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Biomedical subjects

R G Cook

Publications and source records attributed to R G Cook.

At least 73 records · Page 4Linked to original sources

The TL region gene 37 encodes a Qa-1 antigen.

Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains.

Animals↗

Dissociation of the stimulatory activities of staphylococcal enterotoxins for T cells and monocytes.

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.

Amino Acid Sequence↗

Specialized functions of MHC class I molecules. I. An N-formyl peptide receptor is required for construction of the class I antigen Mta.

Maternally transmitted factor (Mtf) is a mitochondrial gene that controls the antigenic polymorphism of the MHC class I maternally transmitted antigen (Mta). Synthetic peptides from the NH2 terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1) mimic Mtf peptide activity in an allele-specific manner. We show that the minimal ND1-alpha peptide length recognized by Mtaa-specific polyclonal CTLs was between 8 and 12 amino acids, while some Mtaa-specific CTL clones recognized a six amino acid peptide. The N-formyl group at the NH2 terminus of ND1 was essential for Mta activity. Competition experiments using N-substituted ND1-alpha peptides showed that an N-formyl peptide receptor on the target cell, which differs from the chemotactic peptide receptor, was required for Mta expression. The specificity of this receptor can account for the distinct immune restriction of Mta in which Mtf peptides are uniquely restricted by Hmt. It is possible that the Hmt gene product is the N-formyl peptide receptor itself and that it represents a class I antigen presentation molecule specialized for binding, transport, and immune presentation of N-formyl-peptide antigens of mitochondrial and prokaryotic origin.

Animals↗

Regulation of expression of TL genes of the mouse Mhc.

The expression of thymus leukemia (TL) antigens and genes in thymocytes and activated T cells was examined by immunoprecipitation, flow cytometric, northern, and nuclear run-off transcription analyses. Cell surface forms of TL were detectable by immunoprecipitation on activated peripheral T cells from Tla haplotypes except Tla(b), in agreement with expression observed on thymocytes. Approximately 40%-50% of concanavalin A (Con A) or anti-CD3-activated T cells were TL+, with expression detected on both the CD4 and CD8 subsets by dual-color analysis. Activated T cells expressed detectable levels of TL mRNA 48 h after stimulation, but no TL transcripts were detectable in unstimulated splenocytes. However, TL mRNA expression in mature activated T cells did not precisely mimic thymocyte expression: the level of expression was considerably lower in activated T cells, and in most haplotypes the transcripts produced in activated T cells appeared to represent a subset of the transcripts produced in thymocytes. By run-off transcription assays in isolated nuclei, TL gene expression was detected in activated but not resting T cells indicating that lack of expression of TL in resting T cells is not due to message instability. These data demonstrate that TL genes are inducible and transcriptionally regulated.

Animals↗

Opiate antagonists enhance the working memory of rats in the radial maze.

Two experiments tested the influence of the opiate antagonists naloxone and naltrexone on the spatial working memory of rats in a 12-arm radial maze. In Experiment 1, ten rats were serially forced to visit six randomly selected arms, then were removed from the maze for delays of either 30, 60, or 240 minutes, and then returned to the maze for a free-choice memory test with all 12 arms available. Five minutes into the delay, rats were injected intraperitoneally (IP) with either physiological saline or naloxone (1 mg/kg). When injected with naloxone the rats revisited forced-choice arms less often than when injected with saline during a subsequent free-choice test. In Experiment 2, twelve rats showed a similar facilitation of working memory when injected with the opiate antagonists naltrexone (0.3 mg/kg) and naloxone (1 mg/kg) in comparison to a saline control condition. These findings demonstrate the beneficial effects that opiate antagonists exert on working memory-based performance in the radial maze. They may also resolve conflicting reports about the influence of opiate antagonists on radial maze performance, by suggesting that the choice of measurement and testing conditions are crucial for detecting these effects in working memory procedures.

Animals↗

Biochemical differences in Qa-2 antigens expressed by Qa-2+,6+ and Qa-2+,6- strains. Evidence for differential expression of the Q7 and Q9 genes.

Cell surface forms of Qa-6 class I molecules are biochemically indistinguishable from Qa-2 although Qa-6 maps telomeric to Qa-2 with the recombinant strain B6.K2. Analysis of appropriate F1 strains did not demonstrate the presence of a trans acting factor that could modify the Qa-2 molecule to produce the Qa-6 determinant. Also, neither a neighboring cell surface molecule nor oligosaccharides were found to block the recognition of the Qa-6 determinant in Qa-2+,6- strains. The 2-D gel profiles of neuraminidase or endoglycosidase treated anti-Qa-2 immunoprecipitates from lysates of cell surface iodinated Qa-2+,6+ strains revealed an additional basic polypeptide which was absent from that of Qa-2+,6- strains. Thus, differential sialylation/glycosylation of Qa-2 molecules masks detection of Qa-2 antigen heterogeneity when cell surface forms are analyzed. Qa-6+ phenotype associated polypeptides were also found at various stages of post-translational processing in cells metabolically labeled in the presence and absence of tunicamycin. Northern analyses using Q7 and Q9 specific oligonucleotide probes revealed appropriate sized transcripts for both genes in the Qa-2+,6+ strain B6 but only Q9 in the Qa-2+,6- strain B6.K2. These data demonstrate that there is structural heterogeneity in Qa-2 antigens expressed by Qa-2+,6+ and Qa-2+,6- strains which results from differential expression of the Q7 and Q9 genes.

Animals↗

Naming, rehearsal, and interstimulus interval effects in memory processing.

Recognition memory was tested for lists of 6 briefly (0.08 s) presented pictures at different interstimulus intervals (ISI) of 0.08, 1, and 4 s. Experiment 1 showed a 16% performance increase (ISI effect) for increasing ISI for travel slide but not kaleidoscope pictures. Experiment 2 showed that learning names for the kaleidoscope pictures then resulted in a substantial (20%) ISI effect, not attributable solely to the added exposure to the pictures. Experiment 3 required names, color evaluations, or blank stares during list-memory presentations. Interviews established that the most effective memory strategy was chaining the names together, followed by repeating the most current name, and in turn followed by reliance upon only the sensory experience. All groups in Experiments 2 and 3, independent of ISI effects, showed U-shaped serial position functions. Rehearsal is shown to be nonessential and cannot be the general cause of the primary effect of the serial position function.

Adolescent↗

DNA sequencing of a mouse liver protein that binds selenium: implications for selenium's mechanism of action in cancer prevention.

Complementary DNA clones for liver protein 56K (SLP-56) were isolated by screening a lambda Zap mouse liver library. The cloned cDNAs represented the complete message. The correct reading frame was verified by alignment of the deduced amino acid sequence with that of peptides sequenced from the purified protein. The primary sequence has not been reported previously since homologous DNA sequences were not found in GenBank. Most importantly, the DNA sequence did not contain an in-frame TGA codon that would code for seleno-cysteine, as occurs in the prototypic selenoprotein, glutathione peroxidase. Hydropathy analysis suggested the protein was not a membrane-spanning protein. SLP-56 was previously localized as a cytosolic-soluble protein on the basis of cell fractionation experiments. The results suggest that SLP-56 is different from proteins whose synthesis and concentration are dependent upon selenium and require TGA to encode for selenocysteine. In this respect, SLP-56 appears to be similar to liver fatty acid binding protein (SLP-14) for which selenium is a ligand. Our working hypothesis is that selenium exerts its inhibitory effects on cell growth by modulating the properties of existing growth regulatory proteins. The two proteins that are readily labeled by selenium in many rodent tissues, SLP-56 and SLP-14, would fit into this category.

Amino Acid Sequence↗

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly.

Amino Acid Sequence↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗