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R G Cook

Publications and source records attributed to R G Cook.

At least 55 records · Page 3Linked to original sources
PubMed1994

Synthetic macrophage activating peptides derived from the N-terminus of human MCF.

Recently, we described the purification and N-terminal sequencing of a novel cytokine termed MCF (Monocyte Cytotoxicity Inducing Factor) (1,2). In order to study the interaction of this cytokine with monocytes, we synthesized a nona-peptide GAAVLEDSQ corresponding to the N-terminus of MCF: two truncated peptides, GAAVL and LEDSQ; and the substituted peptide, GAAVLENSQ. The authentic N-terminal peptide is biologically active in the nanomolar range, while substitution of asparagine for aspartic acid at position 7 diminishes biological activity. Biological activity was observed from the C-terminal fragment LEDSQ, but the N-terminal pentapeptide (GAAVL) was devoid of biological activity. Scatchard analysis revealed a single class of saturable high affinity sites. These studies indicate that the N-terminus of MCF is important in interacting with the binding site on monocytes and it may be possible to design synthetic activators and inhibitors of monocyte/macrophage cytotoxicity.

Amino Acid Sequence↗
PubMed1994

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.

Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.

Amino Acid Sequence↗
PubMed1993

Regulation of TL antigen expression. Analysis of the T18d promoter region and responses to IFN-gamma.

Type I (alpha/beta) and type II (gamma) IFN enhance MHC class I gene expression through an IFN-responsive element (IRE) present in the 5' flanking region of the class I-a genes. Comparison of the 5' sequences between classical class I-a genes and T region class I-b genes reveals little homology except for presence of a potential IRE. We have found that cell surface expression of thymus leukemia Ag (TL) was up-regulated by IFN-gamma to a greater extent than H-2K,D in all TL+ T cell lines tested. In contrast, IFN-alpha/beta, which significantly increased H-2K and H-2D Ag expression, had only minor effects on TL expression. Resting peripheral T cells, which were considered to be TL- from previous studies, were found to express TL at a low level as determined by flow cytometry, immunoprecipitation, as well as polymerase chain reaction; the level of expression also could be elevated by IFN-gamma. To examine the control of TL gene transcription and its regulation by IFN-gamma, varying lengths of the T18d 5' flanking region were analyzed in chloramphenicol acetyl transferase assays. By deletion analysis, promoter activity and IFN-gamma responsiveness were localized to an 86-bp fragment that contains the IRE. Both responses were localized further to a 32-bp fragment that contained the IRE at its 3' end. RNase protection assays revealed two major transcription initiation sites, one immediately 5' of the IRE and another approximately 60 bp downstream. Furthermore, polymerase chain reaction analysis of mRNA from resting T cells, thymocytes, and T cell tumor lines confirmed the RNase protection data. Thus, transcription of T18d initiates much further upstream than the classical class I genes, can utilize an unusual promoter element, and can be elevated by IFN-gamma.

Animals↗
PubMed1993

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

Vegetative cells of the ciliated protozoan Tetrahymena thermophila contain a transcriptionally active macronucleus and a transcriptionally inactive micronucleus. Although structurally and functionally dissimilar, these nuclei are products of a single postzygotic division during conjugation, the sexual phase of the life cycle. Immunocytochemical analyses during growth, starvation, and conjugation were used to examine the nuclear deposition of hv1, a histone H2A variant that is found in macronuclei and thought to play a role in transcriptionally active chromatin. Polyclonal antisera were generated using whole hv1 protein and synthetic peptides from the amino and carboxyl domains of hv1. The transcriptionally active macronuclei stained at all stages of the life cycle. Micronuclei did not stain during growth or starvation but stained with two of the sera during early stages of conjugation, preceding the stage when micronuclei become transcriptionally active. Immunoblot analyses of fractionated macro- and micronuclei confirmed the micronuclear acquisition of hv1 early in conjugation. hv1 staining disappeared from developing micronuclei late in conjugation. Interestingly, the carboxy-peptide antiserum stained micronuclei only briefly, late in development. The detection of the previously sequestered carboxyl terminus of hv1 may be related to the elimination of hv1 during the dynamic restructing of micronuclear chromatin that occurs as the micronucleus enters a transcriptionally incompetent state that is maintained during vegetative growth. These studies demonstrate that the transcriptional differences between macro- and micronuclei are associated with the loss of a chromatin component from developing micronuclei rather than its de novo appearance in developing macronuclei and argue that hv1 functions in establishing a transcriptionally competent state of chromatin.

Amino Acid Sequence↗
PubMed1993

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Streptococcus pyogenes (group A Streptococcus) has re-emerged in recent years as a cause of severe human disease. Because extracellular products are involved in streptococcal pathogenesis, we explored the possibility that a disease isolate expresses an uncharacterized superantigen. We screened culture supernatants for superantigen activity with a major histocompatibility complex class II-dependent T cell proliferation assay. Initial fractionation with red dye A chromatography indicated production of a class II-dependent T cell mitogen by a toxic shock-like syndrome (TSLS) strain. The amino terminus of the purified streptococcal superantigen was more homologous to the amino termini of staphylococcal enterotoxins B, C1, and C3 (SEB, SEC1, and SEC3), than to those of pyrogenic exotoxins A, B, C or other streptococcal toxins. The molecule, designated SSA, had the same pattern of class II isotype usage as SEB in T cell proliferation assays. However, it differed in its pattern of human T cell activation, as measured by quantitative polymerase chain reaction with V beta-specific primers. SSA activated human T cells that express V beta 1, 3, 15 with a minor increase of V beta 5.2-bearing cells, whereas SEB activated V beta 3, 12, 15, and 17-bearing T cells. Immunoblot analysis of 75 disease isolates from several localities detected SSA production only in group A streptococci, and found that SSA is apparently confined to only three clonal lineages as defined by multilocus enzyme electrophoresis typing. Isolates of one of these lineages, (electrophoretic type 2) are strongly associated with TSLS. The data identify SSA as a novel streptococcal superantigen that appears to be more related structurally to staphylococcal enterotoxins than to streptococcal exotoxins. Because abundant SSA production is apparently confined to only three streptococcal clonal lineages, the data also suggest that the SSA gene has only recently been acquired by S. pyogenes.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of an abundant rabbit ovarian protein with 20 alpha-hydroxysteroid dehydrogenase activity.

An abundant 37-kDa protein, which comprises up to 30% of the soluble proteins of the ovary, has been found to have 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity. The steroidogenic enzyme 20 alpha HSD regulates the conversion of progesterone to 20 alpha-hydroxyprogesterone in many mammalian species. Complimentary DNA clones encoding a unique and abundant 20 alpha HSD were isolated from a mature rabbit ovary library using guinea pig antisera generated to the purified 37-kDa protein and from a 5' EcoRI fragment from the initial positive clone. A full-length cDNA clone of 1217 basepairs encoding a 323-amino acid protein with an estimated mol wt of 37 kilodaltons was obtained. Amino acid sequence data indicate a similarity to human chlordecone reductase, bovine lung prostaglandin F synthase, human aldose reductase, human aldehyde reductase, and frog lens rho-crystallin, placing rabbit ovarian 20 alpha HSD in the aldo-keto reductase family of proteins. Northern blot analysis demonstrated a 1.2-kilobase mRNA in the interstitial tissue of mature rabbit ovaries and, to a lesser extent, in corpora luteal tissue. 20 alpha HSD was expressed in bacteria as a recombinant protein and was shown to possess enzymatic activity, preferring NADP as a cofactor. These studies demonstrate that an abundant ovarian protein belonging to the superfamily of NADP-dependent aldo-keto reductases has 20 alpha HSD activity. This is the first example of an abundant crystallin-related protein with known enzymatic activity in a tissue other than the lens.

20-Hydroxysteroid Dehydrogenases↗
PubMed1993

Fractionation of small tryptic phosphopeptides by alkaline PAGE followed by amino acid sequencing.

A novel two-step approach for localizing the site(s) of phosphorylation within intact proteins is described. Phosphorylated (32P-labeled) tryptic peptides are first resolved in a high-percentage polyacrylamide gel that has been optimized for the enrichment and separation of small, negatively charged peptides. Then the resolved peptides are located by autoradiography, excised, eluted and immobilized on a positively charged membrane, Immobilon -N, where they can be sequenced directly. The methods have been developed using a small, basic phosphoprotein (histone H1 from Tetrahymena); however, the approach is probably applicable to a wide variety of phosphoproteins.

Amino Acid Sequence↗
PubMed1993

Laparoscopic inguinal herniorrhaphy in a swine model. Third place winner of the Conrad Jobst Award in the Gold Medal paper competition.

A simplified method of laparoscopic inguinal herniorrhaphy using prosthetic materials was evaluated in a swine hernia model. The goals of this study were to determine 1) effectiveness of repair in a rapidly growing animal, 2) effectiveness and extent of adhesion formation of different prosthetic materials, 3) the effect of repair on testicular growth, and 4) histologic effects on the hernia site and surrounding structures. In a prospective randomized study, 30 juvenile male swine (average, 23 kg) with 35 congenital indirect inguinal hernias underwent laparoscopic herniorrhaphy using one of three prosthetic materials: Group 1 (polytetrafluoroethylene/Gore-Tex), N = 10; Group 2 (polypropylene mesh/Marlex), N = 10; Group 3 (polypropylene mesh/Prolene), N = 10. A standardized laparoscopic herniorrhaphy technique consisting of stapling prosthetic material over the hernia defect without peritoneal dissection was employed. During the 3-month postoperative period, animals were sequentially examined for normal growth and development, normal testicular development, and signs of hernia recurrence. Clinically apparent complications related to herniorrhaphy occurred in five animals (17%) during the observation period (one with repair failure, one with testicular torsion, two with repair failure and bowel obstruction, and one with intestinal obstruction secondary to adhesions). All three animals with bowel obstruction died. At 90 days after surgery all remaining animals (N = 27) were euthanized (Group 1 = 9, Group 2 = 8, Group 3 = 10). Average weight was 84 kg. Necropsy findings included no additional hernia recurrences, and one mesh erosion into the urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Biochemical specificity of H-2M3a. Stereospecificity and space-filling requirements at position 1 maintain N-formyl peptide binding.

The maternally transmitted Ag is a cell surface product of three gene products: 1) H-2M3a (formerly Hmta), a class I MHC heavy chain; 2) beta 2-microglobulin; and 3) maternally transmitted factor (Mtf), the N-terminus of the mitochondrially encoded ND1 subunit of the reduced form of nicotinamide-adenine dinucleotide dehydrogenase. This class I molecule has been shown to be an N-formyl peptide receptor. Although the N-formyl moiety is necessary for binding to M3a, it is not sufficient. We proposed that the R group of the amino acid in position 1 plays a pivotal role in peptide binding to M3a. To test this hypothesis, analogues differing in size and stereospecificity of the R group were synthesized. Substitutions with other hydrophobic amino acids such as N-formyl phenylalanine and N-formyl valine had no significant effect on the ability of these Mtf alpha analogues to sensitize target cells (M3a, Mtf beta) to M3a, Mtf alpha-specific CTL. In contrast, the nonsubstituted, N-formylated, and N-acetylated glycyl analogues of Mtf beta bound equivalently to M3a in a peptide competition assay. Moreover, the alanine analogue bound in an N-formyl-dependent manner. To determine the limitations of the putative N-formyl pocket, peptide analogues were constructed incorporating D-isomer amino acids. When formylated D-alanine or D-methionine replaced the native methionine, these peptide derivatives did not show significant binding to M3a. Therefore, the presence of a space-filling R group (greater than hydrogen) is necessary for an antigenic peptide to bind M3a in an N-formyl-dependent manner. Additionally, the ability of M3a to discriminate between the optical forms of methionine and alanine demonstrates that this N-formyl pocket is stereospecific in its ability to bind peptide. Thus, we have defined three requirements for peptide binding to M3a: an N-formyl moiety at the amino terminus of the peptide, a space-filling R group at position 1 to maintain this N-formyl specificity, and the correct stereoisomer of the first amino acid.

Alanine↗
PubMed1992

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.

Animals↗
PubMed1992

Isolation and characterization of a novel streptococcal superantigen.

The involvement of S. pyogenes (group A Streptococcus) in severe invasive disease, toxic-shock-like syndrome, and episodes of rheumatic fever led us to explore the possibility that these strains produce a novel superantigen. By using a superantigen-specific assay, we purified a 28-kDa protein from culture supernatants that stimulated T cells in an MHC class II-dependent, V beta-specific manner and designated it SSA, streptococcal superantigen. The amino terminus of SSA showed striking resemblance to SEB, SEC1, and SEC3. The structural homology exhibited by SSA to SEB was reflected functionally in that both of these superantigens bound the same class II isotypes. In contrast, SSA differed from SEB and other known bacterial superantigens with respect to its pattern of V beta-specific T-cell activation. SSA stimulated human T cells that expressed V beta 1, 3, 15, and perhaps V beta 5.2. Using SSA-specific antibodies in an immunoblot assay, we screened 26 strains of Lancefield group A Streptococcus and 16 strains of group B, C, and G Streptococcus. We found that SSA was expressed with high frequency in group A strains, but was absent from all other groups tested. These data establish SSA as a novel superantigen secreted by S. pyogenes. Further study of the structure and expression of SSA may reveal a role for this molecule in current episodes of severe streptococcal diseases.

Antibodies, Bacterial↗
PubMed1991

Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin.

The physiological functions of the mouse telomeric major histocompatibility complex (MHC) class I molecules, including Hmt, are unknown. Hmt presents a polymorphic, N-formylated peptide encoded by the mitochondrial gene ND1 forming the cell surface maternally transmitted antigen (Mta). Because the N-formyl moiety is required for Hmt binding, we proposed that Hmt may function generally in presentation of N-formylated antigens. This hypothesis was validated by a competitive binding assay, demonstrating that synthetic N-formyl peptides from other mitochondrial genes also bound Hmt. Bacteria similarly initiate protein synthesis with N-formylmethionine; indeed, we established that Hmt can also present prokaryotic peptides in an N-formyl-dependent manner. These results indicate biochemical specialization of this MHC-peptide interaction and suggest a unique role for Hmt in prokaryotic host defenses.

Amino Acid Sequence↗
PubMed1991

Staphylococcal exotoxin activation of T cells. Role of exotoxin-MHC class II binding affinity and class II isotype.

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 bind directly to class II molecules of the MHC and stimulate T cells based predominantly on the V beta segment used by the TCR. We investigated the relationship between the class II binding affinities of four of these exotoxins, SEA, SEB, SEC1, and toxic shock syndrome toxin-1 and their T cell signaling capabilities. Although the toxins stimulated T cells at concentrations that ranged over more than two orders of magnitude, their affinities for class II (DR1) differed by less than sixfold. The affinities of the toxins predicted their capacity to stimulate resting T cells to proliferate. The binding affinities of the toxins for class II molecules indicated that at concentrations required for T cell stimulation, as few as 0.1% of the class II molecules are complexed with toxin. Finally, the isotype of class II molecules affected the ability of the toxins to bind and use these MHC Ag to stimulate T cells. These data thus demonstrate that of the staphylococcal exotoxins studied, both their potency as T cell mitogens and their ability to function in the presence of single class II isotypes can be attributed in part to their characteristic abilities to bind class II molecules.

Animals↗
PubMed1991

Proteolytic removal of core histone amino termini and dephosphorylation of histone H1 correlate with the formation of condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

During the sexual cycle in Tetrahymena, the germ-line micronucleus gives rise to new macro- and micronuclei, whereas the former somatic macronucleus ceases transcription, becomes highly condensed, and is eventually eliminated from the cell. With polyclonal antibodies specific for acetylated forms of histone H4, immunofluorescent analyses have demonstrated that transcriptionally active macronuclei stain positively at all stages of the life cycle except during conjugation, when parental macronuclei become inactive and are eliminated from the cell. In this report using affinity-purified antibodies to either the acetylated or unacetylated amino-terminal domain of H4, immunofluorescent analyses suggest that the acetylated amino-terminal tails of H4 are proteolytically removed in "old" macronuclei during this period. This suggestion was further confirmed by biochemical analysis of purified old macronuclei that revealed several polypeptides with molecular mass 1-2 kD less than that of intact core histones. These species, which are unique to old macronuclei, are not newly synthesized and fail to stain with either acetylated or unacetylated H4 antibodies. Microsequence analysis clearly shows that these polypeptides are proteolytically processed forms of core histones whose amino-terminal "tails" (varying from 13 to 21 residues) have been removed. During the same developmental period, histone H1 is dephosphorylated rapidly and completely in old macronuclei. These results strongly suggest that the developmentally regulated proteolysis of core histones and dephosphorylation of histone H1 participate in a novel pathway leading to the formation of highly condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

Acetylation↗
PubMed1991

Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence.

HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins. Genomic clones encoding each of these proteins have been sequenced. Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells. The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins. Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids. However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2. Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins. Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box. This finding suggests that at least in T. thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.

Amino Acid Sequence↗
PubMed1991

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.

Amino Acid Sequence↗