeds. PMID:28848591

Phage-displayed specific polypeptide antigens induce significant protective immunity against Trichinella spiralis infection in BALB/c mice.

PubMed

Cui, Jing; Ren, Hui Jun; Liu, Ruo Dan; Wang, Li; Zhang, Zi Fang; Wang, Zhong Quan

2013-02-06

Trichinellosis is a public health problem and is considered an emerging/re-emerging disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects humans, domestic animals and wildlife. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Phage particles are especially ideal vaccine delivery vehicles because they do not interfere with the immune response against the displayed peptide antigens, and, if anything, are more likely to efficiently direct antigen expression to professional antigen-presenting cells. In this study, Tsp10 polypeptide, which was encoded by a cDNA fragment of Trichinella spiralis intestinal infective larvae and was found to bind to normal mouse intestinal cells, was displayed on the surface of T7 phage. Anti-Tsp10 antibodies were able to recognize the native Tsp10 protein mainly localized to the stichosome of T. spiralis. Mice immunized with the recombinant phage T7-Tsp10 showed a 62.8% reduction in adult worms and a 78.6% reduction in muscle larvae following challenge with T. spiralis muscle larvae. Our results demonstrate that the vaccination with Tsp10 polypeptide displayed by T7 phage elicits the Th2-predominant immune responses and produces a significant protection against T. spiralis infection in mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against T. spiralis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transporter Expression in Liver Tissue from Subjects with Alcoholic or Hepatitis C Cirrhosis Quantified by Targeted Quantitative Proteomics

PubMed Central

Wang, Li; Collins, Carol; Kelly, Edward J.; Chu, Xiaoyan; Ray, Adrian S.; Salphati, Laurent; Xiao, Guangqing; Lee, Caroline; Lai, Yurong; Liao, Mingxiang; Mathias, Anita; Evers, Raymond; Humphreys, William; Hop, Cornelis E. C. A.; Kumer, Sean C.

2016-01-01

Although data are available on the change of expression/activity of drug-metabolizing enzymes in liver cirrhosis patients, corresponding data on transporter protein expression are not available. Therefore, using quantitative targeted proteomics, we compared our previous data on noncirrhotic control livers (n = 36) with the protein expression of major hepatobiliary transporters, breast cancer resistance protein (BCRP), bile salt export pump (BSEP), multidrug and toxin extrusion protein 1 (MATE1), multidrug resistance–associated protein (MRP)2, MRP3, MRP4, sodium taurocholate–cotransporting polypeptide (NTCP), organic anion–transporting polypeptides (OATP)1B1, 1B3, 2B1, organic cation transporter 1 (OCT1), and P-glycoprotein (P-gp) in alcoholic (n = 27) and hepatitis C cirrhosis (n = 30) livers. Compared with control livers, the yield of membrane protein from alcoholic and hepatitis C cirrhosis livers was significantly reduced by 56 and 67%, respectively. The impact of liver cirrhosis on transporter protein expression was transporter-dependent. Generally, reduced protein expression (per gram of liver) was found in alcoholic cirrhosis livers versus control livers, with the exception that the expression of MRP3 was increased, whereas no change was observed for MATE1, MRP2, OATP2B1, and P-gp. In contrast, the impact of hepatitis C cirrhosis on protein expression of transporters (per gram of liver) was diverse, showing an increase (MATE1), decrease (BSEP, MRP2, NTCP, OATP1B3, OCT1, and P-gp), or no change (BCRP, MRP3, OATP1B1, and 2B1). The expression of hepatobiliary transporter protein differed in different diseases (alcoholic versus hepatitis C cirrhosis). Finally, incorporation of protein expression of OATP1B1 in alcoholic cirrhosis into the Simcyp physiologically based pharmacokinetics cirrhosis module improved prediction of the disposition of repaglinide in liver cirrhosis patients. These transporter expression data will be useful in the future to predict transporter-mediated drug disposition in liver cirrhosis patients. PMID:27543206

Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

PubMed Central

Wang, A M; Schindler, D; Desnick, R

1990-01-01

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, alpha-N-acetylgalctosaminidase (alpha-GalNAc). The recent isolation and expression of the full-length cDNA encoding alpha-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal alpha-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the alpha-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the alpha-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an alpha-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Images PMID:2243144

Characterization of CCTα and evaluating its expression in the mud crab Scylla paramamosain when challenged by low temperatures alone and in combination with high and low salinity.

PubMed

Yu, Kun; Gong, Jie; Huang, Chencui; Huang, Huiyang; Ye, Haihui; Wang, Guizhong; Zeng, Chaoshu

2015-09-01

Chaperonin containing the T-complex polypeptide-1 (CCT), which is known to be involved in intracellular assembly and folding of proteins, is a class of chaperonin omnipresent in all forms of life. Previous studies showed that CCT played a vital role in cold hardiness of various animals. In order to understand the response of the polypeptide complex to low temperature challenge and other environmental stresses, a subunit of CCT (CCTα) was cloned from the mud crab Scylla paramamosain by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA SpCCTα was of 1972 bp and contained a 1668 bp open reading frame (ORF) encoding a polypeptide of 555 amino acids with four conserved motifs. The messenger ribonucleic acid (mRNA) levels of SpCCTα in ten tissues of adult S. paramamosain was subsequently examined and the highest expression was found in muscle, followed by gill, hepatopancreas, thoracic ganglion, hemocyte, heart, cerebral ganglion, stomach, eyestalk ganglion, and epidermis. The expressions of SpCCTα in the muscle of sub-adult crabs (pre-acclimated to 28 °C) subjected to the challenges of both lower temperatures (25, 20, 15, and 10 °C) alone and low temperatures (15 and 10 °C) in combination with salinity of 35 and 10 were further investigated by fluorescent quantitative real-time PCR (qPCR). It was revealed that when exposed to lower temperatures alone, the mRNA transcripts of the SpCCTα gene in the muscle were generally induced for significant higher expression at 10 °C treatment than the 25, 20, and 15 °C treatments; meanwhile, exposure to 15 °C also frequently led to significantly higher expression than those at 20 and 25 °C. This finding indicated that the up-regulation of SpCCTα was closely related to the cold hardiness of S. paramamosain. The results of an additional experiment challenging the sub-adult crabs with various combinations of low temperatures with different salinity conditions generally demonstrated that at both 10 and 15 °C, the expression of SpCCTα under the high salinity of 35 was significantly lower than that at low salinity of 10, implying that the damages caused by low temperatures with high salinity were less than that under low salinity.

Hydrogenase polypeptide and methods of use

DOEpatents

Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

2016-02-02

Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

PubMed Central

Barbosa, M S; Wettstein, F O

1987-01-01

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons. Images PMID:3039182

Three mouse models of human thalassemia.

PubMed Central

Martinell, J; Whitney, J B; Popp, R A; Russell, L B; Anderson, W F

1981-01-01

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes. Images PMID:6946454

Method of generating ploynucleotides encoding enhanced folding variants

DOEpatents

Bradbury, Andrew M.; Kiss, Csaba; Waldo, Geoffrey S.

2017-05-02

The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

Comparative assessment of the polypeptide profiles from lateral and primary roots of Phaseolus vulgaris L

NASA Technical Reports Server (NTRS)

Westberg, J.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

PubMed

Stefanovic, Branko

2013-01-01

Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

Heat-shock response in a molluscan cell line: characterization of the response and cloning of an inducible HSP70 cDNA.

PubMed

Laursen, J R; di Liu, H; Wu, X J; Yoshino, T P

1997-11-01

Sublethal heat-shock of cells of the Bge (Biomphalaria glabrata embryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass. Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS-PAGE/fluorographic analysis of polypeptides produced by in vitro translation of total RNA from cells subjected to heat shock. Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70. The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da. Comparison of a conserved region of 209 amino acid residues revealed > 80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood fluke Schistosoma mansoni (for which B. glabrata serves as intermediate host) (81%), Drosophila (81%), human (84%), and the marine gastropod Aplysia californica (88%, 90%). In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70. Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed. This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance.

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

PubMed Central

Tamura, A; Ohashi, N; Urakami, H; Takahashi, K; Oyanagi, M

1985-01-01

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera. Images PMID:3922893

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

PubMed Central

Liu, Liyun; Hastings, J. Woodland

2007-01-01

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (≈100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca. PMID:17130452

Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

PubMed

Acosta, O; Mayo, M A

1993-01-01

Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

Lysosomal Membrane Glycoproteins: Properties of LAMP-1 (Lysosome Associated Membrane Protein) and LAMP-2

DTIC Science & Technology

1985-01-01

Biosnthesis of th - Glvcooro ins Precursor forms of L ,MP-1 anc L.,. 2 ann process i’ :o n ecu w er’ examined by pulse -chase Iabeling ann,, recipcian. c t i...in the oresence of deterqent. 4 -",W Studies of the biosynthesis and process inn of the qL/cnDroteins showed that each contained a polypeptide core of...pDroximatelv 43,000 dal tons as identified by use of tunicarnycin and endolvcosidase H. Nascent glycooroteins pulse -labeled for 5 min with [3S

Polypeptides having laccase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

KnotProt: a database of proteins with knots and slipknots.

PubMed

Jamroz, Michal; Niemyska, Wanda; Rawdon, Eric J; Stasiak, Andrzej; Millett, Kenneth C; Sułkowski, Piotr; Sulkowska, Joanna I

2015-01-01

The protein topology database KnotProt, http://knotprot.cent.uw.edu.pl/, collects information about protein structures with open polypeptide chains forming knots or slipknots. The knotting complexity of the cataloged proteins is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. The pattern visible in the matrix gives the knotting fingerprint of a given protein and permits users to determine, for example, the minimal length of the knotted regions (knot's core size) or the depth of a knot, i.e. how many amino acids can be removed from either end of the cataloged protein structure before converting it from a knot to a different type of knot. In addition, the database presents extensive information about the biological functions, families and fold types of proteins with non-trivial knotting. As an additional feature, the KnotProt database enables users to submit protein or polymer chains and generate their knotting fingerprints. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Bacillus subtilis Secreted Protein with a Role in Endospore Coat Assembly and Function

PubMed Central

Serrano, Mónica; Zilhão, Rita; Ricca, Ezio; Ozin, Amanda J.; Moran, Charles P.; Henriques, Adriano O.

1999-01-01

Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response. In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat. Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant. The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores. Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure. Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination. We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW. Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of ςH. When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW. Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward. We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment. Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region. TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation. Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat. PMID:10368135

Polypeptide having an amino acid replaced with N-benzylglycine

DOEpatents

Mitchell, Alexander R.; Young, Janis D.

1996-01-01

The present invention relates to one or more polypeptides having useful biological activity in a mammal, which comprise: a polypeptide related to bradykinin of four to ten amino acid residues wherein one or more specific amino acids in the polypeptide chain are replaced with achiral N-benzylglycine. These polypeptide analogues have useful potent agonist or antagonist pharmacological properties depending upon the structure. A preferred polypeptide is (N-benzylglycine.sup.7)-bradykinin.

Expression of a ripening-related cytochrome P450 cDNA in Cavendish banana (Musa acuminata cv. Williams).

PubMed

Pua, Eng-Chong; Lee, Yi-Chuan

2003-02-13

As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

Transcriptome Analysis in Venom Gland of the Predatory Giant Ant Dinoponera quadriceps: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

PubMed Central

Chong, Cheong-Meng; Leung, Siu Wai; Prieto-da-Silva, Álvaro R. B.; Havt, Alexandre; Quinet, Yves P.; Martins, Alice M. C.; Lee, Simon M. Y.; Rádis-Baptista, Gandhi

2014-01-01

Background Dinoponera quadriceps is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the D. quadriceps venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation. Results We conducted a transcriptome analysis of a cDNA library from the D. quadriceps venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences. Conclusions To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant D. quadriceps. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans. PMID:24498135

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase antibody kit

DOEpatents

Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

2008-12-23

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

2003-02-04

An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1998-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Comparative bioinformatics, temporal and spatial expression analyses of Ixodes scapularis organic anion transporting polypeptides

PubMed Central

Radulović, Željko; Porter, Lindsay M.; Kim, Tae K.; Mulenga, Albert

2015-01-01

Organic anion-transporting polypeptides (Oatps) are an integral part of the detoxification mechanism in vertebrates and invertebrates. These cell surface proteins are involved in mediating the sodium-independent uptake and/or distribution of a broad array of organic amphipathic compounds and xenobiotic drugs. This study describes bioinformatics and biological characterization of 9 Oatp sequences in the Ixodes scapularis genome. These sequences have been annotated on the basis of 12 transmembrane domains, consensus motif D-X-RW-(I,V)-GAWW-X-G-(F,L)-L, and 11 conserved cysteine amino acid residues in the large extracellular loop 5 that characterize the Oatp superfamily. Ixodes scapularis Oatps may regulate non-redundant cross-tick species conserved functions in that they did not cluster as a monolithic group on the phylogeny tree and that they have orthologs in other ticks. Phylogeny clustering patterns also suggest that some tick Oatp sequences transport substrates that are similar to those of body louse, mosquito, eye worm, and filarial worm Oatps. Semi-quantitative RT-PCR analysis demonstrated that all 9 I. scapularis Oatp sequences were expressed during tick feeding. Ixodes scapularis Oatp genes potentially regulate functions during early and/or late-stage tick feeding as revealed by normalized mRNA profiles. Normalized transcript abundance indicates that I. scapularis Oatp genes are strongly expressed in unfed ticks during the first 24 h of feeding and/or at the end of the tick feeding process. Except for 2 I. scapularis Oatps, which were expressed in the salivary glands and ovaries, all other genes were expressed in all tested organs, suggesting the significance of I. scapularis Oatps in maintaining tick homeostasis. Different I. scapularis Oatp mRNA expression patterns were detected and discussed with reference to different physiological states of unfed and feeding ticks. PMID:24582512

Decreased erythrocyte nucleoside transport and hENT1 transporter expression in glucose 6-phosphate dehydrogenase deficiency.

PubMed

Al-Ansari, Mohammad; Craik, James D

2015-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with erythrocyte sensitivity to oxidative damage and hemolytic crises. In β-thalassemia major, where hemoglobin instability imposes oxidative stress, erythrocytes show reduced hENT1 nucleoside transporter expression and decreased nucleoside uptake. This study investigated hENT1 expression and nucleoside transport in G6PD-deficient erythrocytes to determine if decreased hENT1 activity might be a contributory feature in the variable pathology of this enzymopathy. Uptake of (3)H-uridine was measured at room temperature using an inhibitor-oil stop protocol and 5-s incubations. Erythrocyte membranes were analyzed by SDS-PAGE and nucleoside (hENT1), glucose (GLUT-1), and anion exchange (Band 3) transporter polypeptides quantitated on immunoblots. In G6PD-deficient cells, uridine uptake (mean 8.18, 95 % CI 5.6-10.7 vs controls mean 12.35, 95 % CI 9.2-15.5, pmol uridine/gHb/min; P = 0.031) and expression of hENT1 (mean 50.4 %, 95 % CI 38.1-62.7 %, arbitrary units n = 11 vs controls mean 95.23 %, 95 % CI 88.38-102.1 % arbitrary units, n = 8; P < 0.001) were significantly lower; expression of GLUT-1 (mean 106.9 %, vs control mean 99.75 %; P = 0.308) and Band 3 polypeptides (mean 100.1 %, vs control mean 102.84 %; P = 0.329) were unchanged. Nucleoside transporter activity in human erythrocytes sustains intracellular purine nucleotide levels and assists in control of plasma adenosine levels; decreased hENT1 expression and activity in G6PD-deficiency could affect red metabolism and influence a wide spectrum of responses mediated by adenosine receptors.

Potential roles of placental human beta-defensin-3 and apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G in prevention of intrauterine transmission of hepatitis B virus.

PubMed

Bai, Xiaoxia; Tian, Ting; Wang, Peng; Yang, Xiaofu; Wang, Zhengping; Dong, Minyue

2015-03-01

Approximately 5% of newborns were infected by hepatitis B virus (HBV) via intrauterine transmission and this is the main reason for high prevalence of HBV in endemic regions. However, the mechanisms by which intrauterine transmission is avoided in most cases remain elusive and placental natural anti-microbial factors may play a role in the prevention of HBV intrauterine transmission. The expression levels of human β-defensin-3 (HBD-3), apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G (A3G) and mannose binding lectin (MBL) were determined in the placenta of 30 HBV-seronegative pregnant women (controls), 7 HBV-seropositive pregnant women with infants infected via intrauterine transmission (infected group) and 30 HBV-seropositive pregnant women with non-infected infants (non-infected group). The expression of HBD-3, A3G, and MBL of placental trophoblast cell line Swan71 was determined after exposed to HBV. There were significant differences in placental HBD-3 and A3G levels among three groups, but the expression of MBL did not significantly differ. The expressions of HBD-3 and A3G were higher in non-infected group than controls and infected group, but not significantly different between infected group and controls. The exposure to HBV increased significantly the expression of HBD-3, A3G, and MBL by Swan 71. It may be concluded HBV up-regulates HBD-3 and A3G expression in vivo and in vitro in placental trophoblast and lack of this up-regulation is possibly associated with intrauterine transmission of HBV. © 2014 Wiley Periodicals, Inc.

Genetics or environment in drug transport: the case of organic anion transporting polypeptides and adverse drug reactions.

PubMed

Clarke, John D; Cherrington, Nathan J

2012-03-01

Organic anion transporting polypeptide (OATP) uptake transporters are important for the disposition of many drugs and perturbed OATP activity can contribute to adverse drug reactions (ADRs). It is well documented that both genetic and environmental factors can alter OATP expression and activity. Genetic factors include single nucleotide polymorphisms (SNPs) that change OATP activity and epigenetic regulation that modify OATP expression levels. SNPs in OATPs contribute to ADRs. Environmental factors include the pharmacological context of drug-drug interactions and the physiological context of liver diseases. Liver diseases such as non-alcoholic fatty liver disease, cholestasis and hepatocellular carcinoma change the expression of multiple OATP isoforms. The role of liver diseases in the occurrence of ADRs is unknown. This article covers the roles OATPs play in ADRs when considered in the context of genetic or environmental factors. The reader will gain a greater appreciation for the current evidence regarding the salience and importance of each factor in OATP-mediated ADRs. A SNP in a single OATP transporter can cause changes in drug pharmacokinetics and contribute to ADRs but, because of overlap in substrate specificities, there is potential for compensatory transport by other OATP isoforms. By contrast, the expression of multiple OATP isoforms is decreased in liver diseases, reducing compensatory transport and thereby increasing the probability of ADRs. To date, most research has focused on the genetic factors in OATP-mediated ADRs while the impact of environmental factors has largely been ignored.

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

PubMed

Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

2013-01-01

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Cloning and characterization of p52, the fifth subunit of the core of the transcription/DNA repair factor TFIIH.

PubMed Central

Marinoni, J C; Roy, R; Vermeulen, W; Miniou, P; Lutz, Y; Weeda, G; Seroz, T; Gomez, D M; Hoeijmakers, J H; Egly, J M

1997-01-01

TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo. PMID:9118947

Differential effect of genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP) and organic anion-transporting polypeptide 1B1 (OATP1B1) on the uptake of HMG-CoA reductase inhibitors.

PubMed

Choi, Min-Koo; Shin, Ho Jung; Choi, Young-Lim; Deng, Jian-Wei; Shin, Jae-Gook; Song, Im-Sook

2011-01-01

The purpose of this study was to investigate the effect of genetic variations in organic anion-transporting polypeptide 1B1 (OATP1B1) and Na(+)/taurocholate co-transporting polypeptide (NTCP) on the uptake of various statins having different affinities for these transporters. The functional activities and simultaneous expression of NTCP and OATP1B1 were confirmed by the uptake of taurocholate and estrone-3-sulphate as representative substrates for NTCP and OATP1B1, respectively, and by an immunofluorescence analysis. The substrate specificities of NTCP and OATP1B1 for statins and the effects of genetic variations on the uptake of rosuvastatin, pitavastatin, and atorvastatin were measured. Based on the K(m) values and intrinsic clearances of the three statins, pitavastatin was taken up more efficiently than rosuvastatin and atorvastatin by OATP1B1. Consequently, the cellular accumulation of pitavastatin was modulated according to the genetic variation of OATP1B1 (OATP1B1*15), rather than NTCP*2. In contrast, NTCP*2 displayed greater transport of atorvastatin and rosuvastatin, compared with NTCP wild type. Thus, the measurements of decreased rosuvastatin and atorvastatin transport by OATP1B1*15 were confounded by the presence of NTCP and its genetic variant, NTCP*2. In conclusion, the functional consequences of genetic variants of NTCP and OATP1B1 may be different for various statins, depending on the substrate specificity of the OATP1B1 and NTCP transporters.

Elastomeric Polypeptides

PubMed Central

van Eldijk, Mark B.; McGann, Christopher L.

2013-01-01

Elastomeric polypeptides are very interesting biopolymers and are characterized by rubber-like elasticity, large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Their useful properties have motivated their use in a wide variety of materials and biological applications. This chapter focuses on elastin and resilin – two elastomeric biopolymers – and the recombinant polypeptides derived from them (elastin-like polypeptides and resilin-like polypeptides). This chapter also discusses the applications of these recombinant polypeptides in the fields of purification, drug delivery, and tissue engineering. PMID:21826606

Changes on protein expression associated with salinity tolerance in Brassica cell cultures.

PubMed

Martín, J P; Elavummoottil, O C; Moreno, M L

1993-09-01

The synthesis of proteins from salt-tolerant Brassica oleracea L. var. botrytis L. subvar. cauliflora (Gars.) DC. (cauliflower) cell cultures is modified in relation to controls in several features. There are nine newly induced polypeptides in tolerant cultures (absent in control conditions). Some of them are only present under low salt levels (85 mM NaCl). Another group seems to be representative of moderate and high salt levels (170 and 255 mM NaCl), and a third group is present in all the salt conditions tested. On the other hand, the synthesis of most of the polypeptides present in control conditions is modified in salt-tolerant cultures by increasing, decreasing or stopping their synthesis in any of the tested conditions. The relationship between these changes in Brassica and other plant systems is discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinell, J.; Whitney, J.B.; Popp, R.A.

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hba/sup th-J/, appear to be true animal models of human thalassemia. Expression of the ..cap alpha..-globin genes in three stocks of mice, each one heterozygous for one of the ..cap alpha..-globin mutations, was examined at the polypeptide, RNA, and DNA levels. ..cap alpha..-globin polypeptide chains, relative to ..gamma..-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of ..cap alpha..-globin to ..gamma..-globin RNA sequences are also 75 to 80% normal, exactly reflecting the ..cap alpha..-globin to ..gamma..-globin chain ratios. In the case ofmore » mutant 352HB, at least one ..cap alpha..-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their ..cap alpha..-globin genes.« less

Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

DOEpatents

Tsien, Roger Y; Wang, Lei

2015-01-13

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

PubMed

Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

2013-06-01

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.

PubMed Central

Pollock, W B; Loutfi, M; Bruschi, M; Rapp-Giles, B J; Wall, J D; Voordouw, G

1991-01-01

By using a synthetic deoxyoligonucleotide probe designed to recognize the structural gene for cytochrome cc3 from Desulfovibrio vulgaris Hildenborough, a 3.7-kb XhoI genomic DNA fragment containing the cc3 gene was isolated. The gene encodes a precursor polypeptide of 58.9 kDa, with an NH2-terminal signal sequence of 31 residues. The mature polypeptide (55.7 kDa) has 16 heme binding sites of the form C-X-X-C-H. Covalent binding of heme to these 16 sites gives a holoprotein of 65.5 kDa with properties similar to those of the high-molecular-weight cytochrome c (Hmc) isolated from the same strain by Higuchi et al. (Y. Higuchi, K. Inaka, N. Yasuoka, and T. Yagi, Biochim. Biophys. Acta 911:341-348, 1987). Since the data indicate that cytochrome cc3 and Hmc are the same protein, the gene has been named hmc. The Hmc polypeptide contains 31 histidinyl residues, 16 of which are integral to heme binding sites. Thus, only 15 of the 16 hemes can have bis-histidinyl coordination. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc from D. vulgaris Hildenborough suggests that the latter contains three cytochrome c3-like domains. Cloning of the D. vulgaris Hildenborough hmc gene into the broad-host-range vector pJRD215 and subsequent conjugational transfer of the recombinant plasmid into D. desulfuricans G200 led to expression of a periplasmic Hmc gene product with covalently bound hemes. Images PMID:1846136

Protein charge distribution in proteomes and its impact on translation

PubMed Central

Requião, Rodrigo D.; Fernandes, Luiza; de Souza, Henrique José Araujo; Rossetto, Silvana; Domitrovic, Tatiana

2017-01-01

As proteins are synthesized, the nascent polypeptide must pass through a negatively charged exit tunnel. During this stage, positively charged stretches can interact with the ribosome walls and slow the translation. Therefore, charged polypeptides may be important factors that affect protein expression. To determine the frequency and distribution of positively and negatively charged stretches in different proteomes, the net charge was calculated for every 30 consecutive amino acid residues, which corresponds to the length of the ribosome exit tunnel. The following annotated and reviewed proteins in the UniProt database (Swiss-Prot) were analyzed: 551,705 proteins from different organisms and a total of 180 million protein segments. We observed that there were more negative than positive stretches and that super-charged positive sequences (i.e., net charges ≥ 14) were underrepresented in the proteomes. Overall, the proteins were more positively charged at their N-termini and C-termini, and this feature was present in most organisms and subcellular localizations. To investigate whether the N-terminal charges affect the elongation rates, previously published ribosomal profiling data obtained from S. cerevisiae, without translation-interfering drugs, were analyzed. We observed a nonlinear effect of the charge on the ribosome occupancy in which values ≥ +5 and ≤ -6 showed increased and reduced ribosome densities, respectively. These groups also showed different distributions across 80S monosomes and polysomes. Basic polypeptides are more common within short proteins that are translated by monosomes, whereas negative stretches are more abundant in polysome-translated proteins. These findings suggest that the nascent peptide charge impacts translation and can be one of the factors that regulate translation efficiency and protein expression. PMID:28531225

Structure, function and dynamics in adenovirus maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangel, Walter F.; San Martín, Carmen

2014-11-21

Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

PubMed Central

Sesma, F; Gardiol, D; de Ruiz Holgado, A P; de Mendoza, D

1990-01-01

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2117878

Polypeptides having catalase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Distribution of Pancreatic Polypeptide-secreting Endocrine Cells in Nondiabetic and Diabetic Cases.

PubMed

Śliwińska-Mossoń, Mariola; Milnerowicz, Halina

2017-07-01

The aim of the study was to demonstrate the effects of cigarette smoking and ongoing inflammation in chronic pancreatitis on the functioning of pancreatic polypeptide (PP)-secreting cells and to determine the relationship between the occurrence of an increased number of PP cells in the pancreas, the change in their location, and the intensity of their inflammatory changes in the course of pancreatitis and diabetes. Samples of tissues from healthy persons and from patients were verified histopathologically, and then PP was localized by immunohistochemical staining using the monoclonal anti-human PP antibody. The histopathologic evaluation of the hormone expression intensity in tissue sections was carried out using the semiquantitative method and was calculated with digital image analysis. The present study showed a very strong PP expression in the pancreatic tissue (especially in the head of the pancreas) derived from smoking patients with diabetes. The increase in the percentage of cells in the PP islets, between the acinar cells in smoking patients with diabetes and a statistically significant increase in the expression of PP, indicates a pancreatic endocrine dysfunction and suggests that cigarette smoking has a negative impact on the organ's efficiency. Because of its properties, the PP appears to be a useful marker of the endocrine insufficiency of the pancreas and a specific prognostic parameter of developing diabetes due to chronic pancreatitis.

A Novel Dark-Inducible Protein, LeDI-2, and Its Involvement in Root-Specific Secondary Metabolism in Lithospermum erythrorhizon1

PubMed Central

Yazaki, Kazufumi; Matsuoka, Hideaki; Shimomura, Koichiro; Bechthold, Andreas; Sato, Fumihiko

2001-01-01

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities. PMID:11299363

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

PubMed Central

Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

2009-01-01

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors.

PubMed

Adrian, T E; Uttenthal, L O; Williams, S J; Bloom, S R

1986-07-31

Pancreatic polypeptide is often secreted by pancreatic endocrine tumors and is considered a marker for such tumors. To investigate the diagnostic value of this marker, we studied 323 patients with proved pancreatic endocrine tumors. We found plasma concentrations of pancreatic polypeptide to be elevated (more than 300 pmol per liter) in 144 patients (diagnostic sensitivity, 45 percent). However, plasma levels of pancreatic polypeptide can also be elevated in the absence of a pancreatic tumor. To ascertain whether the administration of atropine could distinguish between normal and tumor-associated polypeptide secretion, we studied 30 patients with pancreatic tumors and high plasma levels of pancreatic polypeptide, 18 patients without tumors who had elevated levels of pancreatic polypeptide, and eight normal controls. Polypeptide levels in the 18 patients without tumors were substantially lower than in the 30 patients with tumors. Atropine (1 mg intramuscularly) did not suppress polypeptide levels in patients with tumors, but did suppress plasma levels by more than 50 percent in all subjects without tumors. Thus, although its diagnostic sensitivity is low, pancreatic polypeptide appears to be a useful adjunctive marker of many pancreatic endocrine tumors, and the atropine suppression test can be used to distinguish normal from tumor-related secretion of the polypeptide. Identification of the type of pancreatic endocrine tumor still requires measurement of the hormone that is specific for the tumor.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having beta-glucosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

Provided are isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Zhang, Yu

Provided are isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Shaghasi, Tarana

2016-11-01

The present invention provides hybrid polypeptides having cellobiohydrolase activity. The present invention also provides polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Combinatorial discovery of enzymes with utility in biomass transformation

DOEpatents

Fox, Brian G; Elsen, Nathaniel L

2015-02-03

Methods for the cell-free identification of polypeptide and polypeptide combinations with utility in biomass transformation, as well as specific novel polypeptides and cell-free systems containing polypeptide combinations discovered by such methods are disclosed.

Oncoprotein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

2001-02-27

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1999-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

PubMed Central

German, M S; Moss, L G; Wang, J; Rutter, W J

1992-01-01

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators. Images PMID:1549125

Proteostasis: bad news and good news from the endoplasmic reticulum.

PubMed

Noack, Julia; Brambilla Pisoni, Giorgia; Molinari, Maurizio

2014-01-01

The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms' homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

Synthesis and processing of equine herpesvirus 1 glycoprotein D.

PubMed

Flowers, C C; Flowers, S P; Jennings, S R; O'Callaghan, D J

1995-04-01

Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Shaghasi, Tarana

The present invention relates to hybrid polypeptides having cellobiohydrolase activity. The present invention also relates to polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2015-06-09

Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Duan, Junxin; Tang, Lan

2015-09-22

The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Duan, Junxin

2015-12-15

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Isolation of Polypeptide Sample and Measurement of Its Concentration.

ERIC Educational Resources Information Center

Beanan, Maureen J.

2000-01-01

Introduces a laboratory experiment that isolates a bacterial polypeptide sample and measures the concentration of polypeptides in the sample. Uses Escherichia coli strain MM294 and performs a bio-rad assay to determine the concentration of polypeptides. (YDS)

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-07-14

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Mucin-type O-glycans in Tears of Normal Subjects and Patients with Non-Sjögren’s Dry Eye

PubMed Central

Guzman-Aranguez, Ana; Mantelli, Flavio; Argüeso, Pablo

2009-01-01

Purpose O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study aimed to identify the repertoire of O-glycans in the tear film, and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren’s dry eye. Methods Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium using a glycogene microarray database. Lectin-binding analyses were performed using agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. Results The O-glycan profile of human tears consisted primarily of core 1 (Galβ1-3GalNAcα1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, being α2-6-sialyl core 1 the predominant O-glycan structure in human tears (48%). Four families of glycosyltranferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included thirteen polypeptide-GalNAc-transferases (GALNT), the core 1 β-3-galactosyltransferase (T-synthase), three α2-6-sialyltransferases (ST6GalNAc), and two α2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and dry eye patients, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. Conclusions This study identifies the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren’s dry eye show no alterations in composition and amount of O-glycans in the tear fluid. PMID:19407012

Proteomics of a new esophageal cancer cell line established from Persian patient.

PubMed

Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George

2014-07-18

Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successfulmore » overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.« less

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez de Leon, Alfredo; Rey, Michael

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2012-09-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2010-12-14

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Lopez de Leon, Alfredo [Davis, CA; Rey, Micheal [Davis, CA; Ding, Hanshu [Davis, CA; Vlasenko, Elena [Davis, CA

2012-02-21

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-06-28

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2016-05-31

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-02-10

The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-02-23

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

2013-04-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

2012-10-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-11-20

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-01-27

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-21

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-03-10

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2017-05-02

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-03-31

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-07-14

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Brown, Kimberly [Elk Grove, CA; Harris, Paul [Carnation, WA; Lopez De Leon, Alfredo [Davis, CA; Merino, Sandra [West Sacremento, CA

2007-05-22

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

2013-11-19

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Morant, Marc D.; Harris, Paul

2015-10-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

2012-10-02

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Methods for using polypeptides having cellobiohydrolase activity

DOEpatents

Morant, Marc D; Harris, Paul

2016-08-23

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polynucleotides encoding polypeptides having beta-glucosidase activity

DOEpatents

Harris, Paul; Golightly, Elizabeth

2010-03-02

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2013-06-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

2016-12-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-14

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

2016-05-17

The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Nano polypeptide particles reinforced polymer composite fibers.

PubMed

Li, Jiashen; Li, Yi; Zhang, Jing; Li, Gang; Liu, Xuan; Li, Zhi; Liu, Xuqing; Han, Yanxia; Zhao, Zheng

2015-02-25

Because of the intensified competition of land resources for growing food and natural textile fibers, there is an urgent need to reuse and recycle the consumed/wasted natural fibers as regenerated green materials. Although polypeptide was extracted from wool by alkaline hydrolysis, the size of the polypeptide fragments could be reduced to nanoscale. The wool polypeptide particles were fragile and could be crushed down to nano size again and dispersed evenly among polymer matrix under melt extrusion condition. The nano polypeptide particles could reinforce antiultraviolet capability, moisture regain, and mechanical properties of the polymer-polypeptide composite fibers.

Codominant expression of genes coding for different sets of inducible salivary polypeptides associated with parotid hypertrophy in two inbred mouse strains.

PubMed

López-Solís, Remigio O; Kemmerling, Ulrike

2005-05-01

Experimental mouse parotid hypertrophy has been associated with the expression of a number of isoproterenol-induced salivary proline-rich polypeptides (IISPs). Mouse salivary proline-rich proteins (PRPs) have been mapped both to chromosomes 6 and 8. Recently, mice of two inbred strains (A/Snell and A. Swiss) have been found to differ drastically in the IISPs. In this study, mice of both strains were used for cross-breeding experiments addressed to define the pattern of inheritance of the IISP phenotype and to establish whether the IISPs are coded on a single or on several chromosomes. The IISP phenotype of individual mice was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole saliva collected after three daily stimulations by isoproterenol. Parental A/Snell and A. Swiss mice were homogeneous for distinctive strain-associated IISP-patterns. First filial generation (F1) mice obtained from the cross of A/Snell with A. Swiss mice expressed with no exception both the A/Snell and A. Swiss IISPs (coexpression). In the second filial generation (F2) both parental IISP phenotypes reappeared together with a majority of mice expressing the F1-hybrid phenotype (1:2:1 ratio). Backcrosses of F1 x A/Snell and F1 x A. Swiss produced offsprings displaying the F1 and the corresponding parental phenotypes with a 1:1 ratio. No recombinants were observed among F2 mice or among mice resulting from backcrosses. Thus, genes coding for the IISPs that are expressed differentially in both mouse strains are located on the same chromosome, probably at the same locus (alleles) or at quite closely linked loci (nonalleles). 2005 Wiley-Liss, Inc

Combined Proteomic and Transcriptomic Interrogation of the Venom Gland of Conus geographus Uncovers Novel Components and Functional Compartmentalization*

PubMed Central

Safavi-Hemami, Helena; Hu, Hao; Gorasia, Dhana G.; Bandyopadhyay, Pradip K.; Veith, Paul D.; Young, Neil D.; Reynolds, Eric C.; Yandell, Mark; Olivera, Baldomero M.; Purcell, Anthony W.

2014-01-01

Cone snails are highly successful marine predators that use complex venoms to capture prey. At any given time, hundreds of toxins (conotoxins) are synthesized in the secretory epithelial cells of the venom gland, a long and convoluted organ that can measure 4 times the length of the snail's body. In recent years a number of studies have begun to unveil the transcriptomic, proteomic and peptidomic complexity of the venom and venom glands of a number of cone snail species. By using a combination of DIGE, bottom-up proteomics and next-generation transcriptome sequencing the present study identifies proteins involved in envenomation and conotoxin maturation, significantly extending the repertoire of known (poly)peptides expressed in the venom gland of these remarkable animals. We interrogate the molecular and proteomic composition of different sections of the venom glands of 3 specimens of the fish hunter Conus geographus and demonstrate regional variations in gene expression and protein abundance. DIGE analysis identified 1204 gel spots of which 157 showed significant regional differences in abundance as determined by biological variation analysis. Proteomic interrogation identified 342 unique proteins including those that exhibited greatest fold change. The majority of these proteins also exhibited significant changes in their mRNA expression levels validating the reliability of the experimental approach. Transcriptome sequencing further revealed a yet unknown genetic diversity of several venom gland components. Interestingly, abundant proteins that potentially form part of the injected venom mixture, such as echotoxins, phospholipase A2 and con-ikots-ikots, classified into distinct expression clusters with expression peaking in different parts of the gland. Our findings significantly enhance the known repertoire of venom gland polypeptides and provide molecular and biochemical evidence for the compartmentalization of this organ into distinct functional entities. PMID:24478445

Effect of Yin-Zhi-Huang on up-regulation of Oatp2, Ntcp, and Mrp2 proteins in estrogen-induced rat cholestasis.

PubMed

Zhang, Guoqiang; Zhou, Yan; Rao, Zhi; Qin, Hongyan; Wei, Yuhui; Ren, Jiangxia; Zhou, Liting; Wu, Xin'an

2015-03-01

Yin-Zhi-Huang (YZH), a prescription of traditional Chinese medicine, is widely used to treat neonatal jaundice or cholestasis. This study investigates the regulatory effect of YZH on the localization and expression of organic anion transporting polypeptides 2 (Oatp2), Na(+)-taurocholate co-transporting polypeptide (Ntcp), multidrug-resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) in estrogen-induced cholestasis rats. Cholestasis model rats were induced via subcutaneous injection of estradiol benzoate (EB, 5 mg/kg/d) for 5 d. Other EB-induced rats were treated with saline (2 ml) or YZH (1.5 g/kg, two times a day) for 7, 14, and 21 d. The biochemical and pathologic examinations were performed. Moreover, the localization and expression of Oatp2, Ntcp, Mrp2, and Bsep were determined by immunohistochemisty and Western blotting, respectively. YZH treatment could significantly decrease the serum total bile acids (TBA) (4.9 ± 0.6-2.8 ± 0.8) and direct bilirubin (DBIL) (2.6 ± 0.7-1.0 ± 0.1) levels, improve the histological disorganization, and, respectively, increase the expression of Oatp2 and Ntcp by 46% and 28% compared with saline-treated (p < 0.05) rats at 14 d. The expression of Mrp2 increased by 45% was observed in YZH treated compared with saline-treated (p < 0.05) rats at 7 d. However, there was a little change in the expression of Bsep (p > 0.05) after YZH treatment for 7, 14, and 21 d. In conclusion, the therapeutic effect of YZH to cholestasis could be attributed to the regulation of Oatp2, Ntcp, Mrp2, and Bsep.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Ruizhi; Sang, Qing; Kuang, Yanping

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

Pituitary adenylate cyclase activating polypeptide and PAC1 receptor signaling increase Homer 1a expression in central and peripheral neurons.

PubMed

Girard, Beatrice M; Keller, Emily T; Schutz, Kristin C; May, Victor; Braas, Karen M

2004-12-15

Pituitary adenylate cyclase activating polypeptides (PACAP) and PAC1 receptor signaling have diverse roles in central and peripheral nervous system development and function. In recent microarray analyses for PACAP and PAC1 receptor modulation of neuronal transcripts, the mRNA of Homer 1a (H1a), which encodes the noncrosslinking and immediate early gene product isoform of Homer, was identified to be strongly upregulated in superior cervical ganglion (SCG) sympathetic neurons. Given the prominent roles of Homer in synaptogenesis, synaptic protein complex assembly and receptor/channel signaling, we have examined the ability for PACAP to induce H1a expression in sympathetic, cortical and hippocampal neurons to evaluate more comprehensively the roles of PACAP in synaptic function. In both central and peripheral neuronal cultures, PACAP peptides increased transiently H1a transcript levels approximately 3.5- to 6-fold. From real-time quantitative PCR measurements, the temporal patterns of PACAP-mediated H1a mRNA induction among the different neuronal cultures appeared similar although the onset of sympathetic H1a transcript expression appeared protracted. The increase in H1a transcripts was accompanied by increases in H1a protein levels. Comparative studies with VIP and PACAP(6-38) antagonist demonstrated that the PACAP effects reflected PAC1 receptor activation and signaling. The PAC1 receptor isoforms expressed in central and peripheral neurons can engage diverse intracellular second messenger systems, and studies using selective signaling pathway inhibitors demonstrated that the cyclic AMP/PKA and MEK/ERK cascades are principal mediators of the PACAP-mediated H1a induction response. In modulating H1a transcript and protein expression, these studies may implicate broad roles for PACAP and PAC1 receptor signaling in synaptic development and plasticity.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

DOE PAGES

Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

2016-01-21

BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

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

NASA Technical Reports Server (NTRS)

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 schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

PubMed

Malagnac, Fabienne; Fabret, Céline; Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

2013-01-01

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnorr, Kirk; Kramer, Randall

2017-08-08

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Lan; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo [Davis, CA; Ding, Hanshu [Davis, CA; Brown, Kimberly [Elk Grove, CA

2011-10-25

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul; Golightly, Elizabeth

2012-11-27

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-06-14

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-11-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan [Beijing, CN; Liu, Ye [Beijing, CN; Duan, Junxin [Beijing, CN; Zhang, Yu [Beijing, CN; Jorgensen, Christian Isak [Bagsvaerd, DK; Kramer, Randall [Lincoln, CA

2012-04-03

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin [Beijing, CN; Liu, Ye [Beijing, CN; Tang, Lan [Beijing, CN; Wu, Wenping [Beijing, CN; Quinlan, Jason [Albany, CA; Kramer, Randall [Lincoln, CA

2012-03-27

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2016-11-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2014-09-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding the same

DOEpatents

Spodsberg, Nikolaj [Bagsvaed, DK

2014-01-07

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2014-10-21

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-04-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2007-07-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

2013-10-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2011-06-14

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-04-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

2013-06-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Transgenic overexpression of p23 induces spontaneous hydronephrosis in mice

PubMed Central

Lee, Jaehoon; Kim, Hye Jin; Moon, Jung Ah; Sung, Young Hoon; Baek, In-Jeoung; Roh, Jae-il; Ha, Na Young; Kim, Seung-Yeon; Bahk, Young Yil; Lee, Jong Eun; Yoo, Tae Hyun; Lee, Han-Woong

2011-01-01

p23 is a cochaperone of heat shock protein 90 and also interacts functionally with numerous steroid receptors and kinases. However, the in vivo roles of p23 remain unclear. To explore its in vivo function, we generated the transgenic (TG) mice ubiquitously overexpressing p23. The p23 TG mice spontaneously developed kidney abnormalities closely resembling human hydronephrosis. Consistently, kidney functions deteriorate significantly in the p23 TG mice compared to their wild-type (WT) littermates. Furthermore, the expression of target genes for aryl hydrocarbon receptor (AhR), such as cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1A1) and cytochrome P450, family 1, subfamily B, polypeptide 1 (Cyp1B1), were induced in the kidneys of the p23 TG mice. These results indicate that the overexpression of p23 contributes to the development of hydronephrosis through the upregulation of the AhR pathway in vivo. PMID:21323770

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae*

PubMed Central

Rego, Sara; Heal, Timothy J.; Pidwill, Grace R.; Till, Marisa; Robson, Alice; Lamont, Richard J.; Sessions, Richard B.; Jenkinson, Howard F.; Race, Paul R.; Nobbs, Angela H.

2016-01-01

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans. Complementary crystallographic and biophysical characterization of BspA reveal a novel β-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections. PMID:27311712

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2012-10-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

2007-09-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2013-11-19

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2014-09-30

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2017-09-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2010-06-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

DOEpatents

Brown, Kimberly; Harris, Paul

2013-12-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-12-24

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells.

PubMed

Ghandour, M S; Parkkila, A K; Parkkila, S; Waheed, A; Sly, W S

2000-11-01

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

PubMed Central

Festari, María Florencia; Trajtenberg, Felipe; Berois, Nora; Pantano, Sergio; Revoredo, Leslie; Kong, Yun; Solari-Saquieres, Patricia; Narimatsu, Yoshiki; Freire, Teresa; Bay, Sylvie; Robello, Carlos; Bénard, Jean; Gerken, Thomas A; Clausen, Henrik; Osinaga, Eduardo

2017-01-01

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain. PMID:27913570

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Synergistic administration of photothermal therapy and chemotherapy to cancer cells using polypeptide-based degradable plasmonic matrices

PubMed Central

Huang, Huang-Chiao; Yang, Yoonsun; Nanda, Alisha; Koria, Piyush; Rege, Kaushal

2012-01-01

Aim Resistance of cancer cells to hyperthermic temperatures and spatial limitations of nanoparticle-induced hyperthermia necessitates the identification of effective combination treatments that can enhance the efficacy of this treatment. Here we show that novel polypeptide-based degradable plasmonic matrices can be employed for simultaneous administration of hyperthermia and chemotherapeutic drugs as an effective combination treatment that can overcome cancer cell resistance to hyperthermia. Method Novel gold nanorod elastin-like polypeptide matrices were generated and characterized. The matrices were also loaded with the heat-shock protein (HSP)90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), currently in clinical trials for different malignancies, in order to deliver a combination of hyperthermia and chemotherapy. Results Laser irradiation of cells cultured over the plasmonic matrices (without 17-AAG) resulted in the death of cells directly in the path of the laser, while cells outside the laser path did not show any loss of viability. Such spatial limitations, in concert with expression of prosurvival HSPs, reduce the efficacy of hyperthermia treatment. 17-AAG–gold nanorod–polypeptide matrices demonstrated minimal leaching of the drug to surrounding media. The combination of hyperthermic temperatures and the release of 17-AAG from the matrix, both induced by laser irradiation, resulted in significant (>90%) death of cancer cells, while ‘single treatments’ (i.e., hyperthermia alone and 17-AAG alone) demonstrated minimal loss of cancer cell viability (<10%). Conclusion Simultaneous administration of hyperthermia and HSP inhibitor release from plasmonic matrices is a powerful approach for the ablation of malignant cells and can be extended to different combinations of nanoparticles and chemotherapeutic drugs for a variety of malignancies. PMID:21542685

The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

PubMed

Gardner, B; Anstee, D J; Mawby, W J; Tanner, M J; von dem Borne, A E

1991-06-01

Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

Cellulases, nucleic acids encoding them and methods for making and using them

DOEpatents

Blum, David; Gemsch Cuenca, Joslin; Dycaico, Mark

2013-04-23

This invention relates to molecular and cellular biology and biochemistry. In one aspect, the invention provides polypeptides having cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides cellulase activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or .beta.-glucosidase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts.

Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wogulis, Mark; Sweeney, Matthew; Heu, Tia

The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnorr, Kirk; Kramer, Randall

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Auxin-Regulated Polypeptide Changes at Different Stages of Strawberry Fruit Development 1

PubMed Central

Veluthambi, K.; Poovaiah, B. W.

1984-01-01

The pattern of polypeptides at different stages of strawberry (Fragaria ananassa Duch. cv Ozark Beauty) fruit development was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An 81,000-dalton polypeptide appeared between 5 and 10 days after pollination. Polypeptides with molecular weights of 76,000 and 37,000 daltons were formed after 10 days. The control exerted by auxin in the stage-specific formation of polypeptides was investigated by stopping fruit growth after removing the achenes and reinitiating fruit growth by the application of a synthetic auxin, α-naphthaleneacetic acid (NAA). When the achenes were removed from the 5- and 10-day-old fruits, the fruits failed to grow, the 81,000 dalton polypeptide was not formed between 5 and 10 days, and the 76,000- and 37,000-dalton polypeptides were not formed between 10 and 20 days. Application of NAA to fruits deprived of auxin by removal of achenes resulted in the resumption of growth and also in the appearance of these polypeptides. Removal of achenes of the 5- or 10-day-old fruits and growing them without auxin resulted in the formation of 52,000- and 57,000-dalton polypeptides. These two polypeptides were not formed when NAA was applied to fruits after removal of achenes. Supply of NAA to auxin-deprived fruits 5 days after removal of achenes resulted in resumption of growth and also in the disappearance of these two polypeptides, pointing out their possible relation to the inhibition of fruit growth. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16663624

Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

DTIC Science & Technology

2013-07-01

biology, nanotechnology, and imaging technology, molecular imaging utilizes specific probes as contrast agents to visualize cellular processes at the...This reagent was covalently coupled to the oligosaccharides attached to polypeptide side-chains of extracellular membrane proteins on living cells...website. The normal tissue gene expression profile dataset was modified and processed as described by Fang (8) and mean intensities and standard

Flavivirus RNA Replication: Essential Viral Functions as Targets for Antiviral Therapeutics

DTIC Science & Technology

1991-12-31

We established yellow fever virus ( YFV ) as our study system and assembled all the necessary virologic, molecular biologic, and immunologic reagents for...the detailed study of viral RNA replication. Methodologies for detection, identification, and analysis of YFV polypeptides and RNA species were in...place. Several YFV protesn coding regions were engineered into various surrogate expression systems, and the recombinant proteins thereby produced would

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections

PubMed Central

Durante, Ignacio M.; La Spina, Pablo E.; Carmona, Santiago J.; Agüero, Fernán

2017-01-01

Background The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a ‘mosaic’ structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response. Methods/Principle findings High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection. Conclusions/Significance In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection. PMID:28961244

Organic Anion Transporting Polypeptide 1a1 Null Mice Are Sensitive to Cholestatic Liver Injury

PubMed Central

Zhang, Youcai; Csanaky, Iván L.; Cheng, Xingguo; Lehman-McKeeman, Lois D.; Klaassen, Curtis D.

2012-01-01

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in livers of mice and is thought to transport bile acids (BAs) from blood into liver. Because Oatp1a1 expression is markedly decreased in mice after bile duct ligation (BDL). We hypothesized that Oatp1a1-null mice would be protected against liver injury during BDL-induced cholestasis due largely to reduced hepatic uptake of BAs. To evaluate this hypothesis, BDL surgeries were performed in both male wild-type (WT) and Oatp1a1-null mice. At 24 h after BDL, Oatp1a1-null mice showed higher serum alanine aminotransferase levels and more severe liver injury than WT mice, and all Oatp1a1-null mice died within 4 days after BDL, whereas all WT mice survived. At 24 h after BDL, surprisingly Oatp1a1-null mice had higher total BA concentrations in livers than WT mice, suggesting that loss of Oatp1a1 did not prevent BA accumulation in the liver. In addition, secondary BAs dramatically increased in serum of Oatp1a1-null BDL mice but not in WT BDL mice. Oatp1a1-null BDL mice had similar basolateral BA uptake (Na+-taurocholate cotransporting polypeptide and Oatp1b2) and BA-efflux (multidrug resistance–associated protein [Mrp]-3, Mrp4, and organic solute transporter α/β) transporters, as well as BA-synthetic enzyme (Cyp7a1) in livers as WT BDL mice. Hepatic expression of small heterodimer partner Cyp3a11, Cyp4a14, and Nqo1, which are target genes of farnesoid X receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor 2, respectively, were increased in WT BDL mice but not in Oatp1a1-null BDL mice. These results demonstrate that loss of Oatp1a1 function exacerbates cholestatic liver injury in mice and suggest that Oatp1a1 plays a unique role in liver adaptive responses to obstructive cholestasis. PMID:22461449

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc

2014-01-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

An in vivo library-versus-library selection of optimized protein-protein interactions.

PubMed

Pelletier, J N; Arndt, K M; Plückthun, A; Michnick, S W

1999-07-01

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription.

PubMed

Hu, J C; Gross, C A

1985-01-01

The sigma subunits of bacterial RNA polymerases are required for the selective initiation of transcription. We have isolated and characterized mutations in rpoD, the gene which encodes the major form of sigma in E. coli, which affect the selectivity of transcription. These mutations increase the expression of araBAD up to 12-fold in the absence of CAP-cAMP. Expression of lac is unaffected, while expression of malT-activated operons is decreased. We determined the DNA sequence of 17 independently isolated mutations, and found that they consist of three different changes in a single CGC arginine codon at position 596 in the sigma polypeptide.

Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin.

PubMed

Hou, Hu; Li, Bafang; Zhang, Zhaohui; Xue, Changhu; Yu, Guangli; Wang, Jingfeng; Bao, Yuming; Bu, Lin; Sun, Jiang; Peng, Zhe; Su, Shiwei

2012-12-01

Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen. Copyright © 2012 Elsevier Ltd. All rights reserved.

The singular behavior of a β-type semi-synthetic two branched polypeptide: three-dimensional structure and mode of action.

PubMed

Manzo, Giorgia; Serra, Ilaria; Pira, Alessandro; Pintus, Manuela; Ceccarelli, Matteo; Casu, Mariano; Rinaldi, Andrea C; Scorciapino, Mariano Andrea

2016-11-16

Dendrimeric peptides make a versatile group of bioactive peptidomimetics and a potential new class of antimicrobial agents to tackle the pressing threat of multi-drug resistant pathogens. These are branched supramolecular assemblies where multiple copies of the bioactive unit are linked to a central core. Beyond their antimicrobial activity, dendrimeric peptides could also be designed to functionalize the surface of nanoparticles or materials for other medical uses. Despite these properties, however, little is known about the structure-function relationship of such compounds, which is key to unveil the fundamental physico-chemical parameters and design analogues with desired attributes. To close this gap, we focused on a semi-synthetic, two-branched peptide, SB056, endowed with remarkable activity against both Gram-positive and Gram-negative bacteria and limited cytotoxicity. SB056 can be considered the smallest prototypical dendrimeric peptide, with the core restricted to a single lysine residue and only two copies of the same highly cationic 10-mer polypeptide; an octanamide tail is present at the C-terminus. Combining NMR and Molecular Dynamics simulations, we have determined the 3D structure of two analogues. Fluorescence spectroscopy was applied to investigate the water-bilayer partition in the presence of vesicles of variable charge. Vesicle leakage assays were also performed and the experimental data were analyzed by applying an iterative Monte Carlo scheme to estimate the minimum number of bound peptides needed to achieve the release. We unveiled a singular beta hairpin-type structure determined by the peptide chains only, with the octanamide tail available for further functionalization to add new potential properties without affecting the structure.

Wisteria floribunda agglutinin-sialylated mucin core polypeptide 1 is a sensitive biomarker for biliary tract carcinoma and intrahepatic cholangiocarcinoma: a multicenter study.

PubMed

Shoda, Junichi; Matsuda, Atsushi; Shida, Takashi; Yamamoto, Masakazu; Nagino, Masato; Tsuyuguchi, Toshio; Yasaka, Takahiro; Tazuma, Susumu; Uchiyama, Kazuhisa; Unno, Michiaki; Ohkohchi, Nobuaki; Nakanuma, Yasuni; Kuno, Atsushi; Narimatsu, Hisashi

2017-02-01

Wisteria floribunda agglutinin (WFA)-sialylated mucin core polypeptide 1 (MUC1) was investigated as a new glycoprotein marker for cholangiocarcinoma (CC) using glycoproteomics technologies. In this multicenter study, WFA-sialylated MUC1 levels in serum and bile samples were measured to determine their diagnostic capability in biliary tract carcinoma (BTC) and intrahepatic (Ih) CC. The study included 244 patients with BTC, 59 patients with IhCC, 287 patients with benign biliary tract diseases, and 44 control subjects. Serum WFA-sialylated MUC1 levels were significantly higher in patients with either BTC or IhCC than in control subjects and those with benign biliary tract diseases. Patients with IhCC showed higher WFA-sialylated MUC1 levels than patients with tumors at other sites. No significant differences in WFA-sialylated MUC1 levels were found with regard to cancer stage or tissue type. Receiver operating characteristic curve analysis showed that WFA-sialylated MUC1 was superior to carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) for the diagnosis of benign biliary tract diseases, BTC, and IhCC, as well as for stage I and II carcinomas. Significantly higher levels of biliary WFA-sialylated MUC1 were observed in BTC/IhCC than in benign biliary tract diseases. The diagnostic capability of biliary WFA-sialylated MUC1 was also superior to that of CA19-9, and diagnostic sensitivity was higher than that of biliary cytology for BTC/IhCC. WFA-sialylated MUC1 is a useful novel biomarker for BTC/IhCC. In the future, this measurement should be applied in the clinical setting.

Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2016-02-16

The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well asmore » the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.« less

Polypeptide having swollenin activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

2015-11-04

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

2015-09-01

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having cellobiohydrolase activity and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-09-15

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having acetyl xylan esterase activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having carbohydrate degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

2015-08-18

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Mosaic HIV envelope immunogenic polypeptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korber, Bette T. M.; Gnanakaran, S.; Perkins, Simon

Disclosed herein are mosaic HIV envelope (Env) polypeptides that can elicit an immune response to HIV (such as cytotoxic T cell (CTL), helper T cell, and/or humoral responses). Also disclosed are sets of the disclosed mosaic Env polypeptides, which include two or more (for example, three) of the polypeptides. Also disclosed herein are methods for treating or inhibiting HIV in a subject including administering one or more of the disclosed immunogenic polypeptides or compositions to a subject infected with HIV or at risk of HIV infection. In some embodiments, the methods include inducing an immune response to HIV in amore » subject comprising administering to the subject at least one (such as two, three, or more) of the immunogenic polypeptides or at least one (such as two, three, or more) nucleic acids encoding at least one of the immunogenic polypeptides disclosed herein.« less

Toxicity study of isolated polypeptide from wool hydrolysate.

PubMed

Li, Jiashen; Li, Yi; Zhang, Yu; Liu, Xuan; Zhao, Zheng; Zhang, Jing; Han, Yanxia; Zhou, Dangxia

2013-07-01

The cytotoxicity of wool polypeptide has been evaluated by both cell and animal models. Wool was dissolved in sodium hydroxide solution, the pH value of the solution was adjusted to 5.55 and the precipitate was harvested as wool polypeptide. The spray-dried polypeptide was collected as powders and characterized by SEM, FTIR and TG-DSC. The cell culturing results showed that wool polypeptide had no obvious negative effect on cell viability in vitro. Both acute oral toxicity and subacute 30-day oral toxicology studies showed that wool polypeptide had no influence on body weight, feed consumption, blood chemistry, and hematology at any dose levels. There were no treatment related findings on gross or detailed necroscopy, organ weights, organ/body weight ratios and histology. Our study indicated the absence of toxicity in wool polypeptide and supported its safe use as a food ingredient or drug carrier. Copyright © 2013 Elsevier Ltd. All rights reserved.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-10-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Thermal and acid tolerant beta-xylosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Thompson, Vicki S [Idaho Falls, ID; Schaller, Kastli D [Ammon, ID; Apel, William A [Jackson, WY; Lacey, Jeffrey A [Idaho Falls, ID; Reed, David W [Idaho Falls, ID

2011-04-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose and/or xylobiose using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships

PubMed Central

Levinson, Pauline; Choi, Robert Y.; Cole, Amy L.; Hirbod, Taha; Rhedin, Samuel; Payne, Barbara; Guthrie, Brandon L.; Bosire, Rose; Cole, Alexander M.; Farquhar, Carey; Broliden, Kristina

2012-01-01

Background HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection. Methodology/Principal Findings Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity. Conclusions/Significance These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules. PMID:22389677

Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

NASA Astrophysics Data System (ADS)

Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

1985-01-01

Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

NASA Astrophysics Data System (ADS)

Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

1984-04-01

The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

Compositions and methods for the expression of selenoproteins in eukaryotic cells

DOEpatents

Gladyshev, Vadim [Lincoln, NE; Novoselov, Sergey [Puschino, RU

2012-09-25

Recombinant nucleic acid constructs for the efficient expression of eukaryotic selenoproteins and related methods for production of recombinant selenoproteins are provided. The nucleic acid constructs comprise novel selenocysteine insertion sequence (SECIS) elements. Certain novel SECIS elements of the invention contain non-canonical quartet sequences. Other novel SECIS elements provided by the invention are chimeric SECIS elements comprising a canonical SECIS element that contains a non-canonical quartet sequence and chimeric SECIS elements comprising a non-canonical SECIS element that contains a canonical quartet sequence. The novel SECIS elements of the invention facilitate the insertion of selenocysteine residues into recombinant polypeptides.

Brachytherapy Using Elastin-Like Polypeptides with (131)I Inhibit Tumor Growth in Rabbits with VX2 Liver Tumor.

PubMed

Liu, Xinpei; Shen, Yiming; Zhang, Xuqian; Lin, Rui; Jia, Qiang; Chang, Yixiang; Liu, Wenge; Liu, Wentian

2016-10-01

Brachytherapy is a targeted type of radiotherapy utilized in the treatment of cancers. Elastin-like polypeptides are a unique class of genetically engineered peptide polymers that have several attractive properties for brachytherapy. To explore the feasibility and application of brachytherapy for VX2 liver tumor using elastin-like polypeptides with (131)I so as to provide reliable experimental evidence for a new promising treatment of liver cancer. Elastin-like polypeptide as carrier was labeled with (131)I using the iodogen method. Ten eligible rabbits with VX2 liver tumor were randomly divided into the treatment group (n = 5) and control group (n = 5). The treatment group received brachytherapy using elastin-like polypeptide with (131)I, and in the control group, elastin-like polypeptide was injected into the VX2 liver tumor as a control. Periodic biochemical and imaging surveillances were required to assess treatment efficacy. The stability of elastin-like polypeptide with (131)I in vitro was maintained at over 96.8 % for 96 h. Biochemistry and imaging indicated brachytherapy using elastin-like polypeptide with (131)I for liver tumor can improve liver function and inhibit tumor growth (P < 0.05). Elastin-like polypeptide can be an ideal carrier of (131)I and have high labeling efficiency, radiochemical purity and stability. Brachytherapy using elastin-like polypeptide with (131)I for liver tumor is a useful therapy that possesses high antitumor efficacy advantages.

Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

PubMed

Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

2016-01-01

The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1-5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

Age- and sex-related differences of organic anion-transporting polypeptide gene expression in livers of rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Wei-Yu; Xu, Shang-Fu; Zhu, Qiong-Ni

Organic anion-transporting polypeptides (Oatps) play important roles in transporting endogenous substances and xenobiotics into the liver and are implicated in drug-drug interactions. Many factors could influence their expression and result in alterations in drug disposition, efficacy and toxicity. This study was aimed to examine the development-, aging-, and sex-dependent Oatps expression in livers of rats. The livers from SD rats during development (− 2, 1, 7, 14, 21, 28, 35, and 60 d) and aging (60, 180, 540 and/or 800 d) were collected and total RNAs were extracted, purified, and subjected to real-time PCR analysis. Total proteins were extracted formore » western-blot analysis. Results showed that Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 were all hardly detectable in fetal rat livers, low at birth, rapidly increased after weaning (21 d), and reached the peak at 60 d. The Oatps remained stable during the age between 60–180 d, and decreased at elderly (540 and/or 800 d). After birth, Oatp1a1, Oatp1a4, and Oatp1b2 were all highly expressed in liver, in contrast, Oatp1a5 expression was low. Oatp expressions are male-predominant in rat livers. In the livers of aged rats, the Oatp expression decreased and shared a consistent ontogeny pattern at the mRNA and protein level. In conclusion, this study showed that in rat liver, Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 gene expressions are influenced by age and gender, which could provide a basis of individual variation in drug transport, metabolism and toxicity in children, elderly and women. - Highlights: • Oatp1a1, Oatp1a4, Oatp1a5 and Oatp1b2 expression in livers of rats. • Ontogenic changes of Oatps at − 2, 1, 7, 14, 21, 28, 35, and 60 days. • Age-related changes of Oatps at 60, 180, 540, and 800 days. • Sex-difference of Oatps at the both mRNA and protein levels.« less

Sitagliptin decreases ventricular arrhythmias by attenuated glucose-dependent insulinotropic polypeptide (GIP)-dependent resistin signalling in infarcted rats.

PubMed

Lee, Tsung-Ming; Chen, Wei-Ting; Chang, Nen-Chung

2016-01-25

Myocardial infarction (MI) was associated with insulin resistance, in which resistin acts as a critical mediator. We aimed to determine whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, can attenuate arrhythmias by regulating resistin-dependent nerve growth factor (NGF) expression in postinfarcted rats. Normoglycaemic male Wistar rats after ligating coronary artery were randomized to either vehicle or sitagliptin for 4 weeks starting 24 h after operation. Post-infarction was associated with increased myocardial noradrenaline [norepinephrine (NE)] levels and sympathetic hyperinnervation. Compared with vehicle, sympathetic innervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis of tyrosine hydroxylase, growth-associated factor 43 and neurofilament and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Furthermore, sitagliptin was associated with reduced resistin expression and increased Akt activity. Ex vivo studies showed that glucose-dependent insulinotropic polypeptide (GIP) infusion, but not glucagon-like peptide-1 (GLP-1), produced similar reduction in resistin levels to sitagliptin in postinfarcted rats. Furthermore, the attenuated effects of sitagliptin on NGF levels can be reversed by wortmannin (a phosphatidylinositol 3-kinase antagonist) and exogenous resistin infusion. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation in the non-diabetic infarcted rats. Sitagliptin attenuated resistin expression via the GIP-dependent pathway, which inhibited sympathetic innervation through a signalling pathway involving phosphatidylinositol 3-kinase (PI3K) and Akt protein. © 2016 Authors.

Global and quantitative proteomic analysis of dogs infected by avian-like H3N2 canine influenza virus

PubMed Central

Su, Shuo; Tian, Jin; Hong, Malin; Zhou, Pei; Lu, Gang; Zhu, Huachen; Zhang, Guihong; Lai, Alexander; Li, Shoujun

2015-01-01

Canine influenza virus A (H3N2) is a newly emerged etiological agent for respiratory infections in dogs. The mechanism of interspecies transmission from avian to canine species and the development of diseases in this new host remain to be explored. To investigate this, we conducted a differential proteomics study in 2-month-old beagles inoculated intranasally with 106 TCID50 of A/canine/Guangdong/01/2006 (H3N2) virus. Lung sections excised at 12 h post-inoculation (hpi), 4 days, and 7 days post-inoculation (dpi) were processed for global and quantitative analysis of differentially expressed proteins. A total of 17,796 proteins were identified at different time points. About 1.6% was differentially expressed between normal and infected samples. Of these, 23, 27, and 136 polypeptides were up-regulated, and 14, 18, and 123 polypeptides were down-regulated, at 12 hpi, 4 dpi, and 7 dpi, respectively. Vann diagram analysis indicated that 17 proteins were up-regulated and one was down-regulated at all three time points. Selected proteins were validated by real-time PCR and by Western blot. Our results show that apoptosis and cytoskeleton-associated proteins expression was suppressed, whereas interferon-induced proteins plus other innate immunity proteins were induced after the infection. Understanding of the interactions between virus and the host will provide insights into the basis of interspecies transmission, adaptation, and virus pathogenicity. PMID:25883591

PACAP/VIP and receptor characterization in micturition pathways in mice with overexpression of NGF in urothelium.

PubMed

Girard, Beatrice M; Malley, Susan E; Braas, Karen M; May, Victor; Vizzard, Margaret A

2010-11-01

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting or proliferation in the urinary bladder, produces urinary bladder hyperreflexia, and results in increased referred somatic hypersensitivity. Additional NGF-mediated changes might contribute to the urinary bladder hyperreflexia and pelvic hypersensitivity observed in these transgenic mice such as upregulation of neuropeptide/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific, uroplakin II promoter. In the present study, we examined pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), and associated receptor (PAC1, VPAC1, VPAC2) transcripts or protein expression in urothelium and detrusor smooth muscle and lumbosacral dorsal root ganglia in NGF-overexpressing and littermate wildtype mice using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical approaches. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-immunoreactivity in urothelium of NGF-OE mice whereas PACAP transcript and PACAP-immunoreactivity were decreased in urothelium of NGF-OE mice. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice. VIP transcript expression and immunostaining was not altered in urinary bladder of NGF-OE mice. Changes in PACAP, VIP, and associated receptor transcripts and protein expression in micturition pathways resemble some, but not all, changes observed after induction of urinary bladder inflammation known to involve NGF production.

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

PubMed

Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

1995-03-01

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

PubMed Central

de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

2007-01-01

Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

The cytoplasmic domain of the Drosophila cell adhesion molecule neuroglian is not essential for its homophilic adhesive properties in S2 cells.

PubMed

Hortsch, M; Wang, Y M; Marikar, Y; Bieber, A J

1995-08-11

Drosophila neuroglian is a transmembrane glycoprotein that has strong structural and sequence homology to the vertebrate L1 gene family of cell adhesion molecules (Bieber, A.J., Snow, P.M., Hortsch, M., Patel, N.H., Jacobs, J.R., Traquina, Z.R., Schilling, J., and Goodman, C.S. (1989) Cell 59, 447-460. Two different neuroglian protein forms that are generated by a differential splicing process are expressed in a tissue-specific fashion by embryonic and larval cells (Hortsch, M., Bieber, A.J., Patel, N.H., and Goodman, C.S. (1990) Neuron 4, 697-709). The two neuroglial polypeptides differ only in their cytoplasmic domains. Both of these neuroglian species, when transfected into the expressed in Drosophila S2 cells, induce the calcium-independent, homophilic aggregation of transformed cells. A third artificial neuroglian protein form was constructed by substituting the neuroglian transmembrane segment and cytoplasmic domains with the glycosyl phosphatidylinositol attachment signal of the Drosophila fasciclin I protein. This cDNA construct generates a glycosyl phosphatidylinositol-anchored form of neuroglian, which retains the ability to induce homophilic cell aggregation when expressed in S2 cells, and was able to interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that neuroglian mediates a calcium-independent, homophilic cell adhesion activity and that neither cytoplasmic neuroglian domains nor a direct interaction with cytoskeletal elements is essential for this property.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj; Shaghasi, Tarana

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj; Shagasi, Tarana

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj; Shagasi, Tarana

2015-06-30

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Thermal and acid tolerant beta xylosidases, arabinofuranosidases, genes encoding, related organisms, and methods

DOEpatents

Thompson, David N; Thompson, Vicki S; Schaller, Kastli D; Apel, William A; Reed, David W; Lacey, Jeffrey A

2013-04-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose, xylobiose, and/or arabinofuranose-substituted xylan using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

Polypeptides having beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-05-06

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stringer, Mary Ann; McBrayer, Brett

2016-11-29

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-05-06

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-04-29

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Rab-GDI Complex Dissociation Factor Expressed through Translational Frameshifting in Filamentous Ascomycetes

PubMed Central

Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

2013-01-01

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild. PMID:24069231

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

DOE PAGES

Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.; ...

2017-06-09

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.« less

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Welner, Ditte Hededam; Shin, David; Tomaleri, Giovani P.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, coexpression partners, purification methods, and optimization of protein solubility and stability. Hence researchersmore » are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: Insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.« less

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

PubMed

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-10-17

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells

PubMed Central

Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

2017-01-01

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells. PMID:29156827

Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

PubMed

Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

2016-05-01

Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altabella, T.; Chrispeels, M.J.

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less

Unraveling the Specific Ischemic Core and Penumbra Transcriptome in the Permanent Middle Cerebral Artery Occlusion Mouse Model Brain Treated with the Neuropeptide PACAP38

PubMed Central

Hori, Motohide; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Shioda, Seiji; Numazawa, Satoshi

2015-01-01

Our group has been systematically investigating the effects of the neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain. To do so, we have established and utilized the permanent middle cerebral artery occlusion (PMCAO) mouse model, in which PACAP38 (1 pmol) injection is given intracerebroventrically and compared to a control saline (0.9% sodium chloride, NaCl) injection, to unravel genome-wide gene expression changes using a high-throughput DNA microarray analysis approach. In our previous studies, we have accumulated a large volume of data (gene inventory) from the whole brain (ipsilateral and contralateral hemispheres) after both PMCAO and post-PACAP38 injection. In our latest research, we have targeted specifically infarct or ischemic core (hereafter abbreviated IC) and penumbra (hereafter abbreviated P) post-PACAP38 injections in order to re-examine the transcriptome at 6 and 24 h post injection. The current study aims to delineate the specificity of expression and localization of differentially expressed molecular factors influenced by PACAP38 in the IC and P regions. Utilizing the mouse 4 × 44 K whole genome DNA chip we show numerous changes (≧/≦ 1.5/0.75-fold) at both 6 h (654 and 456, and 522 and 449 up- and down-regulated genes for IC and P, respectively) and 24 h (2568 and 2684, and 1947 and 1592 up- and down-regulated genes for IC and P, respectively) after PACAP38 treatment. Among the gene inventories obtained here, two genes, brain-derived neurotrophic factor (Bdnf) and transthyretin (Ttr) were found to be induced by PACAP38 treatment, which we had not been able to identify previously using the whole hemisphere transcriptome analysis. Using bioinformatics analysis by pathway- or specific-disease-state focused gene classifications and Ingenuity Pathway Analysis (IPA) the differentially expressed genes are functionally classified and discussed. Among these, we specifically discuss some novel and previously identified genes, such as alpha hemoglobin stabilizing protein (Ahsp), cathelicidin antimicrobial peptide (Camp), chemokines, interferon beta 1 (Ifnb1), and interleukin 6 (Il6) in context of PACAP38-mediated neuroprotection in the ischemic brain. Taken together, the DNA microarray analysis provides not only a great resource for further study, but also reinforces the importance of region-specific analyses in genome-wide identification of target molecular factors that might play a role in the neuroprotective function of PACAP38. PMID:27600210

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

PubMed

Muthalif, M M; Rowland, L J

1994-04-01

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins.

Identification of dehydrin-like proteins responsive to chilling in floral buds of blueberry (Vaccinium, section Cyanococcus).

PubMed Central

Muthalif, M M; Rowland, L J

1994-01-01

The level of three major polypeptides of 65, 60, and 14 kD increased in response to chilling unit accumulation in floral buds of a woody perennial, blueberry (Vaccinium, section Cynaococcus). The level of the polypeptides increased most dramatically within 300 h of chilling and decreased to the prechilling level with the initiation of budbreak. Cold-hardiness levels were assessed for dormant buds of Vaccinium corymbosum and Vaccinium ashei after different chilling treatments until the resumption of growth. These levels coincided with the level of the chilling-responsive polypeptides. Like some other previously described cold-induced proteins in annual plants, the level of the chilling-induced polypeptides also increased in leaves in response to cold treatment; the chilling-induced polypeptides were heat stable, resisting aggregation after incubation at 95 degrees C for 15 min. By fractionating bud proteins first by isoelectric point (pI) and then by molecular mass, the pI values of the 65- and 60-kD polypeptides were found to be 7.5 to 8.0 and the pI value of the 14-kD polypeptide was judged to be 8.5. Purification of the 65- and 60-kD polypeptides, followed by digestion with endoproteinase Lys-C and sequencing of selected fragments, revealed similarities in amino acid composition between the 65- and 60-kD polypeptides and dehydrins. Indeed, antiserum to the lysine-rich consensus sequence EKKGIMDKIKEKLPG of dehydrin proteins cross-reacted to all three of the major chilling-responsive polypeptides of blueberry, identifying these as dehydrins or dehydrin-like proteins. PMID:8016270

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

PubMed

Jean, D H; Albers, R W; Koval, G J

1975-02-10

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

Elastin-like polypeptides: the power of design for smart cell encapsulation.

PubMed

Bandiera, Antonella

2017-01-01

Cell encapsulation technology is still a challenging issue. Innovative methodologies such as additive manufacturing, and alternative bioprocesses, such as cell therapeutic delivery, where cell encapsulation is a key tool are rapidly gaining importance for their potential in regenerative medicine. Responsive materials such as elastin-based recombinant expression products have features that are particularly attractive for cell encapsulation. They can be designed and tailored to meet desired requirements. Thus, they represent promising candidates for the development of new concept-based materials that can be employed in this field. Areas covered: An overview of the design and employment of elastin-like polypeptides for cell encapsulation is given to outline the state of the art. Special attention is paid to the design of the macromolecule employed as well as to the method of matrix formation and the biological system involved. Expert opinion: As a result of recent progress in regenerative medicine there is a compelling need for materials that provide specific properties and demonstrate defined functional features. Rationally designed materials that may adapt according to applied external stimuli and that are responsive to biological systems, such as elastin-like polypeptides, belong to this class of smart material. A run through the components described to date represents a good starting point for further advancement in this area. Employment of these components in cell encapsulation application will promote its advance toward 'smart cell encapsulation technology'.

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

PubMed

Potter, J W; Black, C C

1982-08-01

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morant, Marc

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin

2007-04-20

Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfectedmore » with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.« less

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Pituitary adenylate cyclase-activating polypeptide: a novel peptide with protean implications.

PubMed

Pisegna, Joseph R; Oh, David S

2007-02-01

The purpose of this review is to highlight the importance of pituitary adenylate cyclase-activating polypeptide in physiological processes and to describe how this peptide is becoming increasingly recognized as having a major role in the body. Since its discovery in 1989, investigators have sought to determine the site of biological activity and the function of pituitary adenylate cyclase-activating polypeptide in maintaining homeostasis. Since its discovery, pituitary adenylate cyclase-activating polypeptide appears to play an important role in the regulation of processes within the central nervous system and gastrointestinal tract, as well in reproductive biology. Pituitary adenylate cyclase-activating polypeptide has been shown to regulate tumor cell growth and to regulate immune function through its effects on T lympocytes. These discoveries suggest the importance of pituitary adenylate cyclase-activating polypeptide in neuronal development, neuronal function, gastrointestinal tract function and reproduction. Future studies will examine more closely the role of pituitary adenylate cyclase-activating polypeptide in regulation of malignantly transformed cells, as well as in regulation of immune function.

Unimpaired postprandial pancreatic polypeptide secretion in Parkinson's disease and REM sleep behavior disorder.

PubMed

Unger, Marcus M; Ekman, Rolf; Björklund, Anna-Karin; Karlsson, Gösta; Andersson, Chatarina; Mankel, Katharina; Bohne, Katharina; Tebbe, Johannes J; Stiasny-Kolster, Karin; Möller, Jens C; Mayer, Geert; Kann, Peter H; Oertel, Wolfgang H

2013-04-01

Pancreatic polypeptide is released immediately after food ingestion. The release is operated by vagal-abdominal projections and has therefore been suggested as a test for vagal nerve integrity. Pathoanatomical and clinical studies indicate vagal dysfunction in early Parkinson's disease (PD). We assessed the postprandial secretion of pancreatic polypeptide and motilin in healthy controls (n = 18) and patients with idiopathic rapid-eye-movement sleep behavior disorder (iRBD, n = 10), a potential premotor stage of PD, as well as in drug-naive (n = 19) and treated (n = 19) PD patients. The postprandial pancreatic polypeptide secretion showed a physiological pattern in all groups and even an enhanced response in drug-naive PD and iRBD. Motilin concentrations correlated with pancreatic polypeptide concentrations. Postprandial pancreatic polypeptide secretion is not a suitable test for vagal nerve integrity in PD. The unimpaired pancreatic polypeptide response in iRBD and PD might be explained by partially intact vagal-abdominal projections or compensatory mechanisms substituting a defective neuronal brain-gut axis. Copyright © 2012 Movement Disorders Society.

A de novo designed 11 kDa polypeptide: model for amyloidogenic intrinsically disordered proteins.

PubMed

Topilina, Natalya I; Ermolenkov, Vladimir V; Sikirzhytski, Vitali; Higashiya, Seiichiro; Lednev, Igor K; Welch, John T

2010-07-01

A de novo polypeptide GH(6)[(GA)(3)GY(GA)(3)GE](8)GAH(6) (YE8) has a significant number of identical weakly interacting beta-strands with the turns and termini functionalized by charged amino acids to control polypeptide folding and aggregation. YE8 exists in a soluble, disordered form at neutral pH but is responsive to changes in pH and ionic strength. The evolution of YE8 secondary structure has been successfully quantified during all stages of polypeptide fibrillation by deep UV resonance Raman (DUVRR) spectroscopy combined with other morphological, structural, spectral, and tinctorial characterization. The YE8 folding kinetics at pH 3.5 are strongly dependent on polypeptide concentration with a lag phase that can be eliminated by seeding with a solution of folded fibrillar YE8. The lag phase of polypeptide folding is concentration dependent leading to the conclusion that beta-sheet folding of the 11-kDa amyloidogenic polypeptide is completely aggregation driven.

pH sensitive core-shell magnetic nanoparticles for targeted drug delivery in cancer therapy.

PubMed

Lungu, Iulia Ioana; Rădulescu, Marius; Mogoşanu, George Dan; Grumezescu, Alexandru Mihai

2016-01-01

In the last decade, nanobiotechnology has evolved rapidly with an extensive impact on biomedical area. In order to improve bioavailability and minimize adverse effects, drug delivery systems based on magnetic nanocomposites are under development mainly for cancer imaging and antitumor therapy. In this regard, pH sensitive core-shell magnetic nanoparticles (NPs) with accurate controlled size and shape are synthesized by various modern methods, such as homogeneous precipitation, coprecipitation, microemulsion or polyol approaches, high temperature and hydrothermal reactions, sol-gel reactions, aerosol÷vapor processes and sonolysis. Due to their unique combined physico-chemical and biological properties (such as higher dispensability, chemical and thermal stability, biocompatibility), pH responsive core-shell magnetic NPs are widely investigated for controlled release of cytostatic drugs into the tumor site by means of pH change: magnetite@silicon dioxide (Fe3O4@SiO2), Fe3O4@titanium dioxide (TiO2), β-thiopropionate-polyethylene glycol (PEG)-modified Fe3O4@mSiO2, Fe3O4 NPs core coated with SiO2 with an imidazole group modified PEG-polypeptide (mPEG-poly-L-Asparagine), polyacrylic acid (PAA) and folic acid (FA) coating of the iron oxide NP core, methoxy polyethylene glycol-block-polymethacrylic acid-block-polyglycerol monomethacrylate (MPEG-b-PMAA-b-PGMA) attached by a PGMA block to a Fe3O4 core, PEG-modified polyamidoamine (PAMAM) dendrimer shell with Fe3O4 core and mesoporous silica coated on Fe3O4, mostly coated with an anticancer drug. This review paper highlights the modern research directions currently employed to demonstrate the utility of the pH responsive core-shell magnetic NPs in diagnosis and treatment of oncological diseases.

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains.

PubMed

Vidgren, Virve; Ruohonen, Laura; Londesborough, John

2005-12-01

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters. We correlated maltose transport kinetics with the genotypes of some ale and lager yeasts. Maltose transport by two ale strains was strongly inhibited by other alpha-glucosides, suggesting the use of broad substrate specificity transporters, such as Agt1p. Maltose transport by three lager strains was weakly inhibited by other alpha-glucosides, suggesting the use of narrow substrate specificity transporters. Hybridization studies showed that all five strains contained complete MAL1, MAL2, MAL3, and MAL4 loci, except for one ale strain, which lacked a MAL2 locus. All five strains also contained both AGT1 (coding a broad specificity alpha-glucoside transporter) and MAL11 alleles. MPH genes (maltose permease homologues) were present in the lager but not in the ale strains. During growth on maltose, the lager strains expressed AGT1 at low levels and MALx1 genes at high levels, whereas the ale strains expressed AGT1 at high levels and MALx1 genes at low levels. MPHx expression was negligible in all strains. The AGT1 sequences from the ale strains encoded full-length (616 amino acid) polypeptides, but those from both sequenced lager strains encoded truncated (394 amino acid) polypeptides that are unlikely to be functional transporters. Thus, despite the apparently similar genotypes of these ale and lager strains revealed by hybridization, maltose is predominantly carried by AGT1-encoded transporters in the ale strains and by MALx1-encoded transporters in the lager strains.

Histone Deacetylase 2 Is a Component of Influenza A Virus-Induced Host Antiviral Response.

PubMed

Nagesh, Prashanth T; Hussain, Mazhar; Galvin, Henry D; Husain, Matloob

2017-01-01

Host cells produce variety of antiviral factors that create an antiviral state and target various stages of influenza A virus (IAV) life cycle to inhibit infection. However, IAV has evolved various strategies to antagonize those antiviral factors. Recently, we reported that a member of class I host histone deacetylases (HDACs), HDAC1 possesses an anti-IAV function. Herein, we provide evidence that HDAC2, another class I member and closely related to HDAC1 in structure and function, also possesses anti-IAV properties. In turn, IAV, like HDAC1, dysregulates HDAC2, mainly at the polypeptide level through proteasomal degradation to potentially minimize its antiviral effect. We found that IAV downregulated the HDAC2 polypeptide level in A549 cells in an H1N1 strain-independent manner by up to 47%, which was recovered to almost 100% level in the presence of proteasome-inhibitor MG132. A further knockdown in HDAC2 expression by up to 90% via RNA interference augmented the growth kinetics of IAV in A549 cells by more than four-fold after 24 h of infection. Furthermore, the knockdown of HDAC2 expression decreased the IAV-induced phosphorylation of the transcription factor, Signal Transducer and Activator of Transcription I (STAT1) and the expression of interferon-stimulated gene, viperin in infected cells by 41 and 53%, respectively. The role of HDAC2 in viperin expression was analogous to that of HDAC1, but it was not in the phosphorylation of STAT1. This indicated that, like HDAC1, HDAC2 is a component of IAV-induced host innate antiviral response and performs both redundant and non-redundant functions vis-a-vis HDAC1; however, IAV dysregulates them both in a redundant manner.

Dysregulation of retinoic acid receptor diminishes hepatocyte permissiveness to hepatitis B virus infection through modulation of sodium taurocholate cotransporting polypeptide (NTCP) expression.

PubMed

Tsukuda, Senko; Watashi, Koichi; Iwamoto, Masashi; Suzuki, Ryosuke; Aizaki, Hideki; Okada, Maiko; Sugiyama, Masaya; Kojima, Soichi; Tanaka, Yasuhito; Mizokami, Masashi; Li, Jisu; Tong, Shuping; Wakita, Takaji

2015-02-27

Sodium taurocholate cotransporting polypeptide (NTCP) is an entry receptor for hepatitis B virus (HBV) and is regarded as one of the determinants that confer HBV permissiveness to host cells. However, how host factors regulate the ability of NTCP to support HBV infection is largely unknown. We aimed to identify the host signaling that regulated NTCP expression and thereby permissiveness to HBV. Here, a cell-based chemical screening method identified that Ro41-5253 decreased host susceptibility to HBV infection. Pretreatment with Ro41-5253 inhibited the viral entry process without affecting HBV replication. Intriguingly, Ro41-5253 reduced expression of both NTCP mRNA and protein. We found that retinoic acid receptor (RAR) regulated the promoter activity of the human NTCP (hNTCP) gene and that Ro41-5253 repressed the hNTCP promoter by antagonizing RAR. RAR recruited to the hNTCP promoter region, and nucleotides -112 to -96 of the hNTCP was suggested to be critical for RAR-mediated transcriptional activation. HBV susceptibility was decreased in pharmacologically RAR-inactivated cells. CD2665 showed a stronger anti-HBV potential and disrupted the spread of HBV infection that was achieved by continuous reproduction of the whole HBV life cycle. In addition, this mechanism was significant for drug development, as antagonization of RAR blocked infection of multiple HBV genotypes and also a clinically relevant HBV mutant that was resistant to nucleoside analogs. Thus, RAR is crucial for regulating NTCP expression that determines permissiveness to HBV infection. This is the first demonstration showing host regulation of NTCP to support HBV infection. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dysregulation of Retinoic Acid Receptor Diminishes Hepatocyte Permissiveness to Hepatitis B Virus Infection through Modulation of Sodium Taurocholate Cotransporting Polypeptide (NTCP) Expression*

PubMed Central

Tsukuda, Senko; Watashi, Koichi; Iwamoto, Masashi; Suzuki, Ryosuke; Aizaki, Hideki; Okada, Maiko; Sugiyama, Masaya; Kojima, Soichi; Tanaka, Yasuhito; Mizokami, Masashi; Li, Jisu; Tong, Shuping; Wakita, Takaji

2015-01-01

Sodium taurocholate cotransporting polypeptide (NTCP) is an entry receptor for hepatitis B virus (HBV) and is regarded as one of the determinants that confer HBV permissiveness to host cells. However, how host factors regulate the ability of NTCP to support HBV infection is largely unknown. We aimed to identify the host signaling that regulated NTCP expression and thereby permissiveness to HBV. Here, a cell-based chemical screening method identified that Ro41-5253 decreased host susceptibility to HBV infection. Pretreatment with Ro41-5253 inhibited the viral entry process without affecting HBV replication. Intriguingly, Ro41-5253 reduced expression of both NTCP mRNA and protein. We found that retinoic acid receptor (RAR) regulated the promoter activity of the human NTCP (hNTCP) gene and that Ro41-5253 repressed the hNTCP promoter by antagonizing RAR. RAR recruited to the hNTCP promoter region, and nucleotides −112 to −96 of the hNTCP was suggested to be critical for RAR-mediated transcriptional activation. HBV susceptibility was decreased in pharmacologically RAR-inactivated cells. CD2665 showed a stronger anti-HBV potential and disrupted the spread of HBV infection that was achieved by continuous reproduction of the whole HBV life cycle. In addition, this mechanism was significant for drug development, as antagonization of RAR blocked infection of multiple HBV genotypes and also a clinically relevant HBV mutant that was resistant to nucleoside analogs. Thus, RAR is crucial for regulating NTCP expression that determines permissiveness to HBV infection. This is the first demonstration showing host regulation of NTCP to support HBV infection. PMID:25550158

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

PubMed Central

Hwang, Byung Joon; Toering, Stephanie; Francke, Uta; Chu, Gilbert

1998-01-01

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin. PMID:9632823

Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

PubMed Central

He, Cuiwen H.; Black, Dylan S.; Allan, Christopher M.; Meunier, Brigitte; Rahman, Shamima; Clarke, Catherine F.

2017-01-01

Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA) in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB). To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model. PMID:28736527

Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.

PubMed Central

Ellis, R W; Keller, P M; Lowe, R S; Zivin, R A

1985-01-01

The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products. Images PMID:2981365

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

NASA Astrophysics Data System (ADS)

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere.

PubMed

Munegumi, Toratane; Tanikawa, Naoya

2017-09-01

Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dongjing; Wu, Jilin, E-mail: 6296082@qq.com; Liu, Meizhou

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulatedmore » miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the carcinogenesis and progression of HCC. • Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.« less

The expression of 11β-hydroxysteroid dehydrogenase type 1 and 2 in nasal polyp-derived epithelial cells and its possible contribution to glucocorticoid activation in nasal polyp.

PubMed

Kook, Jin Ho; Kim, Hyun Jin; Kim, Kyung Won; Park, Se Jin; Kim, Tae Hoon; Lim, Sae Hee; Kang, Sung Hoon; Lee, Sang Hag

2015-01-01

The actions of glucocorticoids in target tissues depend on the local metabolism of glucocorticoids catalyzed by 11β hydroxysteroid dehydrogenase (HSD) 1 and 2. Glucocorticoids are the most effective anti-inflammatory drugs in the treatment of nasal polyps. However, the mechanisms that underlie the anti-inflammatory effects are unclear. The present study analyzed the expression of 11β-HSD1, 11β-HSD2, and steroidogenic enzymes (cytochrome P450, family 11, subfamily B, polypeptide 1 [CYP11B1]; cytochrome P450, family 11, subfamily A, polypeptide 1 [CYP11A1]) in nasal polyp tissues, and endogenous cortisol levels in nasal polyp-derived epithelial cells. The expression levels and distribution pattern of 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1 were determined in nasal polyp tissues or nasal polyp-derived epithelial cells by using real-time polymerase chain reaction, Western blot, and immunohistochemistry testing. The expression levels of cortisol by using enzyme-linked immunosorbent assay were determined in cultured polyp-derived epithelial cells treated with adrenocorticotrophic hormone (ACTH), 11β-HSD1 inhibitor, or small interfering ribonucleic acid technique. The effect of glucocorticoids on the expression levels of these enzymes was investigated in cultured cells. Expressed in nasal polyp tissues and nasal polyp-derived epithelial cells were 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1. Cortisol production in cultured epithelial cells was decreased in cells treated with 11β-HSD1 small interfering ribonucleic acid or inhibitor, compared with nontreated cells. Cultured cells treated with adrenocorticotropic hormone induced increased cortisol production. 11β-HSD1 expression levels were upregulated in cells treated with glucocorticoid. Analysis of these results indicated that 11β-HSD1 expressed in polyp-derived epithelial cells may be involved in the anti-inflammatory function of glucocorticoid in the treatment of nasal polyps, which contributes to increased levels of endogenous cortisol.

Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex

PubMed Central

Prasad, Bhagwat; Evers, Raymond; Gupta, Anshul; Hop, Cornelis E. C. A.; Salphati, Laurent; Shukla, Suneet; Ambudkar, Suresh V.

2014-01-01

Interindividual variability in protein expression of organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, OATP2B1, and multidrug resistance-linked P-glycoprotein (P-gp) or ABCB1 was quantified in frozen human livers (n = 64) and cryopreserved human hepatocytes (n = 12) by a validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method. Membrane isolation, sample workup, and LC-MS/MS analyses were as described before by our laboratory. Briefly, total native membrane proteins, isolated from the liver tissue and cryopreserved hepatocytes, were trypsin digested and quantified by LC-MS/MS using signature peptide(s) unique to each transporter. The mean ± S.D. (maximum/minimum range in parentheses) protein expression (fmol/µg of membrane protein) in human liver tissue was OATP1B1- 2.0 ± 0.9 (7), OATP1B3- 1.1 ± 0.5 (8), OATP2B1- 1 1.7 ± 0.6 (5), and P-gp- 0.4 ± 0.2 (8). Transporter expression in the liver tissue was comparable to that in the cryopreserved hepatocytes. Most important is that livers with SLCO1B1 (encoding OATP1B1) haplotypes *14/*14 and *14/*1a [i.e., representing single nucleotide polymorphisms (SNPs), c.388A > G, and c.463C > A] had significantly higher (P < 0.0001) protein expression than the reference haplotype (*1a/*1a). Based on these genotype-dependent protein expression data, we predicted (using Simcyp) an up to ∼40% decrease in the mean area under the curve of rosuvastatin or repaglinide in subjects harboring these variant alleles compared with those harboring the reference alleles. SLCO1B3 (encoding OATP1B3) SNPs did not significantly affect protein expression. Age and sex were not associated with transporter protein expression. These data will facilitate the prediction of population-based human transporter-mediated drug disposition, drug-drug interactions, and interindividual variability through physiologically based pharmacokinetic modeling. PMID:24122874

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, David N.; Apel, William A.; Thompson, Vicki S.

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2015-06-02

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2013-10-15

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID; Henriksen, Emily D [Idaho Falls, ID

2012-06-19

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-04-23

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

2010-12-28

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

DOEpatents

Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

2013-07-30

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, David N; Apel, William A; Thompson, Vicki S

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

Functional expression and purification of recombinant Tx1, a sodium channel blocker neurotoxin from the venom of the Brazilian "armed" spider, Phoneutria nigriventer.

PubMed

Diniz, Marcelo R V; Theakston, R David G; Crampton, Julian M; Nascimento Cordeiro, Marta do; Pimenta, Adriano M C; De Lima, Maria Elena; Diniz, Carlos R

2006-11-01

Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.

The influence of co-cultivation on expression of the antifungal protein in Aspergillus giganteus.

PubMed

Meyer, Vera; Stahl, Ulf

2003-01-01

The afp gene of Aspergillus giganteus encodes a small, highly basic polypeptide with antifungal activity, named Antifungal Protein (AFP). The protein is secreted by the mould and inhibits the growth of various filamentous fungi. In this paper we report that co-cultivation of A. giganteus with various microorganisms alters afp expression. It was found that co-cultivation modulates afp expression on the level of transcription, using a reporter system based on the beta-glucuronidase gene. The presence of Fusarium oxysporum triggered afp transcription whereas dual cultures of A. giganteus and A. niger resulted in suppression of afp transcription. Growth tests performed with several carbon and nitrogen sources, revealed that the influence of co-cultivation is strongly dependent on the medium composition.