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Biomedical subjects

F A Robey

Publications and source records attributed to F A Robey.

At least 55 records · Page 3Linked to original sources
PubMed1987

Generation of a murine monoclonal antibody that detects the fos oncogene product.

A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining.

Animals↗
PubMed1987

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.

Amino Acid Sequence↗
PubMed1987

Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens.

A method to incorporate N-chloroacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-chloroacetyl-modified peptides react well with sulfhydryl containing proteins such as 4-mercaptobutyrimide-modified bovine serum albumin to form stable protein-peptide conjugates. By incorporating cysteine into the synthetic peptide, autopolymerization or cyclization of the synthetic peptide occurs by reaction of the free sulfhydryl with the chloroacetyl group. N-Chloroacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens and vaccines.

Acetylation↗
PubMed1986

Interspecies conservation of structure of interphotoreceptor retinoid-binding protein. Similarities and differences as adjudged by peptide mapping and N-terminal sequencing.

Structural properties of the retinal extracellular-matrix glycolipoprotein interphotoreceptor retinoid-binding protein (IRBP) from human, monkey and bovine retinas have been compared. SDS/polyacrylamide-gel-electrophoretic analysis of limited tryptic and Staphylococcus aureus-V8-proteinase digests show virtually identical patterns for the monkey and human proteins, whereas both sets differ considerably from the bovine protein pattern. Time-course digestion shows monkey IRBP to be more readily cleaved than bovine IRBP and also cleaved to smaller fragments. Also, reversed-phase h.p.l.c. of complete tryptic digests of the IRBPs indicate that, although they have in common a similar preponderance of hydrophobic peptides, all three proteins differ extensively in their fine structure. The N-terminal sequences of monkey and bovine IRBPs have been extended beyond those presented in our previous report [Redmond, Wiggert, Robey, Nguyen, Lewis, Lee & Chader (1985) Biochemistry 24, 787-793] to over 30 residues each. The sequences yet show extensive homology, differing at only two positions, although the major monkey sequence has an additional five amino acid residues at its N-terminus ('n + 5' sequence) not observed with bovine IRBP ('n' sequence). The newly determined N-terminal sequence of human IRBP demonstrates the presence of equal amounts of the 'n' and 'n+5' sequences that are qualitatively identical with those of the monkey. The presence of the five-amino-acid-residue extension in primate, but not bovine, IRBP may indicate variation in post-translational processing.

Amino Acid Sequence↗
PubMed1986

Characterization of an anti-peptide antibody that recognizes the murine analogue of the human T cell antigen receptor-T3 delta-chain.

The T cell antigen receptor consists of two disulfide-linked 40,000 to 45,000 dalton glycoproteins (alpha and beta) that contain variable and constant regions analogous to those found in immunoglobulin molecules. The antigen receptor on murine T cells is noncovalently associated with four additional nonpolymorphic structures. We describe an antibody that binds one of these molecules, a 26,000 dalton glycoprotein homologous to the human T3 delta-chain. This antibody immunoprecipitates the entire antigen receptor complex from a T cell hybridoma and from normal murine thymocytes. It represents the first reagent that can immunoprecipitate the antigen receptor complex on all murine T cells.

Animals↗
PubMed1985

C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro.

We have studied the interaction of C-reactive protein (CRP)-chromatin complexes with serum. The amount of chromatin solubilized by serum is directly proportional to the amount of CRP present. Serum minus C3 did not appreciably solubilize chromatin within the time allowed in these experiments regardless of the amount of CRP present. This indicates that, in addition to CRP, complement is critical to the solubilization process. Studies using genetically C2-deficient serum and purified C2 indicate that the classical complement pathway is primarily involved in the solubilization, however, there may be minor involvement by the alternative pathway. We used an enzyme-linked immunosorbent assay to determine the amounts of CRP in plasma from eight patients with systemic lupus erythematosus; two of the eight had levels of CRP far lower than previously reported for normal individuals, and an additional sample had antibodies reactive with CRP. Together, these results suggest that one of the functions of CRP is to mediate the removal of exposed nuclear DNA by complement-dependent solubilization of chromatin. A defect in this mechanism could (a) facilitate the production of antibodies against chromatin components exposed due to tissue damage or (b) contribute to immune complexes containing the chromatin components released from damaged tissue because they are not rapidly cleared.

C-Reactive Protein↗
PubMed1985

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina.

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.

Amino Acid Sequence↗
PubMed1984

Binding of C-reactive protein to chromatin and nucleosome core particles. A possible physiological role of C-reactive protein.

By using a variety of biochemical techniques, chromatin and chromatin fragments have been identified as probable physiological ligands for C-reactive protein. Studies using 14C-labeled C-reactive protein show that binding to chromatin is saturable with a Kd = 8 X 10(-7) M, a value indicating that the affinity of C-reactive protein for chromatin is at least four times its affinity for phosphorylcholine. At saturation, there is approximately one C-reactive protein-binding site for every 160 base pairs of DNA in chromatin. The interaction of C-reactive protein with chicken erythrocyte nucleosome core particle has been studied. Fifty per cent inhibition of the binding of C-reactive protein to phosphorylcholine is obtained at a core particle concentration of 1.25 X 10(-9) M, indicating that the affinity of C-reactive protein for one of the sites on core particles is at least 2400 times greater than the affinity of C-reactive protein for phosphorylcholine. The possibility that C-reactive protein may act as a scavenger for chromatin fragments released from damaged cells is discussed.

Animals↗
PubMed1983

A new sensitive assay for the calcium-dependent binding of C-reactive protein to phosphorylcholine.

A new sensitive assay for the calcium-dependent binding of rabbit C-reactive protein to phosphorylcholine has been developed. The assay involves coating the wells of polyvinylchloride microtiter plates with a bovine serum albumin-phosphorylcholine conjugate followed by the addition of C-reactive protein. The quantity of C-reactive protein is determined by indirect enzyme-linked immunoadsorbent assay using first, affinity purified goat anti-C-reactive protein immunoglobulin followed by a commercial rabbit anti-goat immunoglobulin-alkaline phosphatase conjugate. The binding of rabbit C-reactive protein to the bovine serum albumin-phosphorylcholine conjugate is completely inhibited by free phosphorylcholine, by pneumococcal C-polysaccharide and by calcium chelators but not by high concentrations of neutral, cationic or zwitterionic detergents. The assay is sensitive to 2 ng C-reactive protein.

Animals↗
PubMed1983

Synthesis and use of new spin labeled derivatives of phosphorylcholine in a comparative study of human, dogfish, and Limulus C-reactive proteins.

New spin labeled derivatives of phosphorylcholine have been synthesized. The compounds cause reversible inhibition of the precipitation reactions between pneumococcal C-polysaccharide and the C-reactive proteins from humans, dogfish sharks (Mustelus canis), and horseshoe crabs (Limulus polyphemus). The spin labeled phosphorylcholine derivatives also rival phosphorylcholine as a ligand for the human, dogfish, and Limulus C-reactive proteins. The interactions of the purified C-reactive proteins with the spin labeled derivatives of phosphorylcholine have been studied using electron spin resonance spectrometry. The dramatic decrease in the ESR signal of some of the spin labels is due to immobilization of the label. Only the well known phosphate spin label, 4-phosphate-2,2,6,6-tetramethylpiperidine-1-oxyl could be used for binding studies on human and Limulus C-reactive proteins. Thus, by Scatchard analysis, the human C-reactive protein bound 1 mol of phosphate spin label per mol of protein with a Ka = 3.91 X 10(3) M-1, whereas the Limulus C-reactive protein bound only 0.5 mol of phosphate spin label per mol of protein with an overall Ka = 1.95 X 10(3) M-1. Inhibition studies using purified C-polysaccharide-induced inhibition of the phosphate spin label-human C-reactive protein interaction showed competitive inhibition with a KI of 4.78 X 10(-5) M at 18 degrees C. The phosphate spin label did not bind to dogfish C-reactive protein. However, one new phosphorylcholine spin label did bind and was used for Scatchard and Hill plot analyses. The dogfish C-reactive protein, which exists as a Mr = 50,000 dimer, bound 2 mol of the phosphorylcholine spin label per mol of protein, and this binding exhibited negative cooperativity as indicated by a Hill coefficient of 0.75.

Animals↗
PubMed1983

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component.

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.

Amino Acid Sequence↗
PubMed1982

Synthesis and study of a spin-labeled cyclophosphamide analogue, 3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

Acrolein↗
PubMed1979

Identification of four major classes of sulfhydryl groups in human blood platelets. Ferricyanide titration of spin-labeled platelets.

Human blood platelets have been labeled with the sulfhydryl-specific spin labels, 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl and 3-maleimido-2,2,5,5-tetramethylpyrrolidine-1-oxyl. First, the ESR spectra of platelets labeled with either reagent revealed two classes of sulfhydryl groups, a mobile class and an immobile class. Second, when spin-labeled platelets were titrated with high concentrations of potassium ferricyanide (greater than 10(-3) M), there was a decrease in the peak heights of the mobile class of sulfhydryl groups due to dipole-dipole exchange. Third, plots of peak heights of the mobile class versus ferricyanide concentration revealed three classes of mobile sulfhydryl groups compared to a single immobile class. This technique may be used to show the relative locations of spin-labeled groups on cell surfaces.

Blood Platelets↗
PubMed1979

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.

Adenosine Diphosphate↗
PubMed1978

Kinetic studies on the activation of human factor X. The role of metal ions on the reaction catalyzed by the venom coagulant protein of Viper russelli.

The effect of Ca2+, Mg2+, and Mn2+ on the initial rate of activation of human Factor X by the venom coagulant protein of Vipera russelli has been investigated. Neither Mg2+ nor Mn2+ alone support the reaction. Ca2+ is an essential activator and exhibits cooperative kinetics. Both Mg2+ and Mn2+ enhance the reaction cooperatively when Ca2+ is present at suboptimal concentrations. Similarly, Ca2+ quenches the intrinsic fluorescence of human Factor X in a cooperative manner. While neither Mg2+ nor Mn2+ by themselves affect the fluorescence of human Factor X, they decrease the cooperativity of the Ca2+ binding to the protein as judged by Hill plots of the Ca2+ -induced fluoresence quenching. EPR measurements indicate that there are three high affinity Mn2+ binding sites on human Factor X which can also bind Ca2+. Positive cooperativity was not observed for Mn2+ binding. These data indicate that Ca2+ can cause a conformational change of the Factor X molecule which allows the activation reaction to proceed. We propose that Mn2+ does not support the activation of human Factor X because it cannot induce a necessary conformational change in the absence of Ca2+.

Calcium↗