PubMed Health⌕ Search

Biomedical subjects

T L Murphy

Publications and source records attributed to T L Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism.

Recently, the transcription factor GATA-3 was shown to be selectively expressed in Th2 but not Th1 cells and to augment Th2-specific cytokines. Here, we show that loss of GATA-3 expression by developing Th1 cells requires IL-12 signaling through Stat4 and does not simply result from an absence of IL-4. Moreover, we demonstrate a novel role for GATA-3 in directly repressing Th1 development distinct from its positive actions on Th2-specific cytokines. GATA-3 inhibits Th1 cytokines by a cell-intrinsic mechanism that is not dependent on IL-4 and that may involve repression of IL-12 signaling. Thus, GATA-3 expression and IL-12 signaling are mutually antagonistic, which facilitates rapid dominance of one pathway during early Th development, producing a stable divergence in cytokine profiles.

Animals↗
PubMed1998

Smoking, obesity, and hypertension alter the dose-response curve and test sensitivity of carbohydrate-deficient transferrin as a marker of alcohol intake.

Serum carbohydrate-deficient transferrin (CDT) is a specific and comparatively sensitive marker of excessive alcohol use; however, reports of its sensitivity vary according to the population or patient groups studied and their average alcohol intake. We have characterized the dose-response curve between alcohol intake and CDT concentrations in a study of 1400 men and women from a community-based twin registry. Our results show that mean CDT increases with increasing reported alcohol consumption even within the range of alcohol use considered to be nonhazardous. We found significant effects of sex, age, smoking, previous alcohol dependence, body mass index, and diastolic hypertension on the alcohol-CDT dose-response curve. These variables either affect test sensitivity or require adjustment of reference intervals. The results also provide insight into the physiological and biochemical factors that affect CDT concentration.

Adult↗
PubMed1996

Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway.

Interleukin 12 (IL-12) strongly augments gamma interferon production by natural killer (NK) and T cells. IL-12 also promotes effective cell-mediated immune responses, which are particularly important against intracellular bacteria such as Listeria monocytogenes. While the lipopolysaccharide (LPS) of gram-negative bacteria induces monocyte production of IL-12, the relevant gram-positive components which induce IL-12 production are uncharacterized. We used the human monocytic cell line THP-1 to study IL-12 induction by gram-positive bacteria. Muramyl dipeptides as well as the major muramyl tetrapeptide component of Streptococcus pneumoniae were inactive for inducing IL-12. In contrast, lipoteichoic acid (LTA), a predominant surface glycolipid of gram-positive bacteria, potently induced IL-12 p40 gene expression. A competitive LPS antagonist, Rhodobacter sphaeroides LPS, inhibited LTA-induced IL-12 production, suggesting a common pathway for LPS and LTA in IL-12 activation. Pretreatment of cells with anti-CD14 monoclonal antibody blocked both LPS and LTA induction of IL-12 p40 expression. LTA also induced Thl development in naive CD4 T cells by an IL-12-dependent mechanism, indicating direct induction of physiologic levels of IL-12. Together, these results show that LTA is a potent surface structure of gram-positive bacteria which induces IL-12 in monocytes through a CD14-mediated pathway.

Antibodies, Monoclonal↗
PubMed1995

Effect of intravitreal administration of indomethacin on experimental subretinal neovascularization in the subhuman primate.

OBJECTIVE: To examine the effect of indomethacin, a cyclooxygenase (CO) inhibitor, on laser-induced subretinal neovascularization (SRN) in the monkey. The CO pathway of arachidonic acid metabolism plays an important role in angiogenesis, and the inhibition of CO is known to inhibit angiogenesis in the cornea and in certain tumors. MATERIALS AND METHODS: A cannula was implanted into the vitreous cavity of 11 eyes of six monkeys and connected to an osmotic minipump. Indomethacin (25 micrograms/d) was delivered into the vitreous through the cannula for 14 days (seven eyes). Vehicle alone was injected for 14 days as a control (four eyes). Argon laser photocoagulation was then performed (eight spots at the posterior pole in each eye) to induce SRN. Fundus photographs and fluorescein angiograms were taken periodically to document the evolution of SRN. Light and electron microscopic examination was performed on two eyes of each group 8 weeks after photocoagulation. RESULTS: Subretinal neovascularization developed from 2 to 4 weeks after photocoagulation. The incidence of SRN, indicated by fluorescein leakage, was significantly lower (P < .05) in the group treated with indomethacin (14.3%, eight of 56 lesions) than in the control group (37.5%, 12 of 32 lesions). After 8 weeks, no fluorescein leakage was found in either the control or indomethacin-treated groups. Scar formation was found on histologic examination in both groups. No histologic evidence of indomethacin toxicity was seen in the adjacent retina. CONCLUSIONS: Intravitreal administration of indomethacin inhibits the formation of laser-induced SRN in monkey eyes. This is consistent with the participation of the CO pathway in the process of SRN formation and suggests that this pathway could be a potential target in the treatment of SRN.

Animals↗
PubMed1995

Vessel formation by choroidal endothelial cells in vitro is modulated by retinal pigment epithelial cells.

OBJECTIVE: To elucidate the mechanism of bovine choroidal endothelial (BCE) cell angiogenesis and, in particular, the role of retinal pigment epithelial (RPE) cells by use of an in vitro coculture assay system. METHODS: The BCE cells were isolated from choroidal tissues and cultured. They were embedded in type I collagen gel and incubated. The gel-embedded BCE cells were then covered with a monolayer of RPE cells, pericytes, choroidal fibroblasts, or additional BCE cells on culture day 0 or day 14. The BCE cells in culture formed a meshwork of tubelike structures. The length of the tubelike structures (micrometers per field) was quantified by image analysis, as an indicator of angiogenesis. The effect of RPE cells on normal and growth-arrested BCE cell tube formation was evaluated in this assay system. The mechanism of RPE-induced angiogenesis was studied by parallel experiments with the use of neutralizing antibodies against specific growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor beta). RESULTS: Ultrastructural analysis revealed that the tubelike structures had features typical of choroidal endothelial cells. Cocultures initiated on day 0 revealed that BCE cell angiogenesis was promoted by overlying RPE cells and, to a lesser extent, by pericytes, choroidal fibroblasts, and additional BCE cells when compared with BCE cells without covering cells. In cocultures initiated after BCE tube formation (day 14), there was inhibition of BCE angiogenesis by overlying RPE cells when compared with cultures without overlay or with an overlay of BCE cells. The RPE cells stimulated tube formation of growth-arrested BCE cells less effectively than did normal BCE cells. Neutralizing antibody for basic fibroblast growth factor and vascular endothelial growth factor, but not transforming growth factor beta, inhibited control and RPE-induced tube formation by BCE cells. CONCLUSIONS: Overlying RPE cells stimulate the formation of tubelike structures by choroidal endothelial cells more effectively than do fibroblasts or pericytes but inhibit BCE tube formation in older cultures. The effect involves endothelial proliferation and differentiation. The stimulatory effect of overlying RPE cells can be inhibited by neutralizing antibodies to vascular endothelial growth factor and basic fibroblast growth factor, which suggests that these growth factors play an important role in this phenomenon.

Animals↗
PubMed1995

Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C.

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions, including subretinal neovascularization (SRN) and proliferative vitreoretinopathy (PVR). Therefore, elucidation of the mechanism of RPE migration may be useful in devising effective treatment for these disorders. Since protein kinase C (PKC) has been shown to regulate the migration of other cell types, we studied the effects of PKC agonists and antagonists on RPE migration. We used an in vitro wound healing model in which a small area of a confluent monolayer of bovine RPE cells was denuded with a razor blade. The cultures were subsequently incubated with agents known to stimulate [phorbol 12-myristate 13-acetate (PMA)] or inhibit (calphostin C, staurosporine) PKC. After 20 hr, migration was measured as the number of cells that had entered the denuded area. We also measured the translocation of PKC from the cytosol to the membrane in order to determine the activation or inhibition of PKC by PMA and calphostin C in the cells. The phorbol ester PMA stimulated migration by 41%, and calphostin C and staurosporine inhibited migration by 38% and 31%, respectively, in a medium supplemented with 10% serum. To determine the requirement for serum in this modulation, we also measured the effects of PMA and calphostin C on RPE migration in serum-free medium. Under these conditions, basal migration was greatly decreased, but PMA stimulated migration by 177% and calphostin C inhibited migration by 93%. Since PKC modulation is known to induce the proliferation of cells, we also tested the effects of these agents on growth-inhibited migration by pretreating the cells with the antiproliferative drug mitomycin C. We found that modulation of PKC under these conditions equally affected growth-inhibited and growth-dependent migration. Therefore, based on the increase in RPE migration induced by a PKC agonist, and the decrease in migration caused by PKC antagonists, it is suggested that PKC-mediated signal transduction plays a crucial role in RPE cell migration. This knowledge may be useful in devising effective treatments for SRN and PVR.

Alkaloids↗
PubMed1995

Regulation of interleukin 12 p40 expression through an NF-kappa B half-site.

Interleukin 12 (IL-12) is an inducible cytokine composed of 35- and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-gamma) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence -122GGGGAATTTTA-132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-kappa B (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-kappa B family members in IL-12 p40 activation. Finally, we find that IFN-gamma treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-gamma augmentation of IL-12 production by macrophages.

Animals↗
PubMed1993

Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: roles for NF-Y and NF-ATc.

Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.

Animals↗
PubMed1993

Characterization of a noncontiguous gene deletion of the growth hormone receptor in Laron's syndrome.

Noncontiguous deletion of the GH receptor (GHR) gene has been described as a molecular defect causing Laron's syndrome (LS). The abnormal allele (GHR Del-3,5,6) lacks exons 3, 5, and 6. Exon 4 is retained on variant-sized restriction fragments. We studied DNA from 10 additional LS subjects of Jewish-oriental origin and found this allele in 3 samples. All subjects with this complex deletion allele were from Iran or Iraq and were not clinically distinguishable from other individuals with LS. Multiple restriction enzyme digests and Southern blotting with GHR exon 4-specific probes demonstrated that the allele retains genomic material for at least 9.8 kilobases 5' and 1.8 kilobases 3' to exon 4. Reverse transcription of mRNA, polymerase chain reaction amplification, and cDNA sequencing showed that exon 4 is syntenic and colinear with the other GHR exons. Normal splice sites were used, but the predicted protein was severely truncated due to a frameshift and a premature stop codon in exon 7. The premature stop codon terminated the protein after amino acid 53 and led to loss of the transmembrane and intracellular portions of the receptor. This mutant GHR gene results clinically in severe GH insensitivity.

Alleles↗
PubMed1993

Distribution of body water in patients with cirrhosis: the effect of liver transplantation.

We compared total body water and intracellular-extracellular distribution of body water between male patients with mild liver disease without ascites (n = 9), male patients with severe liver disease and gross ascites (n = 6) and a group of age-, sex-, height- and weight- matched controls (n = 6). In addition, we documented the effects of liver transplantation on intracellular, extracellular and total body water in 12 patients (6 men and 6 women) by means of deuterium oxide dilution and whole-body potassium counting. We saw no significant difference in total body water between the healthy controls, patients without ascites and patients with ascites (46.5 +/- 9.2 kg, 45.4 +/- 6.6 kg and 50.4 +/- 5.1 kg, respectively), although, as expected, extracellular water was increased in patients with ascites compared with healthy controls and cirrhotic patients without ascites (36.9 +/- 6.5 kg vs. 25.4 +/- 4.4 kg, p = 0.005; and 36.9 +/- 6.5 kg vs. 27.0 +/- 5.3 kg, p = 0.002, respectively). We found no difference between non-ascitic patients and healthy controls (25.4 +/- 4.4 kg vs. 27.0 +/- 5.3 kg). However, intracellular water was significantly reduced in patients with severe liver disease compared with that in controls (13.6 +/- 3.3 kg vs. 21.5 +/- 4.2 kg, p = 0.005) or patients without ascites (13.6 +/- 3.3 kg vs. 18.3 +/- 2.9 kg, p = 0.01). The reduction of intracellular water appears to be due to loss of body cell mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Dose-dependent pharmacokinetics of caffeine in humans: relevance as a test of quantitative liver function.

Caffeine clearance was determined in 13 healthy control subjects and in 13 patients with histologically proven cirrhosis. On separate occasions, 70 mg, 200 mg, and 300 mg single doses of anhydrous caffeine were administered orally with decaffeinated coffee to each subject. Subjects were analyzed individually, acting as their own controls, thus reducing interindividual variability. The present study showed that caffeine exhibited dose-dependent pharmacokinetics, particularly in subjects who showed high initial clearance with the low dose (70 mg) of caffeine. There was a significant decrease in caffeine clearance with increasing dose from 70 mg to 300 mg (n = 26, p less than 0.01, Dunnett's test), indicating saturable caffeine metabolism in the dose range tested. These findings imply that if caffeine is to be used as a guide to deteriorating liver function, serial caffeine clearance estimations should be performed in each individual subject, with use of the same dose of caffeine each time.

Administration, Oral↗
PubMed1988

The effect of smoking on caffeine elimination: implications for its use as a semiquantitative test of liver function.

1. The effects of caffeine ingestion and cigarette smoking on caffeine and antipyrine pharmacokinetics were studied using normal subjects as their own controls before and after cessation of smoking in an attempt to minimize genetic and other environmental influences. 2. Moderate caffeine ingestion had no inducing effect on caffeine or antipyrine clearance. 3. Cessation of cigarette smoking significantly reduced clearance of caffeine and antipyrine. 4. These results demonstrate that cigarette smoking significantly affects caffeine pharmacokinetics and this may contribute to the variable results for caffeine kinetics found in patients with liver disease.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

Acetylglucosaminidase↗
PubMed1985

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Animals↗
PubMed1984

Redistribution of a major cell surface glycoprotein during cell movement.

The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (greater than 100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not G0 or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized.

Animals↗
PubMed1983

Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy.

Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

Animals↗