PubMed Health⌕ Search

Biomedical subjects

J G Douglas

Publications and source records attributed to J G Douglas.

At least 19 recordsLinked to original sources
PubMed1992

Development and characterization of rabbit proximal tubular epithelial cell lines.

We have isolated rabbit kidney proximal tubular epithelial cell lines. The selection was based on their ability to form confluent monolayers on porous supports and to maintain receptor-mediated signal transduction and ion transport, characteristic of the proximal tubule. The isolation method consisted of several steps: (1) superficial cortical proximal tubule segments were microdissected and cultured on a matrix-coated porous support until cells formed a confluent monolayer; (2) primary cultures showing hormone-regulated ion transport typical for the proximal tubule were selected and co-cultured with irradiated fibroblasts; and (3) the epithelial cells surviving after several passages were expanded and passaged on porous substrates. Most of the cell lines developed in this manner were obtained by co-culture with irradiated fibroblasts producing a recombinant retrovirus encoding SV40 large T antigen and G418 resistance. However, SV40 T antigen expression was not essential for immortalization, since neither T antigen nor G418 resistance was detected in the isolated cell lines and co-culture with non-producing 3T3 cells gave similar results. One cell line (vEPT) has been characterized in some detail with respect to morphological, biochemical, and ion transport properties. This line forms confluent monolayers with apical microvilli, tight junctions, and convolutions of the basolateral plasma membrane. Once confluent, monolayers maintain conductances of 25 to 32 mS/cm2 for several weeks in culture and possess phlorizin-sensitive short-circuit current (Isc) in glucose containing media, indicative of apical Na(+)-glucose co-transport. vEPT cells also retain receptor and signaling mechanisms for angiotensin II (Ang II). Apical and basal Ang II and 5,6-epoxy-eicosatrienoic acid (5,6-EET) modulate the Isc in a manner similar to primary cultures. The cell lines share with primary cultures expression of the cytokeratins K8, K10/K11, and K19 ("nomenclature" [21]). They also retain several receptor and signal transduction mechanisms. For example, Ang II, arachidonate, bradykinin, 5,6-EET, parathyroid hormone (residues 1 through 34), and purine nucleotides increase cytosolic Ca2+, PTH elevates cAMP levels, and Ang II enhances proximal tubule-specific arachidonic acid metabolism.

Animals↗
PubMed1992

Identification of G protein subtypes in peripheral nerve and cultured Schwann cells.

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.

Adenosine Diphosphate Ribose↗
PubMed1992

Bone density in asthmatic patients taking high dose inhaled beclomethasone dipropionate and intermittent systemic corticosteroids.

BACKGROUND: Asthmatic patients taking low to moderate doses of inhaled topical corticosteroids have been shown to have lower bone density than those taking bronchodilators only. There is little information on bone density in asthmatic patients taking high dose inhaled corticosteroids. METHODS: Bone mass was studied in three age matched groups of asthmatic patients. These comprised: 17 asthmatic patients who had never taken inhaled or systemic corticosteroids (group 1); 20 patients who had taken beclomethasone diproprionate in a dosage of 1000-2000 micrograms daily for at least a year, who had also received courses of systemic corticosteroids in the past (group 2); and 20 patients who were taking both high dose inhaled corticosteroids and regular low dose prednisolone, at a median dose of 7 mg daily (group 3). Vertebral bone density was measured by quantitative computed tomography. Biochemical indices of bone formation and resorption were also measured. RESULTS: Mean bone density in group 2 (127.5(22.6) mg/ml) was similar to that in group 3 (114.5 (36.0) mg/ml). Bone density was significantly lower in both of these groups than in group 1 (160.4 (27.4) mg/ml). There were no significant differences between groups for any of the markers of bone formation and resorption. CONCLUSIONS: Asthmatic patients receiving high dose inhaled beclomethasone and intermittent courses of systemic corticosteroids have reduced vertebral bone density. The bone loss is similar in degree to that seen in patients taking high dose inhaled topical corticosteroids and continuous low dose systemic corticosteroids.

Administration, Inhalation↗
PubMed1992

An audit of the clinical investigation of pleural effusion.

BACKGROUND: Pleural aspiration with pleural biopsy is advised for the investigation of pleural effusion. The clinical investigation of pleural effusion in a group of teaching hospitals was audited with reference to adequacy and diagnostic value of sampling procedures. METHODS: A retrospective review of case records of all patients investigated for pleural effusion during an eight month period was performed. The records of 112 patients, age range 16-91 years, who underwent 150 procedures were reviewed. RESULTS: Microbiology samples were obtained from 137 procedures, of which five provided a positive culture, including one for mycobacteria. Cytology samples were obtained from 145 procedures though approximately two thirds of samples were less than the recommended 30 ml. The pleural biopsy rate was 30%, varying from 0% in general or thoracic surgery to 68% in thoracic medicine (thoracic surgeons carried out thoracoscopy). Twenty nine per cent of pleural biopsy samples were of poor quality. The complication rate was 2% for aspiration alone, and 4% for aspiration plus biopsy. The sensitivity of the first diagnostic procedure for a diagnosis of malignancy or tuberculosis was 53% for cytology alone, 50% for biopsy alone and 72% for cytology plus biopsy. CONCLUSION: The samples obtained from pleural aspiration and biopsy in the initial investigation of pleural effusion are often inadequate. Further education is necessary to improve the quantity and quality of specimens submitted for histological and cytological examination.

Bacterial Infections↗
PubMed1992

Effects of amiloride analogues on AVP binding and activation of V1-receptor-expressing cells.

We tested the interactions between amiloride analogues and V1 vascular arginine vasopressin (AVP) receptors of human platelets, rat glomerular mesangial cells, and A7r5 smooth muscle cells by using radioligand binding techniques, intracellular calcium monitoring, platelet aggregation, and cell contraction techniques. Amiloride analogues were competitive inhibitors of both the agonist [3H]AVP and the tritiated V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyl)tyrosine]AVP ([3H]d(CH2)5Tyr(Me)AVP) binding to V1 AVP receptors in the three different cell types used. The order of potency was ethylisopropyl amiloride (EIPA) greater than Benzamil greater than amiloride. AVP mobilization of intracellular calcium was blocked by the V1 antagonist d(CH2)5Tyr(Me)AVP and was reduced by EIPA in a dose-dependent manner. Moreover, EIPA also inhibited prostaglandin F2 alpha mobilization of intracellular calcium. Alkalinization of the intracellular pH with ammonium chloride reversed the inhibitory effect of EIPA but not that of the V1 antagonist on AVP-induced calcium mobilization. Both amiloride and EIPA blocked AVP-induced aggregation of human platelets and contraction of mesangial cells and glomeruli preparations independently of receptor site antagonism. In conclusion, amiloride analogues interfere with activation of V1 vascular receptors by AVP at different levels including binding to the receptor site, mobilization of intracellular calcium, cell contraction or aggregation, and presumably alteration of intracellular ion transports.

Amiloride↗
PubMed1992

Angiotensin II receptor subtypes in cultured rat renal mesangial cells.

The selective angiotensin (ANG II) antagonists losartan (DuP 753) and PD 123319 have been shown to bind selectively to AT1 and AT2 subtypes, respectively. To characterize ANG II receptor subtypes in mesangial cells, washed membranes were incubated with 0.1 to 0.5 nM 125I-ANG II and increasing concentrations of competitors. The inhibition of 125I-ANG II binding by losartan and PD 123319 was biphasic, and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation comprised 86% of the total and showed high affinity for ANG II and losartan, but low affinity for the AT2 antagonists PD 123319 and CGP42112A, and thus appear identical to the recently cloned AT1 subtype. The remaining 14% of the sites showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD 123319 relative to AT1 sites. However, another AT2-selective antagonist, CGP42112A, showed little affinity for these sites. Both classes of binding sites were inhibited by guanosine 5'-O-(3-thiophosphate) and pertussis toxin treatment. We propose that there are two distinct G protein-coupled ANG II receptor subtypes (AT1A and AT1B) present in renal mesangial cells.

Angiotensin II↗
PubMed1992

Angiotensin II-induced changes in guanine nucleotide binding and regulatory proteins.

Systemic infusion of angiotensin II, a potent agonist, using doses that are initially subpressor, eventually produces sustained blood pressure elevation and reductions in glomerular capillary ultrafiltration coefficient characterized by enhanced signal transduction to angiotensin II and other agonists. In this setting, there is a significant increased affinity of angiotensin II binding to smooth muscle and glomerular mesangial receptors and enhanced sensitivity and magnitude of angiotensin II-induced decrements in cyclic AMP. Since G proteins are important modulators of binding and signal transduction, the present studies were designed to test the hypothesis that differences in the relative amounts of G proteins may be present and have accounted for differences observed. G proteins were identified and quantitated by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radiolabeling in the presence of activated toxins with [gamma-32P]NAD+, immunoprecipitation, and immunoblotting. A 168% and 465% increase in pertussis toxin-catalyzed ADP ribosylation of alpha 40-41 was found in angiotensin II-treated groups over control groups for glomerular and mesenteric membranes, respectively. Immunoblotting revealed a 250% and 35% increase in the levels of the Gi isoforms alpha i-2 and alpha i-3, respectively, and a decrease of 53% in alpha i-1 from the angiotensin II-treated group. No differences were observed in cholera toxin labeling or immunoblotting of Gs. These results demonstrate multiple mechanisms whereby angiotensin-induced signal transduction can be modulated involving both the receptors and G proteins. These observed differences in G proteins in systemic and renal vasculature accompanying angiotensin II infusion suggest the possibility of a regulatory role in the pathophysiology of angiotensin II-induced hypertension and renal disease.

Adenosine Diphosphate Ribose↗
PubMed1992

Malaria in Aberdeen: an audit of 110 patients admitted between 1980-1991.

All 110 patients seen in North East Scotland after contracting malaria from foreign travel were treated in the Regional Infection Unit in Aberdeen. Those patients managed there from January 1980 to March 1991 are described. There were 54 episodes of Plasmodium falciparum malaria (49%) and 26 episodes (23%) of Plasmodium vivax malaria. The remainder had either mixed infection or were diagnosed as malaria on high clinical probability. The majority of the patients were male (80%) and under 40 years of age (84%). Most patients were either caucasians born in the UK (69%) or native Africans (23%) who were students recently arrived for further education or who had returned from visiting their country of origin for summer holidays. The British residents acquired infection either while on oil related business in West or Central Africa (46%) or after travelling on holiday (30%). The peak incidence of presentation was August and September. 93.5% of patients with falciparum malaria had returned or originated from Africa. 42% with vivax malaria had visited Africa and 27% Papua New Guinea. 70% had been prescribed antimalarial prophylaxis but less than half of these took their medication correctly. The majority of patients with falciparum malaria presented within two weeks of arrival in Britain while patients with vivax malaria presented at varying (but generally longer) intervals, 42% being diagnosed more than three months after exposure. Falciparum infection was more severe although there have been no deaths in the unit from malaria. Our experience seemed of interest and worth reporting because of the number of patients whose infection reflected travel related to the off shore oil industry, which is centred in Aberdeen.

Adolescent↗
PubMed1991

Calcium reveals different mechanisms of guanylate cyclase activation by atrial natriuretic factor and ATP in rat lung membranes.

CaCl2 inhibited ATP-stimulated guanylate cyclase activity, but had little effect on basal and atrial natriuretic factor-stimulated guanylate cyclase activity in rat lung membranes. LaCl3 had similar effects as CaCl2 on basal and stimulated guanylate cyclase activity. LiCl and other monovalent salts inhibited ATP-stimulated guanylate cyclase activity more than basal enzyme activity. However, atrial natriuretic factor somehow stabilized the enzyme against the inhibitory effect of LiCl. These results suggest that ATP and atrial natriuretic factor activate the enzyme through different mechanisms. Since the effect of calcium on guanylate cyclase activity is different from that of monovalent salts and can be mimicked by lanthanum, it may be mediated by a specific calcium binding site or binding protein.

Adenosine Triphosphate↗
PubMed1991

Diethylcarbamazine inhibits endothelial and microfilarial prostanoid metabolism in vitro.

Diethylcarbamazine (DEC) rapidly lowers the number of microfilariae in the peripheral circulation. The mechanism of action is unknown, but may involve alterations of arachidonic acid metabolism in vascular tissues. We studied the effects of DEC on arachidonic acid metabolism by bovine pulmonary arterial endothelium monolayers, human platelets and Brugia malayi microfilariae. DEC at a concentration of 2.5 microM, a level achieved in vivo, rapidly decreased prostacyclin, prostaglandin E2 and thromboxane B2 release from endothelial monolayers by 78% (P less than 0.001), 57% (P = 0.05), and 75% (P less than 0.05), respectively. High-pressure liquid chromatography of extracts of endothelial monolayers incubated with DEC showed similar inhibition of these cyclooxygenase pathway products, but exposure to the drug did not result in formation of new eicosanoids. DEC did not inhibit endothelial phospholipase A2-dependent release of arachidonate from membrane stores, whereas prostaglandin H2 synthase activity (cyclooxygenae, EC 1.14.99.1) was reduced to a degree similar to that effected by acetylsalicylic acid. Microfilarial but not platelet synthesis of cyclooxygenase products was also reduced by DEC. These data suggest that the mechanism by which DEC lowers the level of microfilariae in the circulation may in part involve its effects on host endothelial and parasite eicosanoid production.

Animals↗
PubMed1991

Secretion of insulin-like growth factor I and its binding proteins by collecting duct cells.

Insulin-like growth factor I (IGF-I) has been found in the kidney, particularly in the collecting duct in the rat. Since cultured rabbit collecting duct cells constitute a convenient system for in vitro studies, we have examined whether these cells secrete IGF-I. Culture medium conditioned by collecting duct cells was concentrated by reverse phase chromatography and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent molecular weights of 7.5 and greater than 25 kilodaltons (kD) were identified by IGF-I RIA. A smaller amount of 10 kD species was also observed. Further characterization of 7.5 kD IGF-I immunoreactive species by reverse phase HPLC showed that it eluted in a single peak. To determine whether the higher molecular weight species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I (thr59) for two hours at 30 degrees C and applied to a Sephadex G100 column equilibrated in a non-dissociating buffer. The major peak of radioactivity was confined to a high molecular weight region; there was no radioactivity in the fractions corresponding to 7.5 kD. Western ligand analysis of unreduced conditioned medium identified two IGF-I binding species of 25 and 30 kD, similar in size to species observed in normal rabbit serum. 125I-IGF-I binding as assessed in a charcoal adsorption assay could be displaced by IGF-I and IGF-II but not by insulin. Further characterization of the 10 kD peak of IGF-I immunoreactivity indicated that it did not possess IGF-I binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

An open study of teicoplanin in the treatment of gram-positive infections.

Teicoplanin, a new glycopeptide antibiotic similar to vancomycin, was evaluated in treating 36 hospitalized patients suffering from various Gram-positive infections. The 36 patients received teicoplanin once daily as a mean intravenous injection of 550 mg/day (range 200-800 mg/day). Previous antimicrobial therapy was used in 28% of patients. The mean duration of therapy was 7.5 days (range 3-38 days). The overall clinical success rate was 94%. 24/36 patients (66%) had positive microbiology. Elimination of the pathogens was seen in 75% of all evaluable cases. Four patients with early prosthetic valve endocarditis due to coagulase negative Staphylococcus (3 patients) and Propionibacterium acnes (1 patient) had a favorable clinical and microbiological outcome. No adverse drug reactions were observed. Teicoplanin is safe and effective in the therapy of many different infections caused by Gram-positive bacteria.

Adult↗