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J A Cooper

Publications and source records attributed to J A Cooper.

At least 271 records · Page 15Linked to original sources
PubMed1990

Acidic residues at the carboxyl terminus of p60c-src are required for regulation of tyrosine kinase activity and transformation.

Protein phosphorylation sites act to transduce signals into changes in enzymatic activity, representing a point of interaction within a regulatory pathway. The amino acid sequence surrounding a phosphorylation site may well have several functions, including recognition by an appropriate kinase. By generating random mutations in its immediate vicinity, we have examined the sequence requirements of a regulatory tyrosine phosphorylation site, Tyr527, in the proto-oncogene product, p60c-src. The transforming and kinase activities of p60c-src are repressed by phosphorylation of Tyr527. Mutations were made around Tyr527 without changing Tyr527 or the kinase domain. Twenty-nine mutants were sequenced and classified as transforming or nontransforming for Rat-2 cells. Nontransforming mutants contained a surprising variety of COOH-terminal mutations, although acidic residues were present at positions 518 and 524 in all nontransforming mutants. Transforming mutants that contained single-residue changes at Asp518 and Ser522 demonstrated the importance of these residues. Other transforming mutants contained two or more substitutions, but the results are most simply explained if residues Glu524 and Thr523 are also important for normal regulation. Transforming mutations reduced the phosphorylation of Tyr527. We conclude that only a few of the residues in the COOH terminus other than Tyr527 are required to ensure normal phosphorylation and repression of activity in fibroblasts. Other residues may have been conserved during evolution to permit normal function and regulation in other cell types.

Amino Acid Sequence↗
PubMed1990

Pulmonary disease due to antirheumatic agents.

Drug-induced lung disease presents several diagnostic and therapeutic problems to the clinician. This is especially true in the case of lung disease associated with antirheumatic agents in which pulmonary disease may be due to the underlying disorder. Unfortunately, no specific markers exist to differentiate drug-induced lung disease from other pathologic processes. In addition, the numerous drugs often used simultaneously or in close sequence in rheumatic disorders make assignment of toxicity to a specific agent difficult. Six groups of drugs used as anti-inflammatory/antirheumatic agents have been discussed in association with pulmonary damage penicillamine, gold, methotrexate, salicylates, NSAIDs, and colchicine. The major clinical syndromes ascribed to these drugs include hypersensitivity pneumonitis and chronic alveolitis/fibrosis (penicillamine, gold, methotrexate, NSAIDs), pulmonary-renal syndrome (penicillamine), bronchiolitis obliterans (penicillamine, gold), and noncardiogenic pulmonary edema (salicylates, colchicine). Unfortunately, treatment options remain limited. Withdrawal of the offending drug and supportive care are the mainstays of therapy. In cases in which active inflammation causes significant derangement of gas exchange, corticosteroids are warranted. More aggressive management using immunosuppressive drugs has been recommended in cases of refractory PABO and PAGS, but these recommendations are at present based only on isolated case reports.

Anti-Inflammatory Agents↗
PubMed1990

Properties of tripartite chimeras between Src and Lck.

To evaluate the possible regulatory effects of interactions between different domains of Src-family kinases, we have assembled five chimeric molecules containing parts of p60c-src (Src) and p56lck (Lck). Chimeras contained the N-terminal portion, kinase domain or C-terminal tail from either of the parent molecules. None of the four full-length hybrid proteins induced morphological transformation of NIH3T3 cells, nor stimulated phosphorylation of cell proteins on tyrosine residues, suggesting that their protein-tyrosine kinase activities were repressed appropriately in fibroblastic cells. However, two hybrid proteins, SLS and SLL, containing the Src N-terminal region, Lck kinase domain, and either Src or Lck tail, respectively, inefficiently induced anchorage-independent growth. Sites of phosphorylation in these molecules were determined by two-dimensional peptide mapping. SLS and SLL were both phosphorylated at their C-terminal tyrosine residues similarly to the parental molecules. Curiously, both proteins were also significantly phosphorylated at tyrosine 416, a feature of transforming variants of Src and Lck. A modified hybrid, SLO, containing the N-terminal region of Src, kinase domain of Lck and a truncated C-terminus, fully transformed cells and stimulated phosphorylation of cell proteins at tyrosine. Comparison of SLS, SLL and SLO suggests that the full-length hybrids are repressed by their C-terminal phosphorylated tyrosine residues in vivo. Consistent with this, SLO resembled activated Src in being cytoskeletal, and SLS and SLL resembled nontransforming Src and were not cytoskeletal. Hybrids with an Lck N-terminal region were cytoskeletal, like Lck itself, even though they were not transforming, suggesting that cytoskeletal localization of Lck is determined through its N terminus. Curiously, the hybrid molecules appeared not to be regulated in vitro. The specific activities of SLS, SLL and Src were approximately equal, but the specific activity of SLO was not increased, being much less than that of activated Src. Enzymatic dephosphorylation stimulated the in vitro kinase activity of parental Src, but not of SLS or SLL. These observations suggest that chimeric Src-Lck molecules are regulated in the cell, but not in vitro.

Animals↗
PubMed1989

Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin.

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).

Actins↗
PubMed1989

Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins.

We have identified two platelet-derived growth factor (PDGF)-dependent autophosphorylation sites in the beta subunit of the human PDGF receptor (PDGF-R). The major site of phosphorylation (Tyr-857) corresponds to the major autophosphorylation site in many other tyrosine kinases. Tyr-751, which lies within the kinase insert region, is a second in vivo site and the major in vitro site. Immunoprecipitates of wild-type PDGF-Rs prepared from PDGF-treated cells contained a phosphatidylinositol (PI) 3 kinase activity and three specific polypeptides as well as the PDGF-R. Mutation of Tyr-751 to Phe or Gly, or mutation of the catalytic domain to abolish kinase activity, blocked association of the PDGF-R with the PI kinase and the three proteins. These results suggest that autophosphorylation in the kinase insert region triggers the binding of the activated PDGF-R to specific cellular proteins, including a PI kinase whose activity is known to be stimulated by PDGF. Thus autophosphorylation may play a novel role in signal transduction via the PDGF-R.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle.

Three cDNAs encoding the beta chain of Cap Z have been isolated from lambda gt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr = 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z beta. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z beta are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities. Two of the three cDNAs contain the complete coding region. Both coding regions begin with a consensus translation start site; however, their 5'-untranslated regions are different. Northern analysis of whole chicken embryos and adult chicken tissues shows two mRNA species. The embryo and non-muscle tissues have transcripts of 1.35 and 2.00 kilobases, and the muscle tissues have transcripts of 2.15 and 1.45 kilobases. Southern analysis of chicken genomic DNA shows that there are probably two related genes, one more similar to the cDNA than the other.

Actin Capping Proteins↗
PubMed1989

Role of albumin arginyl sites in albumin-induced reduction of endothelial hydraulic conductivity.

We determined the effect of albumin on endothelial hydraulic conductivity (Lp) and the contributions of the positively charged arginyl and lysinyl residues of albumin in mediating the effect. Studies were made using monolayers of cultured sheep pulmonary artery endothelial cells grown to confluence on polycarbonate filters. Water flux was measured as transendothelial hydrostatic pressure was varied from 5 to 20 cm H2O. Lp was calculated from the slope of the relationship of water flux versus pressure. The Lp of endothelial monolayers perfused with albumin-free Hanks Balanced Salt Solution (HBSS) was compared to perfusion with HBSS containing either native albumin, or albumin in which the arginyl residues were modified by a condensation reaction with 1,2-cyclohexanedione (CHD-albumin), or albumin in which the lysinyl residues were modified by a substitution reaction with succinic anhydride (SC-albumin). Baseline Lp at 2.5 mg/ml native albumin was 1.6 +/- 0.1 X 10(-6) cm/s/cm H2O compared to the filter Lp after removing cells of 3.0 +/- 0.3 X 10(-4) cm/s/cm H2O. Endothelial Lp increased by 60% when albumin concentration was decreased from 2.5 mg/ml to 0.5 mg/ml (P less than 0.05), but did not change with an increase in concentration to 10 mg/ml. Albumin-free buffer and CHD-albumin increased endothelial Lp by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold, respectively (P less than 0.05). All endothelial Lp values were restored to baseline when the native albumin concentration was returned to 2.5 mg/ml. Excess l-arginine (2 X 10(-3) M) inhibited the effect of native albumin and increased endothelial Lp 1.5 +/- 0.02-fold (P less than 0.05), but excess l-lysine (4 X 10(-3) in the presence of native albumin had no effect on Lp. None of the perfusates altered the filter Lp value. Neutral dextran (70 kD), in contrast to native albumin, had no effect on endothelial Lp. These results indicate that albumin reduces the hydraulic conductivity of endothelial monolayers in a concentration-dependent fashion and that the arginyl residues of albumin are required for the response. The effect of albumin may be mediated by a charge interaction of albumin with the endothelium.

Albumins↗
PubMed1989

Risk factors for breast cancer by oestrogen receptor status: a population-based case-control study.

Data from a population-based case-control study conducted in Adelaide, South Australia, and involving 451 case-control pairs, were analysed to determine whether the associations of menstrual, reproductive, dietary and other factors with risk of breast cancer differed by oestrogen receptor (ER) status. Data on ER status were available for 380 cases. The proportion of tumours which were ER+ increased with age, and there was a higher proportion of ER+ tumours in post-menopausal than in premenopausal women. Both oral contraceptive use (P = 0.055) and cigarette smoking (P = 0.047) were associated with increased (unadjusted) risk of ER- cancer, while having little association with risk of ER+ cancer. Most dietary factors had little association with risk of either cancer type, the main exception being the reduction in risk of ER- breast cancer with increasing beta-carotene intake (P for trend = 0.017). In general, however, links with the factors examined were not strong enough to suggest different causal pathways for ER- and ER+ breast cancer.

Adult↗
PubMed1989

Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle.

cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The protein predicted by the resulting nucleotide sequence has a Mr of 32,960 and contains four regions that show close homology with four alpha-chymotryptic digestion fragments of the alpha chain. The amino acid composition of the alpha chain of Cap Z and the predicted protein are also similar. Northern blot analysis of whole chicken embryos shows two mRNA species of 1.9 and 2.4 kilobases, respectively, that hybridize with CE2. Three potential polyadenylylation signals in two regions of CE2 460 bp apart are identified, suggesting that the two messages may result from the use of alternative polyadenylylation sites. Comparison of the sequence data with that of other known actin-capping and severing proteins shows no significant homologies, suggesting that Cap Z may be a member of a unique group of capping, nonsevering proteins.

Actin Capping Proteins↗
PubMed1989

Endothelial barrier function.

The endothelial barrier in all organ beds allows the free exchange of water, but is restrictive to varying degrees to the transport of solutes such as albumin. For example, in the brain microvessels, the endothelial barrier restricts the transport of protein, whereas in fenestrated and continuous endothelial cells of the renal and lung endothelial cells, the endothelial barrier is semipermeable. The endothelial monolayer demonstrates selectivity, i.e., the permeation of molecules is inversely related to the molecular weight. Although the "pore" theory has been used to describe the transport across the endothelial barrier, the transport of solutes is also dependent on the charge of solutes and the endothelial cell, and the ability of the solute to bind to or be taken up by endothelial cells. Receptor-mediated trancytosis of albumin may contribute to albumin transport in addition to transport by paracellular pathways (i.e., through a so-called pore). Water permeability across the endothelium is determined by the interaction of albumin with glycocalyx and interstitial components of the endothelium (the "fiber matrix"). Ambient concentration of albumin serves to lower endothelial hydraulic conductivity. Increased endothelial permeability to solutes and water in inflammatory states is dependent on the shape and configuration of endothelial cells as determined by alterations in cytoskeletal elements, such as f-actin, and as regulated by intracellular second messengers such as free cytosolic calcium.

Animals↗
PubMed1989

Structural differences between repressed and derepressed forms of p60c-src.

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Enzyme Activation↗
PubMed1989

Kinetics of cellular influx and histamine release in normal human bronchi.

Inhalation of certain substances can induce bronchial inflammation followed by bronchoconstriction or bronchial hyperreactivity. We have developed a model of airway inflammation and bronchoconstriction induced by an aqueous extract of cotton bracts (CBE) in which the severity of flow rate reduction correlates with the presence of bronchoalveolar neutrophils. In the current study normal human volunteers underwent local bronchial instillation of CBE. Bronchial lavage histamine concentrations and cellular populations were determined at time intervals after local challenge. In addition, bronchial biopsies were obtained after CBE instillation, and the degree of subepithelial inflammation was quantitated. We report that lavage of bronchi locally exposed to CBE contains 1) greater histamine concentrations 4 min after challenge compared with lavage from saline-challenged bronchi and 2) concentrations of eosinophils and macrophages that are greater than control lavage 8 min after exposure. Bronchial biopsies taken after CBE instillation also demonstrated a variable degree of subepithelial granulocyte infiltration that correlated with the global bronchoconstricting response to CBE assessed on a separate day. These studies suggest that inflammation can develop rapidly in normal human bronchi and may precede significant flow rate reductions by greater than 1 h.

Biopsy↗
PubMed1989

Pulmonary transvascular flux of transferrin.

We compared the pulmonary transvascular fluxes of transferrin and albumin in the intact sheep lung. Anesthetized sheep were prepared with lung lymph fistulas. The vascular blood pool was marked with 99mTc-erythrocytes, autologous transferrin was labeled with 113mIn, and albumin was labeled with 125I. Samples of blood, plasma, lymph, and lung were obtained up to 180 min after tracer infusion. Lymph tissue radioactivities were corrected for the intravascular component and expressed as extravascular-to-plasma concentration ratios. Clearance of transferrin and albumin from the plasma space followed a two-compartment model. The clearance rate constant was 2.1 +/- 0.1 x 10(-3) min for albumin and 2.4 +/- 0.1 x 10(-3) min for transferrin (P less than 0.05). Lymph-to-plasma ratios for albumin and transferrin were not different. However, the extravascular-to-plasma ratio for albumin was greater than transferrin (P less than 0.05). The lymph and lung data were deconvoluted for the plasma input function and fit to a two-compartment model. The results indicate that albumin and transferrin have similar permeabilities across the vascular barrier but have different pulmonary circulation to lymph kinetics because the extravascular volume of distribution of albumin is greater than transferrin.

Albumins↗
PubMed1989

Modulation of endoperoxide product levels and cyclophosphamide-induced injury by glutathione repletion.

Glutathione is a tripeptide important in a number of diverse cellular functions including enzymatic reactions involved in prostaglandin endoperoxide metabolism. We have previously reported that cyclophosphamide administration to rats results in acute lung injury manifested by increased bronchoalveolar lavage albumin concentrations. In the current study we examine whether cyclophosphamide treatment affects pulmonary glutathione stores or bronchoalveolar endoperoxide metabolic product levels and whether these effects may be related to acute lung injury caused by the drug. We show that cyclophosphamide treatment causes a dose-dependent reduction in pulmonary glutathione stores 4 h after drug administration. In addition, acute lung injury as the result of cyclophosphamide can be abrogated by coadministration of oxothiazolidine carboxylate, an intracellular cysteine delivery system that also reverses pulmonary glutathione depletion induced by cyclophosphamide in our study. Finally, cyclophosphamide treatment reduces prostaglandin E2 concentrations in bronchoalveolar lavage and alveolar macrophage culture supernatant in a dose-dependent fashion and increases bronchoalveolar thromboxane concentrations in low dose-treated animals. These effects are reversed to a variable degree by coadministration of oxothiazolidine carboxylate. Our study suggests in vivo pulmonary arachidonic acid metabolism and cyclophosphamide-induced acute lung injury are modulated by cellular glutathione stores. These findings may have important implications for the treatment of acute lung injury.

Animals↗