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Biomedical subjects

S Buckley

Publications and source records attributed to S Buckley.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cyclin A expression in normal and transformed alveolar epithelial cells.

The mature adult alveolar epithelial cell (AEC) is a highly differentiated phenotype that does not readily divide and exhibits numerous specialized functions. Yet, transformed AEC proliferate aggressively in certain forms of lung cancer. Normal AEC also proliferate but in a coordinated manner during embryonic growth and fetal development as well as during lung repair. Therefore, biochemical mechanisms regulating the cell cycle in AEC are clearly of fundamental significance for understanding lung development, lung injury, and cancer. Cyclin A is a protein that varies in abundance during the cell cycle and regulates critical transition points through its association with cyclin-dependent protein kinase subunits. We postulated that high expression of cyclin A might be associated with rapid proliferation in transformed AEC. We compared the expression of cyclin A mRNA and protein in primary cultures of fetal and adult rat AEC, in the E1A-T2 neonatal rat AEC, and in the malignant A549 human AEC. We used pharmacologic blockades with mimosine, aphidicolin, and nocodazole for cell cycle synchronization, which was verified by fluorescence-activated cell sorter (FACS) analysis of cellular DNA content. Transformed cells (A549 and E1A-T2) exhibited a much higher level of expression for both cyclin A mRNA and protein than did normal rat AEC. Induction of cyclin A mRNA expression in A549 human AEC and E1A-T2 rat AEC occurred in late G1, prior to the onset of S phase. Fetal and adult rat AEC and rat E1A-T2 AEC expressed two cyclin A mRNA transcripts, whereas human A549 cells in S phase and M phase expressed three cyclin A mRNA transcripts. We conclude that transformed AEC overexpress cyclin A in comparison with primary AEC cultures, while retaining cell cycle-dependent differences in cyclin A expression. We speculate that cyclin A expression is regulated both at the transcriptional and post-transcriptional levels, and that cyclin A may play a key role in the increased proliferation of transformed AEC that is associated with the pathogenesis of lung cancer.

Animals↗
PubMed1993

EGF abrogation-induced fusilli-form dysmorphogenesis of Meckel's cartilage during embryonic mouse mandibular morphogenesis in vitro.

Mutations associated with genes of the EGF superfamily are implicated in facial malformations arising from abnormal development of the first branchial arch. EGF and EGF receptor (EGFr) transcripts are expressed in the mouse embryonic first branchial arch and derivatives from E9 through E15. EGF transcripts are localized to ectomesenchymal cells associated with precartilage, cartilage, bone and tooth-forming cells. EGF and EGFr proteins co-localize to the same cells suggesting an autocrine regulation. To test whether EGF effects the timing and positional information required for Meckel's cartilage (MC) and tooth development, we cultured E10 mandibular explants in serumless, chemically defined medium with either antisense or sense EGF oligodeoxynucleotides. Antisense inhibition of EGF expression produces bilaterally symmetrical Fusilli-form dysmorphogenesis of MC and decreases tooth bud size; these effects are reversed by the addition of exogenous EGF to the culture medium. Tyrphostin RG 50864, which inhibits EGF receptor kinase activity, inhibits EGF stimulation of tyrosine phosphorylation in a concentration-dependent manner and severely retards mandibular development yet increases tooth size. These findings support the hypothesis that endogenous EGF and EGF-like proteins provide signalling to regulate the size and shape both of cartilage and tooth formation during craniofacial morphogenesis.

Animals↗
PubMed1992

Field loss after pan retinal photocoagulation with diode and argon lasers.

Argon and diode laser pan retinal photocoagulation are used in diabetic proliferative retinopathy. This is a prospective study of the effects of each laser type on peripheral field loss. It shows that diabetics have significantly reduced peripheral field prior to laser compared to normals (p = 0.006). With identical pan retinal photocoagulation with either argon or diode laser there is no difference in field loss.

Diabetic Retinopathy↗
PubMed1992

Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture.

Epidermal growth factor (EGF) is known to induce fetal lung maturation and its receptor is present in the lungs of several species. Recently, EGF has been immunolocalized in type II pneumocytes in rat lung. We postulated that EGF is synthesized in type II pneumocytes and that, because of its position-restricted distribution within the alveolus, EGF might act as an autocrine regulator of type II pneumocyte function. Herein, we have tested the hypothesis using adult rat type II pneumocytes in primary culture. In situ hybridization, using an oligonucleotide probe corresponding to amino acid residues 1070 to 1081 of mouse EGF precursor, demonstrated the presence of EGF precursor mRNA. Upon S-200 Sephacryl gel chromatography of type II pneumocyte extracts, EGF-reactive protein eluted as a high-molecular-weight form (greater than 100 kD). EGF immunoreactivity was localized within type II pneumocytes in the periphery of groups of 10 to 15 cells in culture. The type II pneumocytes bound [125I]EGF in a specific manner, indicating the presence of EGF receptors. Scatchard plots gave an apparent affinity constant (Ka) of 1 x 10(9) liters/mol, and the number of receptors was estimated to be 4.8 x 10(11) mg protein (50 per cell). EGF receptor binding specificity was confirmed by the absence of an autoradiographic signal for cells incubated in the presence of a 100-fold excess concentration of transforming growth factor-alpha. Binding of [125I]EGF could also be downregulated 95% by incubation with 0.2 nM transforming growth factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Long-term response of recurrent respiratory papillomatosis to treatment with lymphoblastoid interferon alfa-N1. Papilloma Study Group.

BACKGROUND: We earlier reported that patients with recurrent respiratory papillomatosis responded to six months of treatment with lymphoblastoid interferon alfa-n1. Because another study of patients treated for one year with leukocyte interferon alfa-n3 found that the growth rate of papillomas was slowed in the first six months but returned to base line during months 7 through 12 despite persistent interferon treatment, we now report the long-term results in our original study patients who were followed for a median of four years after the original one-year crossover study. METHODS: After the patients in our study had completed the first study year, their physicians could continue or recommence treatment with lymphoblastoid interferon alfa-n1 in a dose of either 2 MU per square meter of body-surface area per day or 4 MU per square meter every other day. The extent of disease was measured by endoscopy when clinically indicated. RESULTS: Data on late-follow-up were obtained for 60 of the 66 patients. There were 22 complete remissions and 25 partial remissions; 13 patients had no response. The median duration of the complete remissions was 550 days, and 15 patients continued to be in complete remission. The median duration of partial remissions was 400 days and seven patients were still in partial remission. Thirteen of 28 patients responded to a second course of interferon after an interruption in treatment of at least one month. The rate of response in the 11 of 53 patients who had neutralizing antibody to interferon was the same as in the patients without the antibody. CONCLUSIONS: Patients with severe recurrent respiratory papillomatosis may have a sustained or repeated response to treatment with lymphoblastoid interferon alfa-n1. We recommend that patients with recurrent respiratory papillomatosis who require surgery every two to three months be given a six-month trial of interferon alfa-n1.

Adolescent↗
PubMed1991

Protein phosphorylation and dephosphorylation in type II pneumocytes.

Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10(-4) M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, pI 5.8 and 5.7; 25 kDa, pI 5.7). Exposure of cells for 2.5 h to okadaic acid (10(-6) M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, pI 5.7 and 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, pI 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function.

Animals↗
PubMed1991

Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium.

The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 [( 3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1.5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 mumol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (greater than or equal to 100 mumol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 mumol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1-10 mumol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation.

Adenylyl Cyclases↗
PubMed1991

Elevated plasma norepinephrine levels in infants of substance-abusing mothers.

Infants of substance-abusing mothers (ISAM) have significant growth and neurodevelopmental abnormalities. The origin of these abnormalities is unknown. We postulated that ISAM have increased sympathetic nervous system tone and altered catecholamine levels. Therefore, we measured plasma norepinephrine, epinephrine, and dopamine levels and the number and receptor affinity of beta-adrenoreceptor binding sites on lymphocytes and alpha-adrenoreceptor binding sites on thrombocytes in 22 otherwise healthy ISAM (age, 2.1 +/- 0.5 months; mean +/- SD) and 15 healthy controls (age, 2.5 +/- 0.8 months). Norepinephrine levels in venous blood were 1.8-fold higher in ISAM than in control infants (6.30 +/- 3.85 nmol/L vs 3.55 +/- 2.45 nmol/L). There were no differences in plasma epinephrine or dopamine levels. There were no differences in the number of binding sites or receptor affinity for beta- and alpha-adrenoreceptors. We conclude that ISAM have elevated circulating norepinephrine levels compared with controls. We speculate that this is associated with increased sympathetic nervous system tone in ISAM and that the absence of adrenoreceptor down-regulation may create catecholamine suprasensitivity.

Blood Platelets↗
PubMed1989

Potential regulatory role of inflammatory cells on local vascular smooth muscle tone.

A neutrophil-derived relaxing factor (NDRF) from oyster glycogen (OG)-elicited rat PMN, which causes an endothelium independent relaxation of rat aorta, and which is pharmacologically indistinguishable from endothelium-derived relaxing factor (EDRF) has been described. Experiments were designed to evaluate the presence of NDRF in PMN from rat -whole blood, -carrageenan pleurisy, -OG peritonitis, and guinea pig (GP) -OG peritonitis, as well as in OG-elicited rat macrophages (M phi). Significant vascular relaxing activity was found using rat PMN from OG peritonitis and carrageenan pleurisy, as well as from OG-M phi. Little or no activity was found in rat whole blood PMN or PMN from GP-OG peritonitis. These results suggest that NDRF activity may be expressed upon cellular migration to an inflammatory site in the rat, and may not be present in all species. Also, all inflammatory cells examined were capable of reversing EDRF-dependent relaxations when stimulated to produce superoxide anion suggesting a dual regulatory role for these cells on local vascular tone.

Animals↗
PubMed1989

Sublethal oxidant injury inhibits signal transduction in rat type II pneumocytes.

The production of reduced forms of O2 in oxidant injury can lead to lipid peroxidation, which would pose a threat to cell membrane integrity and therefore to signal transduction pathways located in the membrane. We studied the effects of oxidant injury with t-butyl hydroperoxide (tBOOH) on signal transduction in rat type II pneumocytes in culture. Exposure of 1 x 10(6) type II pneumocytes to tBOOH concentrations less than or equal to 100 microM did not cause significant release of lactate dehydrogenase and was therefore termed sublethal. Exposure to sublethal concentrations of tBOOH caused a dose-dependent inhibition of surfactant secretion stimulated both by terbutaline (10(-4)M) and by extracellular ATP (10(-5)M). Adenosine 3',5'-cyclic monophosphate production in response both to terbutaline and to extracellular ATP was also inhibited by exposure to 100 microM tBOOH. In addition, exposure to 100 microM tBOOH inhibited inositol phosphate formation in response to extracellular ATP. We conclude that sublethal oxidant injury inhibits not only receptor-mediated agonist stimulation of surfactant secretion but also receptor-mediated agonist stimulation of second messenger formation in type II pneumocytes in culture. Such inhibition may be important in the pathogenesis of both the adult and neonatal forms of respiratory distress syndrome, in which alveolar surfactant deficiency may be exacerbated by oxidant injury.

Adenosine Triphosphate↗
PubMed1989

P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes.

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.

Adenosine Triphosphate↗
PubMed1989

Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes.

Surfactant from type II pneumocytes prevents the alveolar atelectasis found in both the neonatal and adult forms of respiratory distress syndrome. We have found that verapamil, a phenylalkene with calcium channel and alpha 1-receptor binding properties, has a multiphasic concentration effect on surfactant secretion from [3H]choline-labeled rat type II pneumocytes in culture. Verapamil (10(-8) M) caused a 24% stimulation of surfactant secretion, whereas an 8% inhibition was found at 10(-6) M and a 70% stimulation was found at 10(-4) M. Lactate dehydrogenase release occurred at 5 x 10(-4) M verapamil. Verapamil (10(-4) M) also produced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) in comparison with concentrations of less than or equal to 10(-6) M, an effect that could not be blocked by propranolol (10(-4) M). Verapamil (10(-6) M) increased the total formation of inositol phosphates (IP) by 23% in comparison with IP formation in control cells. Calcium influx was inhibited 15% by 10(-8) M verapamil and 37% by 10(-4) M verapamil. Calcium efflux was stimulated 44% by 10(-5) M verapamil. In combination with 50% effective concentrations (EC50) of terbutaline, phorbol ester, and ATP, the respective effects of verapamil (10(-4) M) on surfactant secretion were approximately additive. We conclude that verapamil has a novel multiphasic concentration effect on surfactant secretion, which appears to involve several signal transduction pathways including cAMP formation, IP formation, inhibition of calcium influx, and stimulation of calcium efflux.

Adenosine Triphosphate↗
PubMed1988

Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay.

We developed a colorimetric assay for determining metabolic activity (viability) of cells exposed to toxic agents. This system is based on the ability of mitochondrial enzymes in viable cells to modify a tetrazolium salt into a blue formazan product that can be detected spectrophotometrically at 570 nm. The assay works equally well for mammalian and insect cell lines and at 48 hr color formation is linear over a cell input range of 1.56-50 X 10(4) cells/ml. The inhibitory effects of T-2 mycotoxin on tetrazolium cleavage in L929 cells is comparable to that observed for protein and DNA synthesis (50% inhibition = 6-8 ng/ml). Using this system to analyze the lethal effect of T-2 toxin on cells from various animal species, it was found that bovine cells were the most sensitive (50% inhibition at 2.2 ng/ml) while hamster cells were the most resistant (50% inhibition at 26.2 ng/ml). Murine cells exhibited intermediate sensitivity (50% inhibition at 10.9 ng/ml). Variable toxin susceptibility was also observed among different cell types. Lymphocytes were 3-fold more sensitive to the T-2 inhibitory effects than comparable tissue culture cell lines. These data indicate that the colorimetric assay system could have broad applications in toxicological studies. Further, the observed differences in species sensitivity may provide insight into the primary mechanism of the T-2 toxin-cell interaction that ultimately leads to cell death.

Animals↗
PubMed1988

Combined effects of corticosteroid, thyroid hormones, and beta-agonist on surfactant, pulmonary mechanics, and beta-receptor binding in fetal lamb lung.

We studied the interactions of corticosteroids, thyroid hormones, and beta-agonist on surfactant phospholipids, pulmonary mechanics, and beta-receptor binding in fetal lambs. We infused cortisol (450 micrograms/h for 48 h), thyrotropin-releasing hormone (TRH) (25 micrograms/h for 48 h), and ritodrine (1.3 micrograms/kg/min for 24 h) independently, and in double (cortisol plus beta-agonist, cortisol plus TRH), and in triple (cortisol plus TRH plus beta-agonist) combinations into chronically catheterized fetal lambs between 0.88 and 0.90 gestation. Infusion of the triple combination of cortisol plus TRH plus beta-agonist resulted in a 20.9-fold increase in the saturated phosphatidylcholine content of fetal lung lavage, in a 5.8-fold increase in the saturated phosphatidylcholine content of whole fetal lung, and in a 13.3-fold increase in the saturated phosphatidylcholine content of fetal tracheal fluid. In addition, lung stability to inflation increased 3-fold, and lung stability to deflation increased 8-fold. The increases in the saturated phosphatidylcholine content of fetal lung lavage and tracheal fluid were greater than the effects of each hormone acting independently, or in the double combinations. The beta-receptor maximal binding capacity was increased 30% by the combined infusion of cortisol and TRH. In addition, the maximal binding capacity after cortisol plus TRH plus beta-agonist infusion was 54% greater than the maximal binding capacity after beta-agonist infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Chronic glucose infusion inhibits development of beta-receptor binding in fetal lamb lung.

We used chronic fetal glucose infusion to test the hypothesis that chronic fetal hyperglycemia and hyperinsulinemia inhibit the development of beta-receptor binding capacity (Bmax) in fetal lamb lung. Glucose was infused (14 +/- 4 mg/kg/h, mean +/- SD) into eight twin and four singleton fetuses from 112 days gestation until death between 118-145 days gestation. The other eight twins and eleven additional singleton fetuses served as controls. Serum glucose levels were elevated 2-fold and serum insulin levels were elevated 3-fold in the glucose-infused fetuses. In the control fetuses beta-receptor Bmax increased 2.5-fold between 130 days gestation and term. However, this increase was attenuated to 1.5-fold in the glucose infused fetuses, p less than 0.01. The 50% inhibition of Bmax was similar in both male and female fetuses, except that the Bmax fell to 30% lower levels in males, p less than 0.01. Chronic glucose infusion also resulted in an 80% reduction in lung lavage saturated phosphatidylcholine content, and an 85% reduction in tracheal fluid saturated phosphatidylcholine content, p less than 0.001. Lung lavage and tracheal fluid saturated phosphatidylcholine content correlated significantly with beta receptor Bmax (r = 0.9, r = 0.85). We conclude that chronic glucose infusion inhibits the development of beta-receptor binding in fetal lamb lung, and that this effect is greater in males than females. Such a mechanism could be a factor in the predisposition of infants of diabetic mothers to develop respiratory distress and could contribute to a male disadvantage in respiratory morbidity.

Animals↗
PubMed1988

Effects of beta-2 agonist on hepatic glycogen metabolism in the fetal lamb.

To determine the effects of fetal beta-2 agonist exposure on fetal hepatic glycogen metabolism, we infused ritodrine at a rate of 1.3 +/- 0.4 microgram/kg/min (mean +/- SD) for 24 h into six chronically catheterized twin fetal lambs starting between 128 and 134 days gestation. The control twins received 0.9% saline at 1.2 +/- 0.12 ml/kg/h. In addition, 15 uncatheterized fetuses were killed between 115 and 148 days gestation as unoperated controls. Ritodrine infusion produced a 1.7-fold elevation in fetal serum glucose level, from 23 +/- 5 to 42 +/- 15 mg/dl, and a 2-fold elevation in serum insulin level, from 16 +/- 5 to 34 +/- 8 mg/ml, p less than 0.01. Hepatic glycogen content increased 7-fold in the uncatheterized controls between 115 and 148 days gestation (r = 0.9, p less than 0.001). Ritodrine infusion reduced hepatic glycogen content by 50% from 179 +/- 19 micrograms/mg in twin controls to 90 +/- 25 micrograms/mg in the ritodrine-infused twins, p less than 0.001. Hepatic glycogen phosphorylase kinase activity was elevated 1.3-fold from 0.149 +/- 0.100 mU/mg protein in control twins to 0.186 +/- 0.007 mU/mg protein in the ritodrine infused twins, p less than 0.001. Glycogen phosphorylase a activity was also increased 1.4-fold from 8.60 +/- 0.76 nM NADPH/min/mg protein in control twins to 11.85 +/- 0.68 nM NADPH/min/mg protein in the ritodrine infused twins, p less than 0.001.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Detection of the lethal effects of T-2 mycotoxin on cells using a rapid colorimetric viability assay.

A colorimetric method of determining cell viabilities in cultured cells is described. The system is based on the ability of mitochondrial enzymes in live but not dead cells to chemically reduce a tetrazolium salt (MTT) into a colored formazan dye which can be detected at 570 nm using a multiwell scanning spectrophotometer. 48 h Chinese hamster ovary (CHO) cell cultures are used in the assay and the amount of colored product formed is directly proportional to cell number over a range of 0.39-12.5 X 10(4) cells/ml. The cytotoxic effects of T-2 mycotoxin can also be detected colorimetrically using this method. The toxin dose which inhibits formazan formation (50% endpoint = 14-16 ng/ml) is very comparable to that which inhibits cell viability (17 ng/ml), or protein and DNA synthesis (10 ng/ml). This system also works well with mitogen-stimulated primary lymphocyte cultures but these cells exhibit a much more sensitive response to T-2 effects having a 50% inhibition endpoint of 2 ng/ml. The assay is rapid to perform and gives a high degree of precision and could serve as a valid alternative to viability assays currently in use.

Animals↗