Biomedical subjects

J A Augustine

Publications and source records attributed to J A Augustine.

11 recordsLinked to original sources

PubMed1999

Inhibition of human umbilical vein endothelial cell proliferation by the CXC chemokine, platelet factor 4 (PF4), is associated with impaired downregulation of p21(Cip1/WAF1).

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E-cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21(Cip1/WAF1), to the cyclin E-cdk2 complex. Analysis of total cellular p21(Cip1/WAF1) showed that in the presence of PF4, p21(Cip1/WAF1) levels were sustained at time points when p21(Cip1/WAF1) was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21(Cip1/WAF1) and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

CDC2-CDC28 Kinases↗

PubMed1998

Molecular mechanism of basic calcium phosphate crystal-induced activation of human fibroblasts. Role of nuclear factor kappab, activator protein 1, and protein kinase c.

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor kappaB and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts. In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications.

3T3 Cells↗

PubMed1998

The p85 and p110 subunits of phosphatidylinositol 3-kinase-alpha are substrates, in vitro, for a constitutively associated protein tyrosine kinase in platelets.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn(2+)-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn(2+)-dependent protein kinase activity of PI3K alpha immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3K alpha showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85 alpha or p110 alpha derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85 alpha and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3K alpha can autophosphorylate on serine and threonine, and both p85 alpha and p110 alpha are substrates for a constitutively-associated protein tyrosine kinase in platelets.

Androstadienes↗

PubMed1992

Penetrating intracranial trauma from a fishhook.

Fishhook injuries rarely pose a true emergency, and only a few cases of posterior ocular injury from fishhooks have been described. We present a case of penetrating ocular, orbital, and cranial trauma produced by a broken fishhook. The morbidity and mortality as well as the initial emergency evaluation of penetrating foreign objects in the orbital-cranial region are discussed.

Brain Injuries↗

PubMed1991

Signal transduction through the T cell antigen receptor. Activation of phospholipase C through a G protein-independent coupling mechanism.

The T cell Ag (Ti-CD3) receptor complex has been proposed to regulate phosphoinositide-specific phospholipase C (PLC) through a cholera toxin (CTX)-sensitive guanine nucleotide-binding (G) protein. In this study, we have used CTX and staurosporine as pharmacologic probes to further define the linkage between the Ti-CD3 receptor and PLC activity in the human T cell line, Jurkat. CTX pretreatment inhibited Ti-CD3 receptor-dependent phosphoinositide hydrolysis and, concomitantly, protein tyrosine kinase activation in intact cells. Studies with electrically permeabilized Jurkat cells revealed that guanosine 5'-(3-O-thio) triphosphate stimulated an increase in PLC activity, that unlike the response to Ti-CD3 receptor ligation, was not affected by cellular pretreatment with CTX. In contrast, the phosphotyrosine phosphatase inhibitors, orthovanadate and molybdate anions, stimulated phosphoinositide hydrolysis in permeabilized cells through a CTX-sensitive mechanism of PLC activation. Additional studies with a known PTK inhibitor, staurosporine, supported the results obtained with CTX. Staurosporine pretreatment inhibited the phosphoinositide hydrolysis induced by anti-CD3 antibodies or phosphotyrosine phosphatase inhibitors, but failed to alter the G protein-dependent PLC activation response to guanosine 5'-(3-O-thio) triphosphate. The results of this study indicate that PLC activity(s) in Jurkat cells are regulated by both G protein- and PTK-dependent coupling mechanisms. However, the differential inhibitory effects of CTX and staurosporine on these PLC activation pathways strongly suggest that a protein tyrosine kinase activation event, rather than a G protein, mediates the functional linkage between the Ti-CD3 receptor and PLC activity in Jurkat cells.

Alkaloids↗

PubMed1991

Interleukin 2- and polyomavirus middle T antigen-induced modification of phosphatidylinositol 3-kinase activity in activated T lymphocytes.

Stimulation of activated T lymphocytes with interleukin 2 (IL-2) results in rapid increases in intracellular protein tyrosine phosphorylation. Both the identity of the protein tyrosine kinase (PTK) activated by IL-2 receptor ligation and the identities of the critical target proteins for this PTK remain largely undefined. In this article, we demonstrate that stimulation of activated murine or human T cells with IL-2 for 10 to 30 min induces two- to threefold increases in the level of phosphatidylinositol (PtdIns) 3-kinase activity present in antiphosphotyrosine (p-Tyr) antibody immunoprecipitates from these cells. Furthermore, substantial levels of PtdIns 3-kinase activity were coprecipitated from IL-2-deprived T cells by antibodies to the src-related PTK p59fyn. Cellular stimulation with IL-2 induced a two- to threefold increase in the level of p59fyn-associated PtdIns 3-kinase activity. To examine the effect of a constitutive increase in PtdIns 3-kinase activity on the growth factor responsiveness of activated T cells, murine CTLL-2 cells were transfected with a polyomavirus middle T antigen (MTAg) expression vector. Anti-p-Tyr and anti-p59fyn immunoprecipitates from MTAg-transfected CTLL-2 cells contained three- to sixfold higher levels of PtdIns 3-kinase activity than wild-type cells. Immune complex kinase assays revealed that MTAg expression concomitantly induced a constitutive threefold increase in the PTK activity of p59fyn in these cells. However, stable MTAg expression did not abrogate the dependence of CTLL-2 cells on exogenous IL-2 for continued growth and proliferation.

1-Phosphatidylinositol 4-Kinase↗

PubMed1990

Differential effects of interleukin-2 and interleukin-4 on protein tyrosine phosphorylation in factor-dependent murine T cells.

Interleukin-2 (IL-2) is a requisite factor for growth and proliferation of IL-2-dependent T cells. At present, the mechanism by which the high-affinity IL-2-IL-2 receptor interaction transmits a mitogenic signal to the cellular interior remains unclear. In this report we have used three murine T cell clones to demonstrate that IL-2 stimulates rapid tyrosine phosphorylation of several proteins. Two of these clones, CTLL-2 and CT6, exhibit a cytotoxic T cell phenotype, while the third, HT-2, was derived from a helper T cell line. All three T cell clones proliferated in response to IL-2 stimulation, but HT-2 cells also proliferated in response to interleukin-4 (IL-4). We comparatively examined the effects of IL-2 and IL-4 on protein tyrosine phosphorylation in these cells by immunoaffinity purification of phosphotyrosyl substrates with an anti-phosphotyrosine monoclonal antibody. Stimulation with concentrations of IL-2 resulting in maximal (10-30 U/ml) or sub-maximal (1-5 U/ml) proliferation caused the rapid tyrosine phosphorylation of 97 and 57 kDa proteins in all three cell lines. The 97 kDa protein was localized in the cytosol, while the 57 kDa protein was detected in both cytosolic and crude membrane fractions. IL-2-dependent tyrosine phosphorylation of an 86 kDa cytosolic protein was observed only in CT6 cells. Tyrosine phosphorylation of 22, 23 and 200 kDa proteins was also observed, but only in the cytotoxic T cell clones. Phosphoamino acid analyses revealed that the 97, 86 and 57 kDa proteins contained phosphotyrosine and phosphoserine residues. Concentrations of IL-2 below the threshold concentration for induction of a proliferative response correspondingly failed to stimulate protein tyrosine phosphorylation. In contrast, growth stimulation of HT-2 cells by IL-4 was not preceded by early changes in protein tyrosine phosphorylation, suggesting that protein tyrosine phosphorylation may not be essential for the induction of IL-4-dependent cell-cycle progression. These results demonstrate that high-affinity IL-2 receptors are coupled to tyrosine kinase activity(s) in T cells. However, the failure of IL-4 to stimulate protein tyrosine phosphorylation in the same cells indicates that enhanced protein tyrosine phosphorylation may not be requisite for growth factor-dependent T cell proliferation.

Amino Acids↗

PubMed1990

Regulatory effects of transforming growth factor-beta on IL-2- and IL-4-dependent T cell-cycle progression.

Previous studies suggested that the potent immunosuppressive activities of transforming growth factor-beta (TGF-beta) were mediated in part through the inhibition of IL-2-dependent S-phase progression and mitosis of activated T cells. To further investigate the mechanism of T cell growth inhibition by TGF-beta, two constitutively activated murine T cell clones were employed as defined model systems for the growth factor-dependent phase of T cell proliferation. The Th cell line, HT-2, proliferated in response to either IL-2 or IL-4, whereas the cytotoxic T cell line, CT6, exhibited strict dependence on IL-2 for growth stimulation. In both cell lines, picomolar concentrations of TGF-beta inhibited S-phase progression stimulated by IL-2 or IL-4. TGF-beta pretreatment decreased the expression of high affinity IL-2R on HT-2 cells, but not on CT6 cells. In contrast, IL-2-stimulated transferrin receptor expression was markedly inhibited by TGF-beta in both T cell lines. Analyses of growth factor-dependent specific mRNA accumulation revealed that TGF-beta exerted selective inhibitory effects on gene expression in HT-2 and CT6 cells. TGF-beta significantly reduced early (1 to 2 h) increases in c-myc mRNA levels stimulated by IL-2 or IL-4 in both cell lines. In HT-2 cells, TGF-beta pretreatment also inhibited the early increase in granulocyte-macrophage CSF mRNA stimulated by IL-2 or IL-4. The inhibition of c-myc and granulocyte-macrophage cyte-macrophage CSF gene expression by TGF-beta was explained, at least in part, by suppression of the growth factor-dependent transcriptional activation of these genes. These studies suggest that inhibition of c-myc gene transcription may play a fundamental role in the antiproliferative effect of TGF-beta on IL-2- or IL-4-stimulated T cells.

Blotting, Northern↗

PubMed1989

A comparison of the effects of cyclosporine (CsA) on hepatic microsomal drug metabolism in three different strains of rat.

1. Male albino Wistar (W), Sprague-Dawley (SD) and Fischer-344 (F344) rats, Rattus norvegicus, were dosed orally once daily with cyclosporine (CsA), 25 mg/kg/day, for 14 days. Control and treated rats were then sacrificed and selected assays were performed to measure differences in hepatic microsomal enzyme activity including: protein (PROT), cytochrome P-450 (CYT-P450) and cytochrome b5 (CYT-b5) levels, NADPH-cytochrome c reductase (CYT-c) activity, ethylmorphine N-demethylase (ETM) and aniline hydroxylase (ANL) activities. 2. Two-way analysis of variance (ANOVA) indicated that the effect of treatment alone showed significant strain-related differences in all parameters examined except CYT-b5 levels, while the effect of strain alone indicated significant differences in PROT, CYT-B5, CYT-c, and ANL. 3. The interaction component of the two-way ANOVA (treatment x strain) indicated strain-related differences in substrate metabolism only (ETM and ANL). 4. F344 rats were found to be significantly different from either Sprague-Dawley or Wistar rats in terms of the effect of CsA on ETM and ANL metabolism.

Animals↗

PubMed1987

Ventilation performance using a self-inflating anesthesia bag: effect of operator characteristics.

Adequate ventilation is required for successful cardiopulmonary resuscitation (CPR). Operator characteristics that influence ventilation performance are not well defined. This study compared ventilation performance and operator characteristics in 74 medical personnel using a self-inflating anesthesia bag. Ventilation device, operator hand size, ventilation technique, average tidal volume, cumulative minute ventilation, and ventilation pressures were recorded during 3 minutes of ventilation. Ventilation volumes and airway pressures were not correlated with hand size or device type. Techniques that used one hand to squeeze the bag resulted in significantly lower average tidal volume than two-handed techniques, with no significant difference in peak or average airway pressure. There was no difference between emergency department and prehospital personnel in average tidal volume delivered. However, prehospital personnel ventilated at significantly higher airway pressures. Emergency department nurses delivered the greatest average tidal volume (923 cc), while emergency department physicians delivered the least (775 cc). Paramedics recorded the highest airway pressures (average, 53 cm H2O; peak, 72 cm H2O), while respiratory therapists recorded the lowest pressures (average, 34 cm H2O; peak, 54 cm H2O). Ventilation during CPR is a complex, learned skill. Large variation exists among different operators. However, appropriate tidal volumes can be delivered using safe airway pressures. Ongoing assessment and retraining of individuals performing ventilation during CPR are essential.

Adult↗

PubMedDate not supplied

The effects of cyclosporin A (CsA) on hepatic microsomal drug metabolism in the rat.

The effects of cyclosporin A (CsA), a powerful immunosuppressant, on the hepatic microsomal mixed function oxidase (MFO) system was studied in male rats. Difference spectroscopy studies indicated that CsA binds to cytochrome P-450 producing a type I spectral change. To investigate potential interactions with the MFO system, CsA was administered orally at doses of either 25 mg/kg or 50 mg/kg once daily for 9 days. Various metabolic parameters were examined, including: levels of microsomal protein, cytochrome P-450, and cytochrome b5, NADPH-cytochrome c reductase activity, N-demethylation of ethylmorphine (ETM), and p-hydroxylation of aniline (ANL). Rats treated with 50 mg/kg showed a 25% or greater decrease over controls in all parameters examined except microsomal protein and cytochrome b5 levels. Rats treated with 25 mg/kg showed a 28% or greater decrease in all parameters except microsomal protein, cytochrome b5, and cytochrome P-450. Kinetic studies of ETM-N-demethylase and ANL-hydroxylase activities were conducted either with microsomes prepared from CsA-treated animals (50 mg/kg/day for 5 days) or with pooled microsomes prepared from untreated animals to which CsA was added directly. Enzyme reaction velocities were measured and apparent KM and apparent Vmax were determined. Studies with CsA-treated animals revealed a 57% decrease in both KM and Vmax for ETM-N-demethylase, and a 46% decrease in KM and a 32% decrease in Vmax for ANL-hydroxylase. Studies involving direct addition of CsA to microsomes at final concentrations of 0.01 mM and 0.10 mM revealed no significant changes in apparent KM or Vmax for either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Hydroxylase↗