Effects of Full-Length Phosphorylated Osteopontin and Constituent Acidic Peptides and Amino Acids on Calcite Dissolution

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.

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Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle

Development ◽

10.1242/dev.104.1.165 ◽

1988 ◽

Vol 104 (1) ◽

pp. 165-173 ◽

Cited By ~ 1

Author(s):

C.H. Barton ◽

G. Dickson ◽

H.J. Gower ◽

L.H. Rowett ◽

W. Putt ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Cell Surface ◽

Protein Sequence ◽

Single Copy ◽

In Vitro Transcription ◽

Full Length ◽

Covalent Attachment ◽

Size Difference

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 × 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 × 10(3). The size difference between the 125 × 10(3). The size difference between the 125 × 10(3) Mr skeletal muscle form and the 120 × 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 × 10(3) which is processed by microsomal membranes to yield a 122 × 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 × 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.

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Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

Journal of Virology ◽

10.1128/jvi.79.15.10032-10039.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 10032-10039 ◽

Cited By ~ 32

Author(s):

William Severson ◽

Xiaolin Xu ◽

Michaela Kuhn ◽

Nina Senutovitch ◽

Mercy Thokala ◽

...

Keyword(s):

Amino Acids ◽

Rna Binding ◽

Mutational Analysis ◽

Essential Amino Acids ◽

Full Length ◽

Hantaan Virus ◽

Deletion Mapping ◽

Mobility Shift ◽

N Protein ◽

Lysine Residues

ABSTRACT The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

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Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6663 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6663-6669 ◽

Cited By ~ 50

Author(s):

L Trieschmann ◽

Y V Postnikov ◽

A Rickers ◽

M Bustin

Keyword(s):

Amino Acids ◽

Structural Features ◽

Binding Domain ◽

Full Length ◽

Modular Structure ◽

Chromosomal Proteins ◽

Nucleosome Core ◽

Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

Journal of Virology ◽

10.1128/jvi.75.13.6095-6106.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6095-6106 ◽

Cited By ~ 212

Author(s):

Stephen J. Polyak ◽

Khalid S. A. Khabar ◽

Denise M. Paschal ◽

Heather J. Ezelle ◽

Gilles Duverlie ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Response ◽

Full Length ◽

Interleukin 8 ◽

Antiviral Resistance ◽

Ns5a Protein ◽

Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

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Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

Infection and Immunity ◽

10.1128/iai.69.12.7839-7850.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7839-7850 ◽

Cited By ~ 39

Author(s):

Gillian D. Pullinger ◽

R. Sowdhamini ◽

Alistair J. Lax

Keyword(s):

Amino Acids ◽

Fusion Proteins ◽

Pasteurella Multocida ◽

Transmembrane Domain ◽

Polyclonal Antibodies ◽

Full Length ◽

Cell Binding ◽

Terminal Fragment ◽

C Terminus ◽

N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

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The C-Terminal of MYH11 Is Required for Cbfb-MYH11 Activity During Embryonic Hematopoiesis and Leukemogenesis

Blood ◽

10.1182/blood.v118.21.2472.2472 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2472-2472

Author(s):

R. Katherine Hyde ◽

Yasuhiko Kamikubo ◽

Ling Zhao ◽

Lemlem Alemu ◽

Lisa Garrett ◽

...

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Transcription Factor ◽

Null Allele ◽

Fusion Gene ◽

Full Length ◽

Primitive Hematopoiesis ◽

Single Allele ◽

Terminal Amino ◽

Definitive Hematopoiesis

Abstract Abstract 2472 RKH, YK, and LZ all contributed equally to this work Inv(16) is found in nearly all patients with acute myeloid leukemia (AML) subtype M4Eo. Inv(16) results in the fusion of the transcription factor gene CBFB, and the MYH11 gene, which encodes Smooth Muscle Myosin Heavy Chain (SMMHC). This results in the fusion gene CBFB-MYH11, which encodes CBFβ-SMMHC. Previously we showed that knock-in mice with a single allele of Cbfb-MYH11 (Cbfb+/MYH11) have severe differentiation defects in primitive hematopoiesis and a total block in definitive hematopoiesis. In addition, chimeric mice generated from Cbfb+/MYH11 ES cells consistently developed leukemia within a few months after treatment with the mutagen N-ethyl-N-nitrosourea (ENU). It is currently not clear which functional domains of CBFβ-SMMHC are responsible for its activity in differentiation and leukemogenesis. In vitro experiments have indicated that CBFβ-SMMHC can form multimeric complexes via the C terminal domain. It has been postulated that this multimerization may be important for the function of CBFβ-SMMHC by resulting in large macromolecular complexes and/or sequestration of its binding partner, the transcription factor RUNX1. To determine the importance of this domain in vivo, we generated knock-in mice expressing a mutant Cbfb-MYH11 allele with a deletion of the 95 C-terminal amino acids (Cbfb+/MYH11ΔC95). In analysis of primitive hematopoiesis, we found that Cbfb+/MYH11ΔC95 and CbfbMYH11ΔC95/MYH11ΔC95 mice had no or very mild differentiation defects, statistically significantly less severe (p<.05) than seen in embryos expressing full-length Cbfb-MYH11. During definitive hematopoesis, there were no observable defects in Cbfb+/MYH11ΔC95 mice, but CbfbMYH11ΔC95/MYH11ΔC95 embryos showed a complete block in definitive hematopoiesis, as seen in mice expressing a single allele of full length Cbfb-MYH11. This indicates that Cbfb-MYH11ΔC95 is less effective in blocking differentiation than the full length fusion gene. Interestingly, both the primitive and definitive embryonic blood phenotypes of the CbfbMYH11ΔC95/MYH11ΔC95 were similar to that observed in embryos lacking functional Cbfb (Cbfb−/−), implying that Cbfb-MYH11ΔC95 may act as a null allele. To test this possibility we used gene expression microarrays to compare gene expression profiles in the peripheral blood from embryonic day 12 CbfbMYH11ΔC95/MYH11ΔC95, Cbfb−/−, and Cbfb+/MYH11, as well as their Cbfb+/+ littermates. Surprisingly, CbfbMYH11ΔC95/MYH11ΔC95 embryos showed deregulated expression of a distinct gene set as compared to both Cbfb−/− and Cbfb+/MYH11 embryos. This implies that Cbfb-MYH11ΔC95 is not a null allele of Cbfb, and likely retains some, but not all, of the neomorph properties of full length Cbfb-MYH11. Consistent with this finding, we observe the accumulation of abnormal myeloid cells in some adult Cbfb+/MYH11ΔC95 mice after ENU treatment, which has not been reported in Cbfb+/− mice. However, we found that Cbfb-MYH11ΔC95 has not retained the most critical of the fusion gene's activities: the ability to induce leukemogenesis. Importantly, none of the Cbfb+/MYH11ΔC95 mice developed leukemia after treatment with ENU. This is in contrast to mice expressing full length Cbfb-MYH11, which all develop leukemia under these conditions. Together, these results indicate that the 95 C-terminal amino acids of CBFβ-SMMHC are required for both embryonic hematopoietic defects and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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A Novel Human Actin-Binding Protein Homologue That Binds to Platelet Glycoprotein Ib

Blood ◽

10.1182/blood.v92.4.1268.416k12_1268_1276 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1268-1276 ◽

Cited By ~ 1

Author(s):

Wen-feng Xu ◽

Zhi-wei Xie ◽

Dominic W. Chung ◽

Earl W. Davie

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Protein ◽

Full Length ◽

Actin Binding ◽

Intracellular Domain ◽

Platelet Surface ◽

Full Length Cdna ◽

Actin Binding Protein ◽

Amino Terminal

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIb. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other. © 1998 by The American Society of Hematology.

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Domain assembly of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3000291 ◽

1994 ◽

Vol 300 (2) ◽

pp. 291-294 ◽

Cited By ~ 30

Author(s):

D L Cope ◽

G D Holman ◽

S A Baldwin ◽

A J Wolstenholme

Keyword(s):

Amino Acids ◽

Binding Sites ◽

Glucose Transporter ◽

Cytochalasin B ◽

Insect Cells ◽

Full Length ◽

Stable Complex ◽

C And N ◽

Terminal Amino ◽

Glut1 Glucose Transporter

A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.

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Non-competitive immunochemiluminometric assay for cardiotrophin-1 detects elevated plasma levels in human heart failure

Clinical Science ◽

10.1042/cs1020411 ◽

2002 ◽

Vol 102 (4) ◽

pp. 411-416 ◽

Cited By ~ 11

Author(s):

Leong L. NG ◽

Russell J. O'BRIEN ◽

Bettina DEMME ◽

Sonja JENNINGS

Keyword(s):

Heart Failure ◽

Amino Acids ◽

Plasma Levels ◽

Limit Of Detection ◽

Linear Increase ◽

Full Length ◽

Human Heart Failure ◽

Competitive Assay ◽

Coefficients Of Variation ◽

Patients With Heart Failure

Cardiotrophin-1 (CT-1) leads to a specific form of ventricular hypertrophy characterized by sarcomeres added in series, and has been reported to be elevated in heart failure. Previous competitive assays for CT-1 necessitate the extraction of plasma and involve prolonged incubations. We describe the development of a non-competitive assay for CT-1 that can measure plasma levels without the need for extraction. Two antibodies specific for the mid-section (amino acids 105-120) and C-terminal (amino acids 186-199) portions of CT-1 were developed in rabbits. One antibody was immobilized and used as the capture antibody. The other antibody was affinity purified and biotinylated. Unextracted plasma was incubated with these antibodies, and detection was with methylacridinium ester-labelled streptavidin. Plasma was obtained from 40 patients with heart failure and 40 normal control subjects. The non-competitive assay demonstrated a linear increase in chemiluminescence (measured as relative light units) with increasing amounts of full-length recombinant CT-1, with no evidence of a hook effect at high concentrations. The lower limit of detection was 2.9 fmol/ml. Intra-assay coefficients of variation ranged from 3.1% to 4.2% in the 10-40fmol/well concentration range, and interassay coefficients of variation ranged from 3.5% to 4.5% in the 550-950fmol/ml range. Measurements of CT-1 levels in patients with heart failure (median 166.5fmol/ml; range 49.5-2788fmol/ml) revealed very significantly elevated levels compared with those in normal controls (median 43.5fmol/ml; range 11.2-258.6fmol/ml; P < 0.0001 by Mann-Whitney test). At a CT-1 concentration of 68fmol/ml, sensitivity and specificity were 95% and 82.5% respectively. Thus this new non-competitive immunochemiluminometric assay for CT-1 could successfully detect full-length recombinant CT-1 in unextracted plasma, and demonstrated that plasma levels of CT-1 were significantly elevated in patients with heart failure.

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