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R G Cook

Publications and source records attributed to R G Cook.

At least 91 records · Page 5Linked to original sources
PubMed1985

Molecular weight diversity among murine class I antigens: both the mature cell surface forms and the unglycosylated polypeptides vary significantly in molecular weight.

The molecular weights of the fully glycosylated cell surface form and the unglycosylated polypeptide of five murine class I antigens (H-2Kb, Db, TL, Qa-1.1, and Qa-2) were compared by SDS-PAGE. Significant molecular weight diversity was observed for both forms among these molecules. The size of the fully glycosylated forms ranged from approximately 52,000 daltons (H-2Db) to 41,000 daltons (Qa-2), whereas the unglycosylated polypeptides ranged from 43,000 daltons (H-2Kb and TL) to 33,000 daltons (Qa-2). The magnitude of the size variation observed in the unglycosylated polypeptides implies that there are differences in the gene organization, RNA processing or post-translational modifications of various class I glycoproteins.

Animals↗
PubMed1985

Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2.

Escherichia coli peptide chain release factors are proteins that direct the termination of translation in response to specific peptide chain termination codons. The mechanisms of codon recognition and peptidyl-tRNA hydrolysis are unknown. We have characterized the genes encoding release factor 1 (RF-1) and release factor 2 (RF-2) to study the structure-function relationships of the proteins and their regulation in the bacterium. In this report, we present the gene structure of RF-1 and RF-2, and a partial peptide sequence of RF-2. RF-1 and RF-2 are highly homologous in their primary structure. In addition, an in-frame premature opal (UGA) termination codon is located within the RF-2 coding region at amino acid position 26. This region of the protein was sequenced by automated Edman degradation to confirm the predicted reading frame, and a second independent isolate of the RF-2 gene was identified and sequenced to confirm the DNA sequence. These results imply that a frameshift occurs prior to the premature termination codon, thus allowing for translation of RF-2 to be completed. This may represent a mechanism of translational control of RF-2 expression. An alternative possible means of translational regulation is discussed.

Amino Acid Sequence↗
PubMed1985

Biochemical characterization of the molecules reactive with Qa-6 antiserum and the monoclonal antibody 20-8-4: evidence for structural similarity with the Qa-2 molecule.

The Qa-6 alloantigen and the molecule that crossreacts with the monoclonal antibody (mAb) 20-8-4 have been shown to be serologically distinct from the Qa-2 alloantigen by strain distribution and tissue distribution, respectively. In this report, we address the biochemical relationships among Qa-2, Qa-6, and the 20-8-4 cross-reactive molecule by using immunoprecipitation and polyacrylamide gel electrophoresis. Each of these molecules had an apparent m.w. of approximately 41K and was associated on the cell surface with beta 2-microglobulin. Removal of N-linked oligosaccharides with endoglycosidase F reduced their apparent m.w. to approximately 33K to 34K. The determinants recognized by anti-Qa-6 and mAb 20-8-4 were shown to reside on the same molecule(s) precipitated by anti-Qa-2 sera by immunodepletion experiments. The mAb 20-8-4 was also shown to preclear the molecules detected by the Qa-6 and Qa-2 antisera. Two-dimensional gel electrophoresis analysis demonstrated complete co-migration of the approximately 41K molecules detected by the three antibodies. By peptide map analysis with V8 protease, all three molecules appeared identical. Also, the determinant recognized by Qa-6 antiserum co-modulated with that recognized by the anti-Qa-2 mAb D3.262. Taken together, these results demonstrate that the molecules recognized by these three antisera and/or mAb are biochemically indistinguishable. These data, in conjunction with the serologic and genetic findings suggest that mAb 20-8-4 recognizes a molecule that is biochemically similar and possibly identical to the Qa-2 antigen. Moreover, although the genetic, serologic, and biochemical data demonstrate that Qa-6 is not controlled by the Qa-2 locus, but rather by a gene telomeric to Qa-2, the molecule bearing the Qa-6 determinant is very similar, if not identical, to the Qa-2 molecule. Several possible explanations for these discrepancies are discussed.

Animals↗
PubMed1985

Differential glycosylation requirements for the cell surface expression of class I molecules.

The importance of asparagine-linked glycosylation in the cell surface expression of several class I molecules was examined. C57BL/6 (B6) T cell blasts were treated with tunicamycin (TM), an antibiotic that inhibits N-linked glycosylation. The levels of various class I molecules on these cells were examined by flow cytometry and were compared to the levels of the same molecules on untreated cells. A 12-hr TM treatment did not significantly alter the levels of H-2Kb, Db, or Qa-2; however, such treatment decreased the surface expression of the Qa-1b allelic product to undetectable levels. A time-course study indicated that a decrease in the level of Qa-1.2 expression was apparent after only 4 hr of TM treatment. An examination of T cell blasts prepared from mouse strains possessing the Qa-1a, Qa-1c, and Qa-1d alleles indicated that all allelic products of this locus demonstrated a marked decrease in cell surface expression on TM treatment, whereas other class I molecules (H-2Ks, TL) exhibited slight or no decrease. Two-dimensional polyacrylamide gel electrophoresis analysis of immunoprecipitates from detergent lysates of surface-iodinated TM-treated B6 blasts revealed the presence of the unglycosylated form of the H-2Kb molecule on the cell surface. No such form of the Qa-1.2 molecule could be detected by similar analysis. To establish that the above observations were not simply a result of the inability of the Qa-1-specific alloantisera to react with the unglycosylated Qa-1 molecule, lysates of surface-iodinated B6 blasts were digested with endoglycosidase F, which cleaves N-linked carbohydrate moieties. Immunoprecipitation analysis indicated that the antisera could react with the unglycosylated form of the Qa-1 molecule. These results indicate that N-linked glycosylation has differential importance in the cell surface expression of class I molecules.

Animals↗
PubMed1985

The Qa-1 alloantigens. III. Biochemical analysis of the structure and extent of polymorphism of the Qa-1 allelic products.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to examine and compare the products of the Qa-1 locus. Analysis of Qa-1 isolated from detergent lysates of surface labeled cells indicated this molecule was a slightly acidic 48,000 to 50,000 dalton glycoprotein that displayed little charge heterogeneity on resting lymphocytes. The level of expression and degree of charge heterogeneity were both increased on activated lymphocytes. Direct comparison of the Qa-1b, Qa-1c, and Qa-1d allelic products by 2-D PAGE revealed that these three molecules could be distinguished from one another on the basis of isoelectric point, indicating that they were distinct at the molecular level. Comparison of Qa-1 isolated from several Qa-1b strains did not detect additional polymorphism. Removal of asparagine-linked oligosaccharides by treatment with endoglycosidase F indicated that carbohydrate contributed 10,000 to 12,000 to the m.w. of these allelic products. Comparative 2-D PAGE analysis could not distinguish between the deglycosylated Qa-1b, Qa-1c, and Qa-1d allelic products, implying that these molecules have similar primary structures. Peptide mapping supported this conclusion. Proteolytic digestion of the deglycosylated Qa-1b and Qa-1c allelic products resulted in identical peptide map patterns; such treatment of the deglycosylated Qa-1d allelic product produced a slightly different pattern. Peptide mapping analysis also demonstrated that the Tlaa and Qa-1a allelic products were distinct from one another, as well as being very different from the other three Qa-1 allelic products.

Alleles↗
PubMed1985

Flexible memory processing by rats: use of prospective and retrospective information in the radial maze.

Four experiments investigated the content of the memory used by rats in mediating retention intervals interpolated during performance in a 12-arm radial maze. The delay occurred following either the 2nd, 4th, 6th, 8th, or 10th choice. A 15-min delay had the greatest disruptive effect when interpolated in the middle of the choice sequence and less of an effect when it occurred either earlier or later. This pattern of results was obtained when either a free- or forced-choice procedure was used prior to the delay and regardless of whether postdelay testing consisted of completion of the maze or two-alternative forced-choice tests. Assuming that the disruptive effect of a delay is a function of memory load, this implies that the rats used information about previously visited arms (retrospective memory) following an earlier interpolated delay but information about anticipated choices (prospective memory) following a delay interpolated late in the choice sequence. There appeared to be a recency effect only in the early and middle delay conditions. This provides converging evidence for the dual-code hypothesis. No evidence for prospective memory was obtained following a 60-min delay.

Animals↗
PubMed1984

Cloned human T cells synthesize Ia molecules and can function as antigen presenting cells.

TNP-specific proliferative cloned human T cell lines were investigated for their synthesis and cell surface expression of HLA-DR molecules and for their capacity to function as antigen presenting cells. Utilizing radioactive amino acid precursors for metabolic labeling, these studies demonstrated endogenous synthesis of HLA-DR molecules by cloned T cells, which by two-dimensional gel electrophoresis were similar to HLA-DR molecules expressed by B cells and monocytes. Moreover, when TNP-modified, the irradiated cloned T cells functioned very effectively to stimulate TNP-specific proliferation by cloned responders; when unmodified they were potent stimulators of allogeneic mixed leukocyte responses. Thus, for haptens covalently attached to cell membrane proteins and for allogeneic HLA antigens, Ia+ cloned T cells can function as effectively for antigen presentation and T cell activation as other Ia+ populations to which such properties have been ascribed.

Antigen-Presenting Cells↗
PubMed1983

Expression of the thymus leukemia antigen by activated peripheral T lymphocytes.

Peripheral T lymphocytes activated in vitro with concanavalin A (Con A) or alloantigens express the thymus leukemia (TL) alloantigen as assessed by staining with the monoclonal antibody TL.m3 and flow cytometric analysis. The determinants detected by TL.m3 on activated cells are encoded within the Tla region and are detected as early as 48 h after activation with Con A. Several long-term cloned cytotoxic T lymphocyte lines were also examined and each expressed TL. By two-dimensional analysis, the TL isolated from activated peripheral cells was indistinguishable from that found on thymocytes and the leukemia cell line ASL-1.

Animals↗
PubMed1983

The Qa-1 alloantigens. II. Evidence for the expression of two Qa-1 molecules by the Qa-1d genotype and for cross-reactivity between Qa-1 and H-2K.

The nature of cell surface determinants detected by Qa-1-specific alloantisera and cytotoxic T lymphocytes (CTL) in mice of the H-2f, Qa-1d genotype was investigated. The H-2f, Qa-1d strains A.CA and B10.M express both Qa-1a and Qa-1b encoded alloantigenic determinants (Qa-1.1 and Qa-1.2, respectively), as defined in the prototypic A (or B6-Tlaa) and C57BL/6 (or A-Tlab) strains, respectively. Both anti-Qa-1.1 and -Qa-1.2 sera immunoprecipitate 46K m.w. glycoproteins from H-2f, Qa-1d strains. In addition to 46K m.w. proteins, anti-Qa-1.1 sera, but not anti-Qa-1.2 sera, precipitate 55 to 75K m.w. proteins; the nature of these proteins and their relationship to Qa-1 is unclear at present. Sequential immunoprecipitation experiments and the analysis of several recombinant strains revealed that anti-Qa-1.1 sera also cross-react with a 46K m.w. H-2f-encoded alloantigen, probably H-2Kf. Both Qa-1.1 and the non-Qa-1.1 cross-reacting determinants were detected by polyclonal anti-Qa-1a CTL on the Qa-1d strains. The Qa-1a encoded but not the cross-reacting determinants were detected by a cloned anti-Qa-1a CTL line. Sequential immunoprecipitation experiments on the recombinant strains B6.AC2 and B10.M(17R), which are Qa-1d but not H-2Kf, revealed that the Qa-1.1 and Qa-1.2 determinants do not reside on the same molecule. Furthermore, although Qa-1b-encoded determinants were detected on these strains with anti-sera and with bulk CTL cultures, cloned anti-Qa-1b CTL lines thus far analyzed have failed to react with Qa-1d targets, indicating that some but not all of the prototypic Qa-1b-encoded determinants are expressed by the Qa-1d strains.

Animals↗
PubMed1982

Qa-1-associated antigens. IV. Evidence for additional Qa-1 polymorphism defined biochemically and by cytotoxic T lymphocyte recognition.

Qa-1-specific, H-2-unrestricted cytotoxic T lymphocytes (CTLs) generated from reciprocally immunized B10.BR (H-2k, Qa-1a) and CBA (H-2k, Qa-1b) mice, and immunoprecipitation of cell surface Qa-1 were used to examined Qa-1 region determinant expression by H-2r and H-2f mice. The H-2b, Qa/Tla congenic mice B6-Tlaa and C57BL/6J (B6) were used as prototype Qa-1a and Qa-1b strains, respectively. Cells from H-2r strains expressed determinants recognized by B10.BR anti-CBA CTLs. Reciprocal cold target inhibition of cytolysis demonstrated that some, but not all, Qa-1b-associated determinants recognized on B6 were expressed by H-2r, Qa-1c cells. In contrast, cells from H-2f strains expressed Qa-1 determinants recognized by both B10.lBR anti-CBA and by CBA anti-B10.BR CTLs. Cold target inhibitions indicated that the H-2f, Qa-1d cells expressed some, but not all, Qa-1a-associated antigens recognized on B6-Tla and did not express the same Qa-1b-associated antigens as B6. Furthermore, H-2f strains expressed cell surface antigens immunoprecipitable by antisera specific for both Qa-1a and Qa-1b-encoded determinants; these molecules were of the same molecular weight as those immunoprecipitated from B6 and B6-Tlaa. These data suggest that CTLs and antisera define the same, closely related Qa-1 determinants encoded by a polymorphic locus.

Animals↗
PubMed1981

The Qa-1 alloantigens. I. Identification and molecular weight characterization of glycoproteins controlled by the Qa-1a and Qa-1b alleles.

Splenocytes from the Qa-Tla congenic strain pairs, A and A-Tlab or B6 and B6-Tlaa, were biosynthetically labeled with 3H-amino acids or cell surface labeled with 125I. Membrane proteins were solubilized with detergent and chromatographed on lentil lectin-Sepharose, and the resulting adherent pools were immunoprecipitated with antisera specific for determinants controlled by the Qa-1a and Qa-1b alleles, Qa-1.1 and Qa-1.2, respectively. Polyacrylamide gel electrophoresis analysis of immunoprecipitates from biosynthetically labeled preparations indicated that both the Qa-1.1 and Qa-1.2 antigens were glycoproteins with a m.w. of approximately 46,000. Qa-1.2 isolated from radioiodinated spleen cells similarly had a m.w. of 46,000. Analysis of anti-Qa-1.1 precipitates from 125I-labeled Qa-1a lysates demonstrated in addition to the 46,000 m.w. component, an electrophoretically heterogeneous protein or series of proteins in the m.w. range of 55,000 to 75,000. The specificity of these reactivities was shown by both antiserum and genetic control immunoprecipitations. These findings indicate that the Qa-1.1 and Qa-1.2 antigens are cell surface glycoproteins that are distinct from the TL antigens, and suggest a further complexity at the Qa-1--Tla locus.

Alleles↗
PubMed1981

Biochemical characterization of Ia alloantigens in guinea pigs. II. Comparative peptide mapping of Ia antigens from B cells, T cells, and macrophages.

Radioactive Ia.4 molecules were prepared from 3H- or 14C-labeled splenocytes, selected PEL, or bronchoalveolar macrophages (M phi). Studies in the accompanying paper indicated that incorporation into Ia.4 in these 3 populations is due to B cells, T cells, and macrophages, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the 58,000 m.w. form of Ia.4. The eluted molecule was then reduced, and the resultant alpha and beta subunits were isolated by a second separation on SDS-PAGE. Alpha (or beta) chains from 1 population labeled with 3H-amino acids was mixed with alpha (or beta) chains obtained from a population representing a different cell type that was labeled with 14C-amino acids and the mixture was digested with trypsin. Double-label (3H/14C) comparative peptide mapping was performed using high-pressure liquid chromatography to separate the peptides. Eighteen to 20 peaks of radioactivity were resolved from alpha chains, and 12 to 15 from beta chains. No reproducible differences were observed when comparing alpha or beta chains of T cells and macrophages, or those of B cells and macrophages. These results indicate that the primary structure of Ia.4 molecules is identical on the 3 cell types in question. The implications of having a T cell bearing the same Ia that it recognized on a macrophage in conjunction with antigen is discussed.

Amino Acids↗
PubMed1979

Organization of the immune response genes.

The I region of the major histocompatibility complex contains immune response genes that display considerable polymorphism; that is, there are many alleles at each locus. These genes regulate the immune response to antigen by mediating intercellular communication among lymphoreticular cells. An analysis of the primary structure of the products of two subregions of (I-A, I-E/C) was undertaken in order to understand the genetic organization of the region, the evolution of the genes and, eventually, their function.

Alleles↗
PubMed1979

Structural studies on the murine Ia alloantigens. V. Evidence that the structural gene for the I-E/C beta polypeptide is encoded within the I-A subregion.

The E/C alpha- and beta-subunits of intra-I-region recombinants were analyzed for primary structural variation by comparative tryptic peptide mapping. The E/C alpha-polypeptides from B10.A, B10.A (3R) and B10.A (5R) showed complete coincident elution of peptides; the E/C beta-chains from B10.A and 3R (or 5R) were approximately 40% different. This suggests that the structural gene for the E/C beta-polypeptide is within the I-A subregion.

Amino Acid Sequence↗
PubMed1979

Surface expression and synthesis of I-A and I-E/C encoded molecules by B lymphocytes and Ig-secreting cells.

Splenocytes from recombinant mice were radiolabeled before or after deletion of subpopulations by cytotoxic anterisera (+C) directed against I-A, I-E/C, IgM, or Ig. Examination of the lysates of the surviving cells by immunoprecipitation demonstrated that 1) virtually all I-A and I-E/C molecules are co-expressed and synthesized by Ig+, IgM+ lymphocytes, 2) I-A, I-E/C, and IgM molecules are present on many of the cells secreting IgM and IgG, and 3) populations of Ig-bearing or Ig-secreting cells that lack detectable I-A and I-E/C antigens can be identified in spleen cell populations. The co-expression of I-A and I-E/C on most cells of the B cell lineage is discussed in terms of our present concepts of Ir gene control of immune responses.

Animals↗