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J A Cooper

Publications and source records attributed to J A Cooper.

At least 127 records · Page 7Linked to original sources
PubMed1997

Mapping of the mouse actin capping protein alpha subunit genes and pseudogenes.

Capping protein (CP), a heterodimer of alpha and beta subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three alpha isoforms (alpha 1, alpha 2, alpha 3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the alpha subunits of mouse CP and found three alpha 1 genes, two of which are pseudogenes, and a single gene for both alpha 2 and alpha 3. Their chromosomal locations were identified by interspecies backcross mapping. The alpha 1 gene (Cappa1) mapped to chromosome 3 between D3Mit11 and D3Mit13. The alpha 1 pseudogenes (Cappa1-ps1 and Cappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The alpha 2 gene (Cappa2) mapped to Chromosome 6 near Ptn. The alpha 3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal from Cappa2 near Kras2. One mouse mutation, de, maps in the vicinity of the alpha 1 gene. No known mouse mutations map to regions near the alpha 2 or alpha 3 genes.

Actin Capping Proteins↗
PubMed1997

Mouse disabled (mDab1): a Src binding protein implicated in neuronal development.

Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks.

Amino Acid Sequence↗
PubMed1997

Vertebrates have conserved capping protein alpha isoforms with specific expression patterns.

Capping protein (CP), a ubiquitous actin binding protein composed of an alpha and a beta subunit, is important for actin assembly and cell motility. Lower organisms have one gene and one isoform of each subunit. Chickens have two very similar alpha-subunit isoforms. To determine if vertebrates in general contain multiple alpha isoforms and if those alpha isoforms have conserved sequences, we isolated and analyzed alpha subunit cDNA's in mice and humans. Both mice and humans also have two alpha isoforms. Phylogenetic analysis of the alpha isoform sequences reveals that vertebrates have two highly conserved subfamilies, alpha1 and alpha2. The alpha1 and alpha2 subfamilies are very similar to each other but can be defined and distinguished from each other by a small number of key amino acid residues. In addition, 3' untranslated cDNA sequences are conserved within the isoform subfamilies. To investigate the function of the alpha isoforms, we examined their expression in mouse cells and tissues. Endothelial cells contain only the alpha2 isoform, and erythrocytes contain almost exclusively the alpha1 isoform. Most tissues have both alpha1 and alpha2 isoforms but the ratio of alpha1:alpha2 varies widely. Together, these findings support the hypothesis that the CP alpha isoforms have conserved, unique and essential roles in vertebrates.

Actin Depolymerizing Factors↗
PubMed1997

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins.

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.

Amino Acid Sequence↗
PubMed1997

Apal polymorphism in insulin-like growth factor II (IGF2) gene and weight in middle-aged males.

In 1474 healthy Caucasoid men aged 45-65 y, insulin-like growth factor II (IGF2) Apal AA homozygotes showed a mean body weight 4 kg lower than Apal GG homozygotes (77.6 +/- 10.9 kg vs 81.6 +/- 11.5 kg, P = 0.003) with heterozygotes (GA) intermediate (80.1 +/- 11.9 kg). The mean serum IGF-II concentration in 44 Apal AA individuals was significantly higher than in 48 Apal GG individuals (683.3 +/- 146.9 ng/ml vs 614.0 +/- 124.0 ng/ml, P = 0.01). An INS Pstl polymorphism showed no association with weight and it was also found to be in minimal linkage disequilibrium with the IGF2 Apal site (coefficient 0.016). The IGF2 Apal AA genotype is therefore associated with lower mean body weight but higher serum IGF-II concentrations than the GG genotype. Apal GG homozygotes incur a 1.67-fold risk of pathological Body Mass index (BMI) (> 30 kg/m2) compared with AA homozygotes.

Base Sequence↗
PubMed1997

The casein kinase II beta subunit binds to Mos and inhibits Mos activity.

Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation. Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK). We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos. The Mos-interacting region of CKII beta was mapped to the C terminus. Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta as well as in unfertilized Xenopus eggs. CKII beta inhibited Mos-mediated MAPK activation in rabbit reticulocyte lysates and repressed MKK activation by v-Mos in a coupled kinase assay. In addition, microinjection of CKII beta mRNA into Xenopus oocytes inhibited progesterone-induced meiotic maturation and MAPK activation, presumably by binding of CKII beta to Mos and thereby inhibiting MAPK activation. Moreover, this inhibitory phenotype could be rescued by another protein that binds to CKII beta, CKII alpha. The ability of ectopic CKII beta to inhibit meiotic maturation and the detection of a complex between endogenous Mos and CKII beta suggest that CKII beta may act as an inhibitor of Mos during oocyte maturation, perhaps setting a threshold beyond which Mos protein must accumulate before it can activate the MAPK pathway.

Amino Acid Sequence↗
PubMed1997

A common mutation in the lipoprotein lipase gene promoter, -93T/G, is associated with lower plasma triglyceride levels and increased promoter activity in vitro.

Single-strand conformational polymorphism analysis of the lipoprotein lipase promoter identified a T-->G transition at position -93. The frequency in healthy white men was 3.4% (n = 1575). There was an 83% allelic association between -93T-->G and Asp9-->Asn (D9N); all N9 mutations occurred on a -93G allele, but not all -93G mutations occurred on an N9 allele. It was thus possible to assess the effect on plasma triglyceride (Tg) levels of the rare -93G mutation in the presence of the wild-type D9. Carriers of the -93G, with genotype TG/DD, had significantly lower Tg levels than TT/DD individuals (1.36 versus 1.78 mmol/L, P = .01); carriers of both mutations (TG/DN) had the highest Tg levels (1.93 mmol/L). When the group was stratified above and below the sample mean for body mass index (BMI), carriers of the -93G on a D9 allele (TG/DD) were "protected" against the Tg-raising effect of obesity, as assessed by BMI. In Afro-Caribbeans (n = 91), the carrier frequency of -93G was 18-fold higher (63%), with weaker (17%) allelic association between -93G and N9. In vitro, the -93G promoter had 24% higher activity than the -93T in a rat smooth muscle cell line and 18% higher activity in a human adrenal cell line. A protein identified by band-shift assays bound to the -93G but not to the -93T allele, which may explain the lower Tg levels in -93G carriers.

Adult↗
PubMed1997

Risk of coronary heart disease and activation of factor XII in middle-aged men.

Increased activity is known to be present in the extrinsic, intrinsic, and final common pathways of the hemostatic system in men at high risk of coronary heart disease (CHD), but the status of the contact system of coagulation in this condition is uncertain. Plasma levels of activated factor XII (XIIa), the initial product of contact activation, have therefore been measured by ELISA in 2464 men aged 51 to 62 years, clinically free of CHD, who were taking part in a prospective cardiovascular survey based in general medical practices. Statistically significant, independent, and positive associations of XIIa were found with serum cholesterol and triglyceride concentrations, blood pressure, body mass index, factor VII activity, plasma fibrinogen concentration, and tobacco smoking, all associated with CHD. Plasma XIIa also increased with recent alcohol intake. Men in the highest quintile of risk according to their conventional risk factors had a mean XIIa of 2.07 ng/mL (95% confidence interval 1.99-2.16), 31% higher than that of men in the lowest quintile (1.58; 95% confidence interval 1.51-1.65). Thus, the contact system of coagulation appears to be activated when CHD risk is increased. Furthermore, the independent associations of XIIa with the major conventional CHD risk factors and its broad range of values in the general population (0.1 to 12.5 ng/mL), combined with a relatively low day-to-day variability in individuals (the within-person component of its total variation being 14.7%), suggest its potential usefulness as a marker of atherosclerotic vascular damage.

Coronary Disease↗
PubMed1997

In-phase and out-of-phase MR imaging of bone marrow: prediction of neoplasia based on the detection of coexistent fat and water.

OBJECTIVE: The purpose of this study was to determine if gradient-echo MR imaging with TEs selected with fat and water in phase and out of phase can help predict the likelihood of neoplastic or nonneoplastic lesions in bone marrow. SUBJECTS AND METHODS: Thirty consecutive patients with 31 suspected bone marrow lesions underwent MR imaging, including two spoiled gradient-echo sequences identical in all parameters except TE, which was chosen such that fat and water were either in phase or out of phase. Relative ratios of the abnormal bone marrow signal intensity and a control site on the in-phase and out-of-phase images were expressed. The images were also assessed independently by two reviewers who were unaware of the patients' identities and clinical histories. Reviewers assessed decreased marrow signal intensity relative to control sites on the out-of-phase and in-phase images. Pathologic confirmation was obtained in 16 patients (17 lesions); the remainder of patients had either established diagnoses or determination of benignity based on stability of findings at 1 year. Relative ratios were compared with the Student's t test and receiver operating characteristic (ROC) curve analysis, and the reviewers' scores were evaluated with ROC curve analysis. RESULTS: The relative signal-intensity ratios were 1.03 +/- 0.13 for the neoplastic group and 0.62 +/- 0.13 for the nonneoplastic group (p < .0001). ROC curve analysis of the signal-intensity ratios showed a z-score of .99. A ratio cutoff value of 0.81 resulted in a 95% sensitivity and a 95% specificity for detection of neoplasm. Both reviewers achieved 100% sensitivity and 94-100% specificity for detection of neoplasms. CONCLUSION: In-phase and out-of-phase gradient-echo MR imaging of bone marrow signal-intensity abnormalities can help predict the likelihood of neoplastic or nonneoplastic lesions.

Bone Marrow↗
PubMed1997

Platelet counts and aggregation measures in the incidence of ischaemic heart disease (IHD).

Although studies in those who have already experienced clinical episodes of ischaemic heart disease (IHD) have suggested properties of platelets influencing recurrence, there is limited information on the value of platelet tests in predicting first episodes of IHD. One study has suggested that a raised platelet count and increased aggregability in response to adenosine diphosphate (ADP) may increase IHD incidence but the numbers of IHD events involved were small. The larger Northwick Park Heart Study (NPHS) included platelet count and both ADP and adrenaline-induced aggregation and this paper presents their associations with subsequent IHD. Platelet counts were performed in 1369 white NPHS men aged between 40 and 64 at recruitment, of whom 181 subsequently experienced a major episode of IHD over a follow-up period of 16.1 years. Platelet count was unrelated to the incidence of IHD. ADP-induced aggregation was performed in a random sample of 740 men in whom 66 IHD events occurred during the subsequent 10.1 years, aggregability being measured both as ED50, the ADP dose at which aggregation occurred at half its maximum velocity, and also as EMR, the maximum rate of aggregation achieved. Neither measurement showed any association with IHD incidence, nor did similar measurements in 460 men in whom adrenaline-induced aggregation was also carried out. There are at least three possible explanations for the lack of any association between the measures of aggregability used and IHD. First, the large within-person variability of platelet aggregation tests may make the demonstration of any associations difficult, though the study had reasonable power to show effects with ADP. Secondly, the tests used may not be a valid index of the contribution of platelet function to thrombosis and IHD. However, the clear effect of several personal and demographic influences associated with IHD on the tests used brings this explanation into question. Thirdly, the role of platelets in thrombogenesis may be determined mainly by plasma influences such as fibrinogen, rather than by intrinsic properties of platelets themselves. Platelet counts within the physiological range and the aggregation tests used in this and in some other studies are of no value as indices of the risk of first episodes of IHD.

Adult↗
PubMed1996

A peptide derived from a B cell epitope of Plasmodium falciparum rhoptry associated protein 2 specifically raises antibodies to rhoptry associated protein 1.

Mice immunised with a recombinant form of malarial antigen rhoptry-associated protein 2 (RAP2) produce antisera which recognise the native protein by indirect immunofluorescence and immunoblotting. Purified IgG components of the antisera partially inhibit erythrocyte invasion in vitro. This response was obtained only if the recombinant immunogen was presented to the mice in the presence of reducing and denaturing agents. An 8-mer epitope in RAP2 was recognised by antibodies in three of the antisera: E25TEFSKLY32. Immunisation with this octapeptide raised antibodies which strongly recognised reduced RAP2 in seven out of eight mice. However, this antisera either failed to recognise (five out of eight mice), or only weakly (three out of eight mice) recognised nonreduced RAP2. Examination of disulphide bonds in native RAP2 showed that the 4 cysteines of RAP2 form two disulphide bridges: Cys24-Cys88, and Cys277-Cys376. One of these (Cys24-Cys88) is adjacent to the octapeptide in the native protein. Surprisingly, seven out of eight mice immunised with the octapeptide also raised antibodies against native rhoptry-associated protein 1 (RAP1). The raising of antibodies which recognise RAP1 was induced specifically by the RAP2 octapeptide rather than the carrier protein used for immunisation. The epitope in RAP1 recognised by the antibodies was identified and shown not to be the result of any shared contiguous homologous sequence between the two proteins, but to shared homologous amino acids in critical positions within the epitope. Purified IgG components from the antisera of mice immunised with the octapeptide gave partial inhibition of erythrocyte invasion in vitro.

Amino Acid Sequence↗
PubMed1996

Regulation, substrates and functions of src.

Src is the best understood member of a family of 9 tyrosine kinases that regulates cellular responses to extracellular stimuli. Activated mutants of Src are oncogenic. Using Src as an example, and referring to other Src family members where appropriate, this review describes the structure of Src, the functions of the individual domains, the regulation of Src kinase activity in the cell, the selection of substrates, and the biological functions of Src. The review concentrates on developments in the last 6-7 years, and cites data resulting from the isolation and characterization of Src mutants, crystallographic studies of the structures of SH2, SH3 and tyrosine kinase domains, biochemical studies of Src kinase activity and binding properties, and the biology of transgenic and knockout mouse strains.

Amino Acid Sequence↗
PubMed1996

Activation of factor VII during alimentary lipemia occurs in healthy adults and patients with congenital factor XII or factor XI deficiency, but not in patients with factor IX deficiency.

Factor VII activity (FVIIc), a risk marker for coronary heart disease, is increased during postprandial lipemia. Factor VII activation accompanies lipolysis of triglyceride-rich lipoproteins, but the nature of this association and whether it is causal remain uncertain. To explore this issue, four patients with homozygous factor XII deficiency, four with complete factor XI deficiency, six with factor IX deficiency, and their respective age- and sex-matched controls were given two isocaloric dietary regimens, one providing on average 136 g fat and the other 19 g fat. Blood was taken before breakfast, immediately before lunch at 195 minutes, and at completion of the study at 390 minutes. All samples for each subject and matched control were assayed as one batch for FVIIc, activated factor VII, and factor VII antigen (FVIIag). Activation of factor VII was observed with the high-fat regimen but not with the low-fat regimen in all controls, factor XII-deficient patients, and factor XI-deficient patients. No factor VII activation was observed during either regimen in factor IX-deficient patients, but a normal postprandial responsiveness of factor VII to dietary fat was restored in one patient who replicated the study after factor IX therapy. Plasma FVIIag was not altered postprandially in either regimen in any group of patients or controls. Factor IX apparently plays an obligatory role in the postprandial activation of factor VII, although the mechanism remains to be determined.

Adult↗
PubMed1996

Use of the two hybrid system to detect the association of the protein-tyrosine-phosphatase, SHPTP2, with another SH2-containing protein, Grb7.

SHPTP2 is a ubiquitously-expressed SH2-containing tyrosine phosphatase that is tyrosine phosphorylated in response to activation of various receptor and nonreceptor tyrosine kinases. SHPTP2 associates with the platelet-derived growth factor (PDGF) receptor after ligand stimulation, and binding of SHPTP2 to this receptor promotes tyrosine phosphorylation of SHPTP2. The yeast two-hybrid system was modified to identify partners of tyrosine-phosphorylated proteins. Using SHPTP2 as bait and supplying an exogenous tyrosine kinase gene to the yeast cells, we have found that SHPTP2 interacts with another signaling protein, Grb7. We have localized the region of interaction to tyrosine 580 in the carboxyl end of SHPTP2 and to the SH2 domain in the carboxy-terminus of Grb7. We demonstrate that Grb7 binds to SHPTP2 in vitro under conditions where the latter is tyrosine-phosphorylated. These experiments show that this modified two hybrid technique may be useful for the identification of proteins involved in tyrosine kinase signal transduction cascades.

Base Sequence↗
PubMed1996

Movement of cortical actin patches in yeast.

In yeast, actin forms patches associated with the plasma membrane. Patch distribution correlates with polarized growth during the cell cycle and in response to external stimuli. Using green fluorescent protein fused to capping protein to image actin patches in living cells, we find that patches move rapidly and over long distances. Even patches in clusters, such as at the incipient bud site, show movement. Patches move independently of one another and generally over small distances in a local area, but they can also move larger distances, including through the mother-bud neck. Changes in patch polarization occur quickly through the cell cycle. These observations provide important new parameters for a molecular analysis of the regulation and function of actin.

Actins↗