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J A Cooper

Publications and source records attributed to J A Cooper.

At least 55 records · Page 3Linked to original sources
PubMed2000

Mapping of the mouse actin capping protein beta subunit gene.

BACKGROUND: 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 isoforms of CPbeta produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. RESULTS: We isolated genomic clones corresponding to the beta subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. CONCLUSIONS: The CPbeta gene (Cappb1) mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the beta gene.

Journal Article↗
PubMed2000

TOR signaling regulates microtubule structure and function.

The functional diversity and structural heterogeneity of microtubules are largely determined by microtubule-associated proteins (MAPs) [1] [2]. Bik1p (bilateral karyogamy defect protein) is one of the MAPs required for microtubule assembly, stability and function in cell processes such as karyogamy and nuclear migration and positioning in the yeast Saccharomyces cerevisiae [3]. The macrocyclic immunosuppressive antibiotic rapamycin, complexed with its binding protein FKBP12, binds to and inhibits the target of rapamycin protein (TOR) in yeast [4] [5]. We report here that TOR physically interacts with Bik1p, the yeast homolog of human CLIP-170/Restin [6] [7]. Inhibition of TOR by rapamycin significantly affects microtubule assembly, elongation and stability. This function of TOR is independent of new protein synthesis. Rapamycin also causes defects in spindle orientation, nuclear movement and positioning, karyogamy and chromosomal stability, defects also found in the bikDelta mutant. Our data suggest a role for TOR signaling in regulating microtubule stability and function, possibly through Bik1p.

Fungal Proteins↗
PubMed2000

Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae.

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.

Actins↗
PubMed2000

The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton.

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin beta family, ASAP1 (also known as centaurin beta4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

3T3 Cells↗
PubMed2000

Attenuation of interleukin-8 production by inhibiting nuclear factor-kappaB translocation using decoy oligonucleotides.

Interleukin-8 (IL-8), a monocyte-derived neutrophil chemoattractant factor, is a polymorphonuclear neutrophil chemotaxin that is involved in a number of inflammatory disorders. Transcription of the IL-8 gene is controlled by regulatory proteins, including nuclear factor-kappaB (NF-kappaB), a family of proteins that is important in the transcriptional control of a number of genes. When cells are activated, NF-kappaB translocates from the cytoplasm to the nucleus, where it activates transcription by binding to a specific sequence within the 5' untranslated region of the gene. During translocation, NF-kappaB is potentially susceptible to diversion by oligonucleotides that contain the binding sequence for this protein. In the current study, we produced phosphorothioate-modified oligonucleotides containing the specific DNA sequence that NF-kappaB binds within the IL-8 gene. We then investigated the effects of transfection of monocytes with these oligonucleotides on interleukin-1beta (IL-1beta)-stimulated IL-8 production, IL-8 mRNA expression, and NF-kappaB binding activity. We found that transfection with these oligonucleotides significantly inhibited monocyte IL-8 production. A single-stranded oligonucleotide with two copies of the NF-kappaB-binding sequence was the most potent of those tested. This single-stranded oligonucleotide also inhibited IL-1beta-induced translocation of NF-kappaB to the nucleus and reduced IL-8 mRNA expression. These studies demonstrated that monocyte production of IL-8 can be attenuated using a single-stranded oligonucleotide that binds a transcriptional activating protein before it translocates to the cell nucleus. This approach ultimately may be useful in the control of inflammation involved in a number of diseases.

Binding, Competitive↗
PubMed2000

Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro.

The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization.

Actin Depolymerizing Factors↗
PubMed2000

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury.

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.

Adenoviridae↗
PubMed2000

Control of actin assembly and disassembly at filament ends.

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.

Actin Cytoskeleton↗
PubMed2000

Actin assembly at membranes controlled by ARF6.

The small GTPase, ADP-ribosylation factor-6 (ARF6), has been implicated in regulating membrane traffic and remodeling cortical F-actin. Using real-time video analysis of actin assembly in living cells, we investigated the function and mechanism of ARF6 in control of actin assembly. Expression of an activated form of ARF6 that mimicks the GTP-bound form of the GTPase induced actin assembly resulting in the movement of vesicle-like particles, some of which contain markers for pinosomes. Activated ARF6 also stimulated actin assembly at foci on the ventral surface of the cell and stimulated fluid phase pinocytosis. Particle motility induced by ARF6 involved Arp2/3 complex, tyrosine kinase activity, phospholipase D (PLD) and D3-phosphoinositides, but not phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We conclude that ARF6 regulates actin assembly for pinosome motility and at foci on the ventral cell surface.

ADP-Ribosylation Factor 6↗
PubMed2000

The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling.

The actin cytoskeleton seems to play two critical roles in the activation of T cells. One of these roles is T cell shape development and movement, including formation of the immunological synapse. The other is the formation of a scaffold for signaling components. This review focuses on the recent convergence of cell biology and immunology studies to explain the role of the actin cytoskeleton in creating the molecular basis for immunological synapse formation and T cell signaling.

Actins↗
PubMed2000

Evaluation of a needle-free injection system for local anaesthesia prior to venous cannulation.

We evaluated a single-use, disposable, carbon-dioxide-powered, needleless injector (J-Tip, National Medical Products Inc., CA, USA), which is claimed to deliver a virtually painless, subcutaneous injection. Seventy-two patients undergoing various types of surgery had a large-bore intravenous cannula inserted prior to induction of general anaesthesia. Three minutes beforehand, a subcutaneous injection of 0.3 ml of 1% plain lidocaine was administered. Subjects were randomly allocated to receive the lidocaine either by the needleless injector or from a conventional syringe and a 25 G needle. Pain scores were recorded on injection of the lidocaine and on insertion of the cannula. There was significantly less pain on injection with the needleless injector than with the 25 G needle (p < 0.001) but, surprisingly, there was more pain on cannulation (p < 0. 001). We conclude that the device certainly delivers a less painful subcutaneous injection than a 25 G needle, but perhaps provides less effective skin anaesthesia for venous cannulation at sites where the subcutaneous space is small; its use might be better suited to areas where the subcutaneous space is deeper.

Anesthesia, Local↗
PubMed2000

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin.

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.

Actins↗
PubMed2000

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae.

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

Actin Cytoskeleton↗
PubMed2000

Autonomic dysfunction is related to impaired pancreatic beta cell function in patients with coronary artery disease.

OBJECTIVE: To assess the role of beta cell failure in the development of autonomic dysfunction in patients with coronary artery disease. DESIGN: Autonomic function was measured by standard clinical methods and by heart rate variability in 24 type II diabetic and 24 non-diabetic subjects with coronary artery disease. Quantitative estimates of pancreatic beta cell function (%beta) and insulin resistance were made from basal plasma glucose and insulin concentrations using a computer solved model. Fasting proinsulin levels provided an independent measure of beta cell function. RESULTS: The circadian rhythm of sympathovagal balance (ratio of low to high frequency spectral components of heart rate variability) was significantly attenuated in patients with below median (%beta </= 63. 3) compared with above median pancreatic beta cell function. Multiple measures of autonomic function showed positive associations with %beta (low frequency: r = 0.41, p = 0.004; high frequency: r = 0.27, p = 0.07; lying/standing fall in systolic pressure: r = -0.30, p = 0.04) and negative associations with proinsulin (low frequency: r = -0.35, p = 0.03; high frequency: r = -0.36, p = 0.02; standard deviation of RR intervals: r = -0.43, p = 0.007). Associations tended to be stronger in non-diabetic than diabetic patients. In contrast, there was no association between insulin resistance and either autonomic function or the circadian rhythm of sympathovagal balance, regardless of diabetes status. CONCLUSIONS: The data are consistent with the hypothesis that beta cell failure plays a pathogenic role in the development of autonomic dysfunction in patients with coronary artery disease.

Adult↗
PubMed2000

Haemostatic and lipid determinants of prothrombin fragment F1.2 and D-dimer in plasma.

The determinants of plasma levels of prothrombin fragment F1.2 (F1.2) and D-dimer in different populations are unclear and this may complicate their interpretation as predictors of thrombotic risk, particularly in the case of D-dimer. We therefore measured F1.2 and D-dimer levels together with a number of other haemostatic and lipid variables in a cross-sectional community-based study of 150 healthy adults (73 male, 77 female), age range 23-80 years, identified from the list of a general practice by stratified random sampling within sex and decade of age. Plasma F1.2 was significantly higher in females than males and was independently and positively associated with age, factor VII activity (FVIIc) and C1 inhibitor, and inversely associated with high density lipoprotein (HDL) cholesterol. Plasma D-dimer showed a quadratic association with age (p <0.0001). In those < or =55 years D-dimer was inversely associated with dilute clot lysis time (DCLT) and activated protein C (APC) ratio. In those >55 years it was significantly higher in females than males and associated positively with age, fibrinogen and, in males, activated factor XII (FXIIa). In a multiple-linear model which combined both age groups, F1.2 and D-dimer were independently associated with each other (r = 0.22, p = 0.03). Thus, thrombin generation and fibrin turnover/fibrinolysis are associated in healthy subjects. HDL cholesterol (inversely) and FVIIc are associated with basal thrombin generation (i.e. F1.2). Determinants of D-dimer differ according to age and interpretation of the biological significance of D-dimer levels in epidemiological studies may therefore not be straightforward.

Adult↗
PubMed2000

Prothrombin activation is increased among asymptomatic carriers of the prothrombin G20210A and factor V Arg506Gln mutations.

The risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50-61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G202 IOA and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment FI+2 [mean +/- SD, 0.88 +/- 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 +/- 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 +/- 0.24 among non-carriers for either mutationl. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.

Blood Coagulation Factors↗