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Biomedical subjects

E Cunningham

Publications and source records attributed to E Cunningham.

At least 19 recordsLinked to original sources
PubMed2000

HLA and Mooren's ulceration.

BACKGROUND: Mooren's ulcer is a progressive intractable destructive peripheral ulceration of the cornea, probably of autoimmune aetiology. The disease is rare in the northern hemisphere but is more common in southern and central Africa and the Indian subcontinent. Although rare, its predominance in certain racial groups and their second generation migrants worldwide indicates a genetic as well as a geographic predisposition. The highly polymorphic human leucocyte antigens (HLA) confer genetic susceptibility to several autoimmune disorders. Therefore, a possible link between Mooren's ulcer and HLA type was investigated. METHODS: Patients (n=22) with non-infective destructive peripheral corneal inflammatory disease were recruited worldwide. Differential diagnosis confirmed Mooren's ulceration in 12 cases. HLA typing (HLA-A, B, C, DRB, DQB) was performed by serology and PCR using sequence specific primers. The patients came from varied ethnic backgrounds and their HLA typing results were compared with published data from ethnically matched control populations. RESULTS: Of the 12 patients with Mooren's ulcer, 10 (83%) were HLA-DR17(3) positive (including all nine patients of Asian, Indonesian, and black African origin), and 10 (83%) were HLA-DQ2 positive. The frequency of HLA-DR17(3) and DQ2 was higher in the Mooren's ulcer group compared to published data from ethnically matched control populations, where the expected antigen frequencies range between 5% and 40%. CONCLUSION: These results suggest a possible association between HLA-DR17(3) and/or DQ2 and susceptibility to Mooren's ulcer.

Adolescent↗
PubMed1997

Evidence for platelet-activating factor receptor subtypes on human polymorphonuclear leukocyte membranes.

Platelet-activating factor (PAF) is a potent phospholipid mediator that acts through specific cell surface receptors. The existence of PAF receptor subtypes has been suggested by functional and radioligand binding studies in a variety of cells and tissues. This report addresses this issue more directly and demonstrates differences between specific PAF receptors in human polymorphonuclear leukocytes (PMNs) and COS-7 cells transfected with the cloned human PAF receptor gene. The presence of more than one receptor in human PMNs is supported by three different studies. First, the Kd from the saturation isotherms for the binding of [3H]WEB 2086 on PMNs was 7-fold larger (Kd = 29.2 nM) than the kinetic Kd (4.2 nM). Second, the pseudo-Hill slope determined from the saturation experiments with PMNs was significantly lower than unity (0.69 +/- 0.05 SEM), and the saturation Kd values for transfected COS-7 (Kd = 9.6 nM) and PMN membranes were significantly different. These results contrasted with those for the transfected COS-7 cells, which showed a Kd from the saturation isotherms similar to that of the kinetic Kd (3.2 nM) and a pseudo-Hill slope that was not different from 1.0. Third, when the radiolabeled ligand [3H]WEB 2086 was increased in concentration from 10 to 50 nM in inhibition experiments with the human PMN membranes, the Ki increased, indicative of binding mainly to receptors with lower affinity. These results suggest that PAF receptor subtypes exist in human PMNs based on distinct radioligand binding characteristics from the human cloned PAF receptor.

Animals↗
PubMed1996

ADP-ribosylation factor 1-regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells.

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

Animals↗
PubMed1996

ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis.

BACKGROUND: In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca(2+)-regulated secretion. Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells. RESULTS: We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 microM Ca2+ with regard to secretion and PLD activity. Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity. The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PITP). Unexpectedly, PITP was also found to restore GTP gamma S-dependent secretion. Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPITP alpha and rPITP beta. The rARF1 protein restored both secretory function and PLD activity, whereas PITP only restored secretory function. However, both ARF and PITP were capable of stimulating phosphatidylinositol bis phosphate (PIP2) synthesis. CONCLUSIONS: ARF and PITP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+. We have previously shown that PITP participates in the synthesis of PIP2. In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5 kinase, the enzyme responsible for PIP2 synthesis. We propose that ARF and PITP both restore exocytosis by a common mechanism-promoting PIP2 synthesis.

ADP-Ribosylation Factor 1↗
PubMed1995

An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system.

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5'-[gamma-thio]triphosphate-mediated phospholipase C beta activity.

3T3 Cells↗
PubMed1995

Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2.

BACKGROUND: Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C-beta (PLC-beta) that is regulated by GTP-binding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLC-beta. RESULTS: We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-beta, in the presence and absence of exogenous PI-TP. Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'[gamma-thio]-triphosphate (GTP gamma S) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC-beta present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2. CONCLUSIONS: The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC-beta from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLC-beta is not the lipid that is resident in the membrane but that provided through PI-TP.

Animals↗
PubMed1995

Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling.

Stimulation of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis is a widespread mechanism for receptor-mediated signaling in eukaryotes. Cytosolic phosphatidylinositol transfer protein (PITP) is necessary for guanosine triphosphate (GTP)-dependent hydrolysis of PIP2 by phospholipase C-beta (PLC-beta), but the role of PITP is unclear. Stimulation of phospholipase C-gamma (PLC-gamma) in A431 human epidermoid carcinoma cells treated with epidermal growth factor (EGF) required PITP. Stimulation of PI-4 kinase in cells treated with EGF also required PITP. Coprecipitation studies revealed an EGF-dependent association of PITP with the EGF receptor, with PI-4 kinase, and with PLC-gamma.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

ARF1(2-17) does not specifically interact with ARF1-dependent pathways. Inhibition by peptide of phospholipases C beta, D and exocytosis in HL60 cells.

The small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N-terminal peptide ARF1(2-17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-depleted HL60 cells was inhibited by ARF1(2-17). In the presence of endogenous cytosol, ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD and exocytosis. GTP-gamma-S-stimulated phospholipase C-beta (PLC-beta) was also inhibited by ARF(2-17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2-17) inhibited the reconstitution of GTP-gamma-S-stimulated PLC-beta activity with exogenously-added PLC-beta 1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2-17) peptide inhibits both ARF-independent (i.e. PLC-beta) and ARF-dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.

ADP-Ribosylation Factor 1↗
PubMed1994

Phospholipase D: a downstream effector of ARF in granulocytes.

Activation of the phospholipase D (PLD) pathway is a widespread response when cells are activated by agonists that bind receptors on the cell surface. A 16-kD cytosolic component can reconstitute guanosine triphosphate (GTP)-mediated activation of phospholipase D in HL60 cells depleted of their cytosol by permeabilization. This factor was purified and identified as two small GTP-binding proteins, ARF1 and ARF3. Recombinant ARF1 substituted for purified ARF proteins in the reconstitution assay. These results indicate that phospholipase D is a downstream effector of ARF1 and ARF3. The well-established role of ARF in vesicular traffic would suggest that alterations in lipid content by PLD are an important determinant in vesicular dynamics.

ADP-Ribosylation Factor 1↗
PubMed1993

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling.

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.

Amino Acid Sequence↗
PubMed1991

P.r.n. medications in child psychiatric patients: a pilot placebo-controlled study.

BACKGROUND: The administration p.r.n. (as needed) of sedative medications is a widespread practice in the management of acute dyscontrol of child psychiatric inpatients. Its efficacy, however, has never been tested in a controlled clinical trial. METHOD: Twenty-one male inpatients, aged 5-13 years, participated in a double-blind, placebo-controlled study of the p.r.n. use of diphenhydramine, a sedative antihistaminic often used in child psychiatry wards. The patients' DSM-III-R diagnoses were conduct disorder, attention-deficit hyperactivity, and major depression. Each patient in acute dyscontrol blindly received either oral or intramuscular doses of diphenhydramine 25-50 mg (N = 9) or placebo (N = 12). The Conners Abbreviated 10-Item Teacher Rating Scale and the Clinical Global Impressions scale were completed before and 0.5, 1, and 2 hours after the dose. RESULTS: Repeated measures ANOVA showed significant time effects, but no difference due to drug. The intramuscular route tended to be more effective than the oral, regardless of whether active drug or placebo was given. CONCLUSION: The data indicate that if p.r.n. administrations are effective, this is a placebo effect. Likewise, intramuscular administrations are more effective because of a route effect ("the needle") and not because of a specific pharmacologic activity.

Administration, Oral↗
PubMed1990

Extracorporeal regional complexing haemodialysis treatment of acute inorganic mercury intoxication.

A 70-year-old white female presented approximately 24 h after ingesting three 475 mg tablets (1.425 g) of mercuric chloride in a suicide attempt. Acute renal failure necessitated the initiation of haemodialysis approximately 4 d after the ingestion. Treatment with BAL (2,3-dimercaptopropanol) resulted in only small increases in mercury output into dialysate. A new procedure involving the extracorporeal infusion of the chelating agent dimercaptosuccinic acid (DMSA) into the arterial blood line during haemodialysis was initiated. This procedure of Extracorporeal Regional Complexing Haemodialysis (ERCH) had been effective in increasing methylmercury removal in patients poisoned by contaminated grain. The first DMSA-ERCH procedure was performed 6 d after poisoning. There was a dramatic increase in mercury output into the dialysate. During three treatment sessions of 80 min each, 1189 micrograms of mercury were removed from the patient. The dialysed mercury represented the only mercury output since the patient was anuric and not producing faeces. DMSA-ERCH appears to be much more effective than BAL and haemodialysis in the treatment of acute inorganic mercury poisoning. The long interval between poisoning and initiation of treatment probably contributed to the patients ultimate demise, 28 d after poisoning. Efficacy of the DMSA-ERCH procedure for inorganic mercury poisoning is likely to be improved as the interval between exposure and treatment is reduced.

Aged↗
PubMed1990

The influence of alpha-adrenergic agents on tonic accommodation.

This study has examined the effect of an alpha-1 adrenergic agonist (phenylephrine HCl, 2.5%) and a non-selective alpha antagonist (thymoxamine HCl, 0.5%) on tonic accommodation (TA). TA was measured at 1 min intervals throughout a 45 min period using an objective infra-red optometer (Canon Autoref R-1). Additionally pupil diameter and the near-point of accommodation (NPA) were monitored in order to assess the efficacy of the instilled drug. A double-blind protocol was adopted between thymoxamine, phenylephrine and a saline control for 7 emmetropic subjects. There was no significant difference between the values of TA obtained under the three pharmacological conditions. However, both adrenergic agents induced changes in pupil diameter and NPA. Furthermore in all conditions TA increased by approximately 0.25D while the subject sat passively in darkness. The data indicates that alpha-adrenergic innervation alters the closed-loop accommodative response independently of TA.

Accommodation, Ocular↗
PubMed1989

The Pocket Speech Aid.

A rapid-access but limited vocabulary communication aid for the non-vocal and speech impaired is useful in some well defined situations. The design and construction of such a device is described. Three distinct situations have been identified where an aid is essential and an initial evaluation has shown the Pocket Speech Aid to be very successful.

Biomedical Engineering↗