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P Adamson

Publications and source records attributed to P Adamson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Use of PCR in endophthalmitis.

In eyes with suspected endophthalmitis, early diagnosis and appropriate treatment have been noted to be associated with a better visual outcome. Currently, however, confirmation of the diagnosis of endophthalmitis (bacterial and/or fungal) is dependent on conventional techniques of microbiological isolation of organisms which require between one and twelve days. Furthermore, many samples prove to be culture-negative. In order to improve the rate of microbiological diagnosis, PCR technology has been successfully applied to the detection of bacteria and fungi in ocular samples. Specific oligonucleotide primers have been used to detect the presence of pathogens, which have been subsequently identified using RFLP analysis, DNA sequencing, and/or cloning techniques. Results demonstrated that PCR-based methods are rapidly able to confirm the presence of pathogens with high specificity and sensitivity. PCR-based techniques have also been used to rule out with confidence the presence of pathogens, a unique advantage of this methodology. The use of molecular methods has significantly increased the number of intraocular samples from which a confirmed diagnosis is made and reduced the time to laboratory diagnosis. PCR-based methods promise to be useful diagnostic tools in the management of these patients, especially those from whom ocular samples prove to be culture-negative.

Aged↗
PubMed1999

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.

ADP Ribose Transferases↗
PubMed1999

Cytokine production and mRNA expression by conjunctival T-cell lines in chronic allergic eye disease.

BACKGROUND: Activated CD4+ T cells, mast cells and eosinophils are the main cytokine-producing cell-types infiltrating the conjunctiva during chronic allergic eye diseases. Interactions between these cells are thought to play an important immunopathogenic role in these disorders (giant papillary conjunctivitis; vernal keratoconjunctivitis; atopic keratoconjunctivitis). OBJECTIVE: The objective was to compare the cytokine profiles of conjunctival T-cell lines from patients with different forms of chronic allergic eye disease. METHODS: T cells were isolated from conjunctival biopsies and non-specifically expanded into lines. The lines were immunophenotyped by flow cytometry. Cytokine production was quantified by immunoassays and more sensitive molecular techniques were used to investigate cytokine mRNA expression to identify the presence of interleukin (IL) -2, IL-4 and interferon (IFN) -gamma transcripts. RESULTS: Following four to six rounds of stimulation, the conjunctival T-cell populations were CD3+ (> 93%), with variable levels of CD4 and CD8 expression. All were HLA-DR+ (> 80%) with some HLA-DQ expression. Conjunctival T-cell lines from atopic keratoconjunctivitis produced selective increases in IFN-gamma, IL-10 and IL-13 (P<0.01), those from vernal keratoconjunctivitis produced increased IL-5 (P<0.01) whereas T-cell lines from giant papillary conjunctivitis produced only low levels of cytokines. IL-4 was only detected at the mRNA level and was expressed in four out of five T-cell lines in the vernal keratoconjunctivitis group. In contrast there was moderate to strong expression of IFN-gamma in five out of six T-cell lines in atopic keratoconjunctivitis. CONCLUSION: Different patterns of T-cell cytokine profiles were observed for each disease, with low-level, non-polarized cytokine production in giant papillary conjunctivitis, a TH2-like profile in vernal keratoconjunctivitis and a shift towards a TH1-like profile in atopic keratoconjunctivitis.

Adult↗
PubMed1999

Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion.

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.

DNA, Bacterial↗
PubMed1999

Dexamethasone regulation of P-glycoprotein activity in an immortalized rat brain endothelial cell line, GPNT.

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

ICAM-1 signaling pathways associated with Rho activation in microvascular brain endothelial cells.

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls leukocyte adhesion and trafficking to the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we report here that ICAM-1 cross-linking induces tyrosine phosphorylation of three cytoskeleton-associated proteins: focal adhesion kinase, paxillin, and p130Cas (Cas), which are found to associate as complexes. Tyrosine-phosphorylated Cas associates with the adaptor protein Crk and the GTP exchange factor C3G. In the same conditions the small G protein Rho was activated, as shown by the increase in its GTP loading. In addition, tyrosine phosphorylation of focal adhesion kinase, paxillin, and Cas as well as triggering of the Crk signaling pathway are blocked by pretreatment of the cells with the exoenzyme C3, a specific Rho inhibitor. C3-sensitive activation of the c-Jun N-terminal kinase in response to ICAM-1 cross-linking is also observed, whereas no significant activation of Ras or of the extracellular signal-regulated kinase was detected. In conclusion, these results suggest that through coupling to Rho activation and phosphorylation of cytoskeletal proteins and transcription factors, ICAM-1 cross-linking participates in the cell shape changes and gene regulation that may accompany lymphocyte migration through the blood-brain barrier.

Animals↗
PubMed1998

Expression of G-protein subtypes in cultured cerebral endothelial cells.

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Animals↗
PubMed1998

Polymerase chain reaction and restriction fragment length polymorphism mediated detection and speciation of Candida spp causing intraocular infection.

PURPOSE: To determine the usefulness of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in the identification and speciation of Candida spp that causes ocular infection. METHODS: Oligonucleotide primers based on the cytochrome P450 L1 A1 demethylase gene were used to successfully amplify by PCR a single 1.0-kb and a single 500-bp DNA fragment from C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. pelliculosa genomic DNA. RFLPs within the PCR product were identified after restriction enzyme digestion. RESULTS: The sensitivity of the amplification reaction after two rounds of PCR was 10 fg genomic C. albicans DNA or one copy of the gene. No amplification product was obtained when DNA from C. guilliermondii, Aspergillus fumigatus, Fusarium solani, human leukocytes, or 10 species of bacteria was used as a template. Experiments with spiked normal vitreous demonstrated equal sensitivity as long as the volume of vitreous did not exceed 20% of the total PCR volume. RFLP analysis of the PCR product generated from each species obtained from the first- and second-round amplification products enabled species identification after digestion with specific endonucleases. Application of the technique to four clinical samples was successful. CONCLUSIONS: It is expected that the simplicity of the DNA extraction technique allied with the broad specificity of the outer primers for all ophthalmically relevant Candida spp and the sensitivity of the second-round PCR will aid in the detection of fungal DNA in small intraocular samples. PCR-RFLP analysis has great potential in the rapid detection and identification of Candida spp and in the provision of a useful laboratory tool for the future.

Aqueous Humor↗
PubMed1997

Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent and -independent pathways.

Endothelin-1 (ET-1) has been shown to induce DNA synthesis in primary astrocytes by stimulating the extracellular signal-regulated kinase (ERK) pathway. To clarify the mechanisms responsible for the anchorage-dependent growth of astrocytes, the relationships between cell adhesion and ERK activation were investigated. Here it is reported that ET-1 promotes the formation of stress fibers and focal adhesions and the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, as well as Src activation and association of phosphorylated FAK with Grb2. Pretreatment of astrocytes with cytochalasin D or C3-transferase, which inhibits actin polymerization or Rho activity, respectively, prevented the activation/phosphorylation of Src, FAK, and paxillin after ET-1 stimulation; by contrast, the ERK pathway was not significantly affected. This differential activation of FAK/Src and ERK pathways was also observed with astrocytes 10 and 60 min after replating on poly-L-ornithine-precoated dishes. Collectively, these findings indicate that activation of FAK and Src is dependent on actin cytoskeleton integrity, Rho activation, and adhesion to extracellular matrix, whereas ERK activation is independent of these intracellular events and seems to correlate with activation of the newly identified protein tyrosine kinase PYK2. Induction of DNA synthesis by ET-1, however, was reduced dramatically in astrocytes pretreated with either cytochalasin D or C3-transferase. This study provides a demonstration of Rho- and adhesion-dependent activation of FAK/Src, which collaborates with adhesion-independent activation of PYK2/ERK for DNA synthesis in ET-1-stimulated astrocytes.

Actins↗
PubMed1997

Paroxetine increases heart rate variability in panic disorder.

Panic patients have decreased heart rate variability, a risk factor for sudden cardiac death, and increased rates of cardiac death and stroke. Imipramine has been found to further reduce heart rate variability in panic. This study uses power spectral analysis to compare autonomic components of heart rate variability in 16 unmedicated control subjects and 17 panic patients before and after treatment with paroxetine at 20 mg/day for 4 weeks. Patients had higher predrug reclining and standing sympathetic activity than control subjects. After drug, patients' total sympathetic activity decreased. Predrug patients failed to increase sympathetic activity on orthostasis, lacking the normal baroreflex response found in control subjects. After drug, patients normalized this sympathetic component of the baroreflex response. Before drug, patients' parasympathetic reclining and standing activity did not differ from control subjects, and patients showed the normal orthostatic parasympathetic decrease. After drug, patients' total parasympathetic activity increased, whereas the baroreflex response was preserved. Nine medicated patients had more than a 50% reduction of panic attacks. In view of paroxetine's increase of heart rate variability, potential benefits of selective serotonin reuptake inhibitors in decreasing cardiac mortality in panic disorder are discussed.

Adult↗
PubMed1996

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Animals↗
PubMed1996

Protein tyrosine kinase inhibitors act downstream of IL-1 alpha and LPS stimulated MAP-kinase phosphorylation to inhibit expression of E-selectin on human umbilical vein endothelial cells.

HUVEC exposed to IL-1 alpha, TNF alpha or LPS showed a time dependent increase in E-selectin expression which was maximal at between 4-6h after stimulation. Stimulation of HUVEC with IL-1 alpha, TNF alpha or LPS for between 2 and 6h followed by removal or neutralisation of IL-1 alpha, TNF alpha or LPS and incubation in new media up to 6h resulted in identical levels of E-selectin expression at 6h, as cells which had been continuously stimulated for 6h with IL-1 alpha, TNF alpha or LPS. These studies demonstrated that HUVEC were committed to the induction of E-selectin following a 2 hr incubation with either IL-1 alpha, TNF alpha or LPS. The protein tyrosine kinase (PTK) inhibitors ST271, ST638 or genistein (0-100M) were ineffective in reducing cytokine or LPS stimulated E-selectin expression during a 2h cytokine or LPS stimulation of cells, in which inhibitors were either coincubated with cytokine/LPS or previously preincubated with the PTK inhibitors. However when PTK inhibitors were present during both agonist activation (2h) and subsequent expression of E-selectin after removal of agonist (4h) the PTK inhibitors resulted in a dose dependent reduction in both IL-1 alpha and LPS stimulated E-selectin expression (IC50 = 50M). Moreover when PTK inhibitors were only incubated with cells for the 4h after cytokine or LPS activation of cells, PTK inhibitors resulted in a more effective dose dependent reduction in IL-1 alpha or LPS stimulated E-selectin expression (IC50 = 10M). Determination of total and surface expressed E-selectin showed that the reduction in E-selectin expression represented a reduction in E-selectin protein and analysis of E-selectin mRNA by RT-PCR demonstrated that inhibition of E-selectin protein synthesis reflected reduced E-selectin mRNA. Other cytokine or LPS signalling pathways such as the activation of MAP-kinase (ERK-2) was unaffected by pre and coincubation with the PTK inhibitors. These studies suggest that HUVEC can become committed to the induction of E-selectin after removal of the stimulus and that protein tyrosine kinase inhibitors do not effect initial (5-30 min) cytokine or LPS signals which result in E-selectin expression but can inhibit the expression of cytokine/LPS induced E-selectin expression at a step distal to MAP-kinase activation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Ultrastructural localization of ras-related proteins using epitope-tagged plasmids.

To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy. For ultrastructural localization of these tagged proteins, a method was devised to process microinjected cells in situ into low-temperature resin. The proteins were localized on the sections using 9E10 detected with colloidal gold conjugates. Ha-ras protein was localized almost exclusively on the cell membranes. Rho-A and rho-C were predominantly associated with the submembraneous actin network, and rho-B was found in association with multivesicular bodies. Rac1 protein induces the formation of large pinocytotic vesicles and was detected on the cytoplasmic face of these vacuoles. These experiments demonstrate the successful use of this approach for detection of de novo synthesized proteins from microinjected plasmids by both light and electron microscopy on a small (< 50 cells) sample size.

Animals↗
PubMed1993

Cellular responses regulated by rho-related small GTP-binding proteins.

Rho-related proteins are members of the ras superfamily of small GTP-binding proteins. Their function in fibroblasts has been analysed using microinjection of living cells. Rho appears to link plasma membrane receptors to the assembly of focal adhesions and actin stress fibres. The closely related protein rac, on the other hand, links receptors to the polymerization of actin at the plasma membrane to form membrane ruffles and pinocytotic vesicles. In phagocytic cells, rac has been shown to be required for activation of a membrane-bound NADPH oxidase in response to receptor activation. These systems provide the basis for a working model for the mechanism of action of the rho family of small GTPases.

Animals↗
PubMed1992

Post-translational modifications of p21rho proteins.

Post-translational modifications of the ras proteins, which are required for plasma membrane localization and biological function of the proteins, have been shown to include prenylation and carboxymethylation at the carboxyl terminal cysteine residue of the cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) box. In addition, p21Ha-ras and p21N-ras, but not p21K-ras (B), are palmitoylated. The three mammalian rho proteins (A, B, and C) are also members of the ras superfamily but have distinct biological activities and different intracellular distributions from p21ras. Analysis showed all three rho proteins are modified by a COOH-terminal carboxymethylation similar to p21ras, whereas p21rhoC labeled with [3H]mevalonic acid in vivo revealed the presence of a C20 prenoid, similar to that already described for p21rhoA. However, in vivo and in vitro studies of p21rhoB showed this protein to be modified by both C15 and C20 prenoids. Mutation of C193 in the CAAX box abolished prenylation, whereas mutation of the adjacent C192 resulted in a significant reduction in the amount of the C20, but not C15 prenoid, recovered from p21rhoB. In vivo labeling studies with [3H]palmitic acid and mutational analysis showed that both cysteine residues at 189 and 192 upstream of the CAAX box in p21rhoB are sites for palmitoylation. We conclude that there are different populations of post-translationally modified p21rhoB in the cell and that the sequence specificity for geranylgeranyl- and farnesyltransferases may be more complicated than previously proposed.

Amino Acid Sequence↗
PubMed1992

Intracellular localization of the P21rho proteins.

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

Amino Acid Sequence↗