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R G Cook

Publications and source records attributed to R G Cook.

At least 37 records · Page 2Linked to original sources
PubMed1990

Superantigens: interaction of staphylococcal enterotoxins with MHC class II molecules.

We have shown that the staphylococcal enterotoxins and TSST specifically bind to MHC class II molecules. This binding to class II molecules is a prerequisite for the function of these bacterial exotoxins as T cell mitogens in vitro. While SEA bound all class II molecules tested with respect to isotype and allotype, the other enterotoxins were limited in binding by the class II isotype. In contrast to conventional antigen, the nature of enterotoxin interactions with MHC enables them to stimulate class I-restricted CD8+ T cells, most likely due to the ability of SEs to engage the T cell receptor based solely on V beta usage. Finally, in addition to activating adjacent T cells, the enterotoxins and TSST can evoke responses from the class II-bearing cells to which they bind. Enterotoxin/TSST effects on cells that bear class II molecule "receptors", in addition to their induction of T cell hormones such as interleukin-2 and interferon-gamma, provide possible explanations for some of the symptomatology seen with these bacterial exotoxins and also implicate MHC class II molecules as signal-transducing receptors.

Antigens, Bacterial↗
PubMed1989

A 14-kilodalton selenium-binding protein in mouse liver is fatty acid-binding protein.

In a previous study, we purified three selenium-binding proteins (molecular masses 56, 14, and 12 kDa) from mouse liver using column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aim of the present study was to determine the amino acid sequence of the 14-kDa protein thereby establishing any relationship with known proteins. Although the amino terminus of the 14-kDa protein was blocked, separate in situ digestions of the protein with endoproteinases Glu-c and Lys-c gave overlapping peptides that provided a continuous sequence of 93 amino acids. This sequence exhibited a 92.5% sequence homology with rat liver fatty acid-binding protein. In situ enzymatic digestion and partial sequencing of a 12-kDa selenium-binding protein revealed identical homology to the 14-kDa protein. The 14-kDa protein bound specifically to an oleate-affinity column from which the protein and 75Se coeluted. Delipidation or sodium dodecyl sulfate treatment failed to remove 75Se from the protein, indicating that the selenium moiety was tightly bound to the protein. These observations confirm that the mouse liver selenium-binding 14-kDa protein is a fatty acid-binding protein. The nature of the selenium linkage to the protein still needs to be explored.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of a smooth muscle caldesmon.

Caldesmon is a smooth muscle and nonmuscle regulatory protein that interacts with actin, myosin, tropomyosin, and calmodulin. Two overlapping clones, isolated from a chicken oviduct cDNA plasmid library and a chicken gizzard cDNA lambda NM1149 library, were used to generate a 4108-base pair sequence coding for one caldesmon. Expression of the coding sequence confirms this is one of the large smooth muscle caldesmons. The deduced protein molecular weight is 86.974, significantly less than the molecular weights estimated by sodium dodecyl sulfate gel electrophoresis. The protein has a high content of Gly, Lys, Arg, and Ala; there are two cysteine residues, one at either end of the molecule. Comparison with the Protein Identification Resource database demonstrates a similarity with a tropomyosin binding domain of troponin T, but none with any calmodulin or actin binding proteins. The center of the protein has an 8-fold repeat of a 13 amino acid sequence whose general motif is -Glu3-(Lys/Arg)2-Ala2-Glu2-(Lys/Arg)1-X-(Lys/Arg)1-Ala1-, where X is Glu, Gln, or Ala. Comparison with peptide sequences from a chymotryptic fragment that binds actin and calmodulin places this domain on the C terminus of caldesmon adjacent to the troponin T similarity. A tentative map of the major binding domains is proposed on the basis of available data.

Amino Acid Sequence↗
PubMed1989

COUP transcription factor is a member of the steroid receptor superfamily.

The COUP (chicken ovalbumin upstream promoter) transcription factor (COUP-TF) exists in a number of different tissues and is essential for expression of the chicken ovalbumin gene. It binds to the ovalbumin promoter and, in conjunction with a second protein (S300-II), stimulates initiation of transcription in vitro. COUP-TF also binds specifically to the rat insulin promoter element, although the two binding sites share little sequence similarity. Here we report the isolation of a human complementary DNA clone encoding COUP-TF. Comparison of the amino-acid sequence of COUP-TF with known sequences reveals that it is a member of the steroid/thyroid hormone/vitamin receptor superfamily. Consequently, it is the first member of this family that has been shown to function in a cell-free transcription system. We conclude that this superfamily of gene regulators contains proteins which bind and activate distal promoter elements of eukaryotic genes.

Amino Acid Sequence↗
PubMed1989

Class II MHC molecules are specific receptors for staphylococcus enterotoxin A.

T cell proliferation in response to stimulation with Staphylococcus enterotoxin A (SEA) requires accessory cells that express class II major histocompatibility complex (MHC) molecules. Murine fibroblasts transfected with genes encoding the alpha and beta subunits of HLA-DR, DQ, or DP were used to show that the proliferative response of purified human T cells to SEA is dependent on class II molecules but is not restricted by the haplotype of the responder. Binding of fluoresceinated SEA to class II transfectants and precipitation of class II heterodimers with SEA-Sepharose show that the proliferative response is a result of SEA binding to class II molecules. The binding is specific for class II molecules and is independent of class II allotype or isotype. The ability of SEA to bind class II molecules may be a general characteristic of this class of antigens, now called "superantigens".

Animals↗
PubMed1989

Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation.

GMP-140 is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, it is rapidly redistributed to the plasma membrane. The cDNA-derived primary structure of GMP-140 predicts a cysteine-rich protein with multiple domains, including a "lectin" region, an "EGF" domain, nine tandem consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Some cDNAs also predict a soluble protein with a deleted transmembrane segment. The domain organization of GMP-140 is similar to that of ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils. This similarity suggests that GMP-140 belongs to a new family of inducible receptors with related structure and function on vascular cells.

Amino Acid Sequence↗
PubMed1989

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein.

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.

Amino Acid Sequence↗
PubMed1989

Synthesis and characterization of a bovine hexokinase 1 cDNA probe by mixed oligonucleotide primed amplification of cDNA using high complexity primer mixtures.

Hexokinase (EC 2.7.1.1) catalyzes the first step in glucose metabolism, using ATP for the phosphorylation of glucose to glucose 6-phosphate. A portion of the HK1 gene was cloned by mixed oligonucleotide primer amplification of cDNA using primers of high complexity. The amino acid sequence for a partial fragment of bovine cardiac muscle HK was determined and used to create primer mixtures of 256- and 1024-fold complexity. Two products were generated from bovine cardiac muscle cDNA which show 82% nucleotide and 93% amino acid identity with a region of rat brain HK1 and cDNA. This work demonstrates that extension and amplification of cDNA probes may be successful even when amino acid sequence data indicate substantial codon degeneracy.

Amino Acid Sequence↗
PubMed1989

Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena.

In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly.

Acetylation↗
PubMed1989

Relational and absolute stimulus learning by monkeys in a memory task.

Three experiments showed stimulus control by either the absolute properties of probe stimuli, relational properties of the probe-list relationship, or both in a serial probe recognition memory task in which a four-item memory list was followed by a single probe (test) item. In Experiment 1, 3 rhesus monkeys received 39 to 75 repetitions of the same 24-trial stimulus sequence. Special tests showed stimulus control by the absolute properties of the probe stimuli. Retention of previous relational control was demonstrated by the good transfer (83%) to novel list and probe stimuli at the beginning of Experiment 2. During Experiment 2, control by absolute properties of the probe stimuli gradually reoccurred. Only a small measure of control by list stimuli could be detected or promoted. In Experiment 3, 4 monkeys were shown to have largely lost their ability to perform on the basis of the list-probe relationship, and were performing primarily on the basis of the absolute properties of the probe stimuli. Over the next 15 weeks, these monkeys were transferred to new stimuli at the beginning of each week. Control by the relational aspects of the task gradually returned. As transfer performance increased, control by the absolute properties of the probe stimuli was eliminated. The results are discussed in terms of stimulus control and performance strategies used by the monkeys.

Animals↗
PubMed1988

Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive.

Hydropathy analysis of the Mr 27,000 rat liver gap junction protein sequence deduced from a cDNA clone has suggested the presence of four transmembrane segments (Paul, D. L. (1986) J. Cell Biol. 103, 123-134). In the present report, several features of the molecular topology of the protein were investigated by microsequence analysis of peptides generated by treatment of isolated gap junctions with a variety of proteases. Under the experimental conditions used, the proteases had access only to the portion of the Mr 27,000 protein that was originally (in vivo) the cytoplasmic surface of the gap junction. Microsequencing of the peptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the amino terminus of the protein is disposed at or near the cytoplasmic surface of the gap junction, and that this surface also contains a protease-hypersensitive hydrophilic sequence between residues 109 and 123, presumably connecting the second and third transmembrane segments. Immunocytological localization of binding of monoclonal antipeptide antibodies demonstrates that the carboxyl terminus of the protein is also localized to the cytoplasmic surface of the gap junction. No protease sensitivity was found in the hydrophilic sequences thought to connect either the first and second transmembrane segments or the third and fourth segments, supporting the model's prediction that these sequences face the narrow intercellular gap which cannot be penetrated by proteases.

Amino Acid Sequence↗
PubMed1988

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels.

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.

Amino Acid Sequence↗
PubMed1988

Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase.

Urate oxidase (E.C. 1.7.3.3) catalyzes the oxidation of uric acid to allantoin in most mammals except humans and certain primates. The amino-terminal amino acid sequence for porcine urate oxidase was determined and used in a novel procedure for generating complementary DNA (cDNA) probes to this amino acid sequence. The procedure is based on the polymerase chain reaction and utilizes mixed oligonucleotide primers complementary to the reverse translation products of an amino acid sequence. This rapid and simple cDNA cloning procedure is generally applicable and requires only a partial amino acid sequence. A cDNA probe developed by this procedure was used to isolate a full-length porcine urate oxidase cDNA and to demonstrate the presence of homologous genomic sequences in humans.

Amino Acid Sequence↗
PubMed1988

Polymorphic Bgl II restriction sites of DR alpha demarcate a novel HLA-DR1 antigen.

Mechanisms to account for the unusual properties of a DR1 alpha beta complex (designated DRgp50) that is resistant to dissociation under normal conditions utilized were investigated. Expression of this DRgp50 complex is highly correlated with the failure of cells from certain DR1 individuals (DR1x) to stimulate specific DR1-restricted or alloreactive T-cell clones. Pulse/chase experiments demonstrated that this DRgp50 complex was not detectable until approximately 1 h of chase. The DR1 alpha and beta chains associated into the heterodimer in the absence of glycosylation and alterations in the number of oligosaccharides or sialylation of cell surface forms were not evident when compared with normal DR1 alpha and beta chains. Restriction fragment length polymorphism patterns of DR beta genes from normal (DR1n) and DR1x individuals were indistinguishable. However, a difference in the alpha chain genes between DR1n and DR1x individuals was revealed using Bgl II. This Bgl II restriction site mapped to the 3' untranslated region of DR alpha and represents a new genomic marker to distinguish this functional and biochemical variant of DR1.

Bacterial Proteins↗
PubMed1988

The detection of conventional class I and class II I-E homologue major histocompatibility complex molecules on feline cells.

The presence on feline cells of class I and class II I-E type major histocompatibility complex (MHC) homologues was demonstrated using cross-reacting monoclonal antibodies (mAb). The feline class I antigen homologues were detected with both immunofluorescent and biochemical techniques, using the anti-human class I mAb W6/32. The class I antigens were detected on in vitro cultured feline fibroblasts and lymphoid cells, but not on fresh lymphoid cells, apparently as a result of the association of bovine beta-2 microglobulin with feline class I heavy chains which generated the determinant(s) recognized by mAb W6/32. Class II I-E-like molecules could be detected with immunofluorescent techniques using the species cross-reactive anti-mouse I-E antibody 40D only when peripheral blood mononuclear cells were activated, for example, with the mitogens staphylococcus enterotoxin A or lipopolysaccharide. The predominant expression of I-A-like molecules by resting class II-positive feline cells could explain some of the functional difference we have seen in comparison with those of most other mammalian species.

Animals↗
PubMed1988

Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4.

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.

Acetylation↗
PubMed1988

Characterization of phosphorylation sites in histone H1 in the amitotic macronucleus of Tetrahymena during different physiological states.

Histone H1 is highly phosphorylated in transcriptionally active, amitotic macronuclei of Tetrahymena during vegetative growth. However, the level of H1 phosphorylation changes dramatically in response to different physiological conditions. H1 is hyperphosphorylated in response to heat shock and during prezygotic stages of conjugation. Conversely, H1 is largely dephosphorylated during prolonged starvation and during elimination of parental macronuclei during conjugation. Mapping of phosphorylation sites within H1 indicates that phosphorylation occurs at multiple sites in the amino-terminal portion of the molecule, predominantly at threonine residues. Two of these sites have been identified by compositional analyses and microsequencing of tryptic peptides. Interestingly, two major sites contain the sequence Thr-Pro-Val-Lys similar to that contained in the sites recognized by growth-associated histone kinase in other organisms. No new sites are detected during the hyperphosphorylation of H1 which occurs during heat shock or in early stages of conjugation, and no sites are preferentially dephosphorylated during starvation or later stages of conjugation. Therefore, changes in the overall level of H1 phosphorylation, as opposed to phosphorylation or dephosphorylation at particular sites, appear to be important in the regulation of chromatin structure under these physiological conditions. Further, since no cell division or DNA replication occurs under these conditions, changes in the level of H1 phosphorylation are best correlated to changes in gene expression during heat shock, starvation, and conjugation. We suggest that, at least in Tetrahymena, H1 hyperphosphorylation is used as a rapid and transient mechanism for the cessation of transcription under conditions of cellular stress.

Amino Acid Sequence↗