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P Proulx

Publications and source records attributed to P Proulx.

At least 19 recordsLinked to original sources
PubMed1997

Bestatin-mediated inhibition of leucine aminopeptidase may hinder HIV infection.

Bestatin, an inhibitor of leucine aminopeptidase (LAPase), significantly decreased HIV infection as reflected by a reduced number of positive immunofluorescent cells, p24 levels, reverse transcriptase activity and the number of proviral copies found in Bestatin-treated cells. Cellular and extracellular LAPase activity in infected cells was higher than the LAPase activity found in uninfected cells. However, cellular and extracellular LAPase activity as well as total protein kinase C activity was lower in Bestatin-treated cells. Conversely, the incubation of human lymphocytic HUT78 cells with LAPase promotes HIV infectivity. The possible role of LAPase in the pathophysiology of HIV was assessed by determining LAPase serum levels in HIV infected patients. LAPase activity levels were three orders of magnitude greater in sera obtained from HIV patients than those detected in sera of uninfected individuals. Although Bestatin reduced HIV infection, a moderate decrease in the reverse transcriptase activity of chronically-infected H9 human T-lymphocytic cells was observed. Based on the higher levels of LAPase present in the serum of HIV patients and on the combined inhibitory effect of Bestatin on LAPase and on protein kinase C activities, we suggest that LAPase may play an important role in the early events of HIV infection such as viral entry.

Anti-HIV Agents↗
PubMed1995

Effect of retinoic acid on Nm/23 nucleoside diphosphate kinase and components of cyclic adenosine monophosphate-dependent signalling in human neuroblastoma cell lines.

The effects of retinoic acid on components of the cAMP-dependent signalling system were examined in two related human neuroblastoma cell lines SK-N-SH-F (SHF) and SK-N-SH-N (SHN). Retinoid treatment for a week significantly increased the concentration of intracellular cAMP and the levels of activity of protein kinase A and adenylate cyclase in both cell lines. Retinoic acid treatment also caused a very marked translocation of nucleoside diphosphate kinase from the cytosol to the membrane fraction. The increases in cyclic nucleotide and protein kinase A activity were observed to occur as early as within 1 and 2 days respectively and preceded or were concurrent with the onset of observable morphological differentiation. Results also indicated that agents which elevated intracellular cAMP caused neuronal differentiation and blunted retinoic acid-induced melanocytic differentiation in SHF cells. However, increases in cAMP brought about by treatment of SHF cells with retinoic acid alone were several-fold smaller and thus insufficient to induce neuritogenesis in these cells. The results as a whole indicate that one overall effect of retinoic acid treatment is to upgrade the activity of components of the cAMP-dependent signalling system in both neuroblastoma cell lines. However, retinoic acid causes the SH-F and SH-N cell lines to differentiate along different routes which means that the upgrading responses may be related to more general aspects of differentiation rather than to specific phenotype expression.

Adenylyl Cyclases↗
PubMed1994

Transfer of arachidonyl groups within the lipids of two human neuroblastoma cell lines.

The incorporation and mobilization of [3H]arachidonic acid in lipids of human neuroblastoma cell lines, SK-N-SHF and LA-N-5, was studied. Essentially similar results were obtained with these two cell lines. Except for phosphatidylinositol which displayed the highest specific activity, the incorporation patterns within phospholipid classes tended to reflect phospholipid composition initially. However at later stages, counts in the acid-stable phosphatidylcholine plateaued and/or decreased while those of plasmenylethaniolamine and acid-stable phosphatidylethanolamine increased steadily. When cells were pulse-labelled with [3H]arachidonic acid and chased with fresh medium, there was a movement of label from diacyl (acid-stable) phosphatidylcholine to plasmenylethanolamine and diacyl (acid-stable) phosphatidylethanolamine. Plasmenylcholine did not appear to be involved in the arachidonyl group transfer. Under these chase conditions there was extensive turnover in the 32P-labelled polar headgroup of phosphatidylinositol but not in that of the other phospholipids. In both incorporation and chase studies involving [3H]arachidonic acid, a movement of arachidonyl groups from triacylglycerol to phospholipid could be observed. The results indicated that the patterns of incorporation and redistribution of arachidonic acid in human neuroblastoma cells were effectively regulated to favor lipids such as phosphatidylinositol and the subclasses of phosphatidylethanolamine. Possible mechanisms involved in these enrichment processes are discussed.

Arachidonic Acid↗
PubMed1992

Retinoic acid- and staurosporine-induced bidirectional differentiation of human neuroblastoma cell lines.

The differentiation pattern of two related human neuroblastoma cell lines, SK-N-SHF and SK-N-SHN, induced by retinoic acid and staurosporine was studied. Immunohistochemical and electron microscopic examination of the cells indicated that the SHF variant could undergo differentiation along a melanocytic route when treated with retinoic acid and to neuronal cells when treated with retionic acid and staurosporine together. Treatment of SHN cells with either or both these agents caused neuronal differentiation. The melanocytic pathway was characterized in part by the flattening of the cells, the appearance of melanocytic antigens and various forms of melanosomes, an increase in tyrosinase activity, and the absence of neuronal marker proteins. The neuronal route was typified by the development of long neuritic processes containing microtubules and numerous neurosecretory granules as well as by immunohistochemical reactions for neural cell adhesion molecule, synaptophysin, and neurofilament proteins. The significance of these results is discussed in terms of the differentiation responses of neuroblastoma cells to chemical agents as well as some of the factors involved in the regulation of phenotype expressions of these cells.

Alkaloids↗
PubMed1990

Further characterization of the diacylglycerol-phosphatidylethanolamine exchange reaction catalyzed by cell-free extracts of Escherichia coli.

The conditions for phosphatidylethanolamine (PE)-diacylglycerol (DAG) exchange catalysed by cell-free extracts of Escherichia coli were studied using 14C- or 3H-analogues of both these lipids. The reaction, examined with either labelled PE or labelled DAG, occurred without co-factor addition and was inhibited by Ca2+ and Mg2+. Detergents such as Triton X-100 greatly enhanced the activity; however, the optimal concentration of this agent depended on the lipid substrate concentration. The exchange-catalysing enzyme involved in these extracts appeared to be very specific for DAG and PE, since no other labelled phospholipid or acylglycerol derivative formed radioactive product under the assay conditions tested. Again, endogenous [3H]PE present in the enzyme source, but no other endogenous lipid, was converted to labelled DAG in the presence of added 1,2-dioleoyl-sn-glycerol. The Vmax value for the conversion of labelled PE to DAG was very similar to the Vmax value found for the conversion of labelled DAG to PE as would be expected in the case of an exchange reaction being responsible for both conversions. However, the Km value for PE was appreciably larger than that for DAG. The enzyme involved, displayed a broad acyl chain specificity as could be judged from: (1) the ability of various species of DAG and PE to stimulate the exchange; (2) the suitability of lipid substrates prepared from widely different biological sources; and (3) the interchange of acyl groups that occurred between dimyristoyl PE and dilauroylglycerol. As would be expected for an exchange reaction, the incorporation of lauroyl groups into PE occurred without an increase in the total fatty acid content of this phospholipid. The results of the present study confirm and further characterize the PE-DAG exchange reaction of E. coli.

Calcium↗
PubMed1990

Effects of retinoic acid and staurosporine on the protein kinase C activity and the morphology of two related human neuroblastoma cell lines.

Studies on the involvement of protein kinase C in retinoic acid-induced differentiation of human neuroblastoma were carried out with two variants of the SK-N-SH cell line namely the SH-F subline, which differentiates to give a fibroblast-like phenotype, and the SH-N subline, which develops into the typical neuronal phenotype. In SH-F, a substantial increase in protein kinase C activity accompanied morphological differentiation. Accordingly, after 7 days of retinoic acid treatment, EDTA-extracted, cytosolic protein kinase C activity increased by slightly more than 2-fold over vehicle-treated controls. Again, detergent-extracted activity, representing membrane-bound or total protein kinase C, showed a similar 2.6- to 5.1-fold increase in treated cells. A time-course study revealed an earliest increase in total activity after two days of retinoic acid treatment which continued linearly for the first 6 to 8 days, and then levelled off. A study of the effect of retinoic acid on the protein kinase C in vitro with SH-F cell extracts showed only a slight increase in activity (of 25%) at the relatively high concentration of 10(-4) M; however, no significant differences were observed at lower concentrations. In contrast, the SH-N cell line responded to retinoic acid by a 45% decrease in EDTA-extractable, and a 63% decrease in detergent-extractable protein kinase C activity. Added to SH-F cell cultures, 15 nM staurosporine was found to inhibit protein kinase C in vivo and to a lesser extent, the protein kinase A. Present together with retinoic acid, staurosporine not only prevented the augmentation but caused a marked decrease of protein kinase C activity in this cell line. Morphological studies indicated that when SH-N cells are treated with staurosporine, or staurosporine and retinoic acid together, a neuronal phenotype similar to that produced by retinoic acid alone is observed. In contrast, when the SH-F cell line is treated with staurosporine or staurosporine and retinoic acid together, the flattened fibroblast-like cell type normally induced by retinoic acids is not observed. Instead, these cells display much smaller cell bodies and elaborate extensions resembling the neuronal phenotype produced by retinoic acid induced differentiation of the SH-N variant. These results suggest that changes in the protein kinase C activity may be involved in regulating the expression of the phenotype during cell differentiation.

Alkaloids↗
PubMed1989

Diacylglycerol-phosphatidylethanolamine exchange in Escherichia coli.

The conversion of [3H]- or [14C]diacylglycerol to labelled phosphatidylethanolamine by dialysed, particulate fraction of Escherichia coli was studied. The reaction occurred in the presence of hydroxylamine, under which conditions, the synthesis of [14C]phosphatidylethanolamine from CDP-diacylglycerol and [14C]serine did not occur. The conversion was enhanced by addition of dilauroyl- or dioleoylphosphatidylethanolamine. A conversion of [3H]- or [14C]phosphatidylethanolamine to labelled diacylglycerol could also be readily demonstrated provided unlabelled diacylglycerol was added. Double-labelled [acyl-3H, 32P]phosphatidylethanolamine was converted to labelled diacylglycerol without formation of labelled water-soluble products. The formation of double-labelled phosphatidylethanolamine from [3H]diacyl[14C]glycerol or of double-labelled diacylglycerol from [acyl-3H,glycerol-14C]phosphatidylethanolamine occurred without significant change in isotope ratio. When [acyl-3H]phosphatidylethanolamine was incubated with increasing concentrations of [acyl-14C]diacylglycerol, correspondingly increasing concentrations of [14C]phosphatidylethanolamine were formed which matched the concentrations of [3H]diacylglycerol produced concurrently. It was concluded that E. coli extracts can catalyze an exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol with complete sparing of the phosphoethanolamine moiety.

Diglycerides↗
PubMed1989

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187.

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.

Animals↗
PubMed1989

Studies on the effects of vitamin E on neuroblastoma N1E 115.

The effects of DL-alpha-tocopherol and DL-alpha-tocopherol succinate on neuroblastoma N1E 115 cells were studied. Tocopherol had no growth-arresting properties, whereas its succinate ester derivative inhibited growth at concentrations greater than or equal to 20 microM. The succinate derivative was taken up somewhat more readily than free tocopherol; however, for any equal uptake of both forms of vitamin E, only the succinate derivative could affect growth. Tocopherol succinate was taken up without marked conversion to tocopherol. Following uptake, plasma membrane and organelle fractions contained most of the vitamin E derivatives; however, the particulate and membrane fractions were about twice as enriched in the succinate derivative as in free tocopherol. On the other hand, a proportionally higher amount of unconjugated vitamin E was recovered in the cytosol fraction. Fluorescence polarization studies indicated no differences in the overall fluidity of the plasma membranes treated or not treated with either form of vitamin E. The data point to the functionality of the free carboxyl group of the succinate derivative as a basis for the difference in potency of the two forms of vitamin E.

Animals↗
PubMed1987

Relation between Ca2+ uptake and fluidity of brush-border membranes isolated from rabbit small intestine and incubated with fatty acids and methyl oleate.

The rate of incorporation of oleic acid into isolated brush-border membranes was found to be considerably faster than methyl oleate incorporation under similar experimental conditions. The effects of fatty acids and methyl oleate incorporation on Ca2+ uptake and fluidity were monitored. Whereas treatment with 0.01-0.05 mM oleic acid corresponding to incorporations smaller than 90 nmol/mg protein enhanced Ca2+ transport, exposures to higher concentrations of this fatty acid corresponding to incorporations larger than 150 nmol/mg protein, decreased uptake of this cation. On the other hand, treatment with 0.01-0.2 mM methyl oleate corresponding to incorporations of up to 220 nmol/mg protein had only a stimulatory effect on the Ca2+ uptake. Oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with diphenylhexatriene in a dose-dependent manner. In contrast, palmitic acid had little or no effect on the diphenylhexatriene-reportable order of the membrane within the range of concentrations used. Monitored as a function of temperature, the anisotropy values showed a gradual melting for both the control and lipid-treated membranes. The results support the concept that saturated and cis-unsaturated fatty acids dissolve in different lipid domains and this in itself appears to be an important factor defining whether the biological function of the membrane is affected by the uptake. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca2+ uptake process increases Ca2+ uptake in concert with increased diphenylhexatriene-monitored fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surface causes inhibition of Ca2+ uptake.

Animals↗
PubMed1987

The uptake of phosphatidylcholine by brush-border membranes isolated from rabbit small intestine.

The uptake of [H3] dipalmitoyl phosphatidylcholine by isolated brush border membranes from rabbit small intestine was studied. The process was found to be stimulated by Ca2+ in a manner which was influenced by the protein -Ca2+ concentration ratio. At higher ratios the enhancement by divalent cation was diminished. Preincubation of the membranes with Ca2+ followed by their transfer to a Ca2+-free medium did not result in enhancement of uptake. The stimulation by Ca2+ could be seen only when this cation was present in the incubation medium. The uptakes of [1-14C] palmitic acid, and di-[1-14C] palmitoylglycerol were also examined and found to be comparable in extent to that of labelled phosphatidylcholine but more pronounced than that of 2-[1-14C] palmitoylglycerol. The incorporation of labelled palmitic acid, phosphatidylcholine or monoglyceride in brush border membranes prepared from jejunum and ileum was similar. The results indicate that brush border membranes are capable of incorporating a variety of exogenous lipids especially in the presence of Ca2+ and the possible consequences of such uptake on the structure and function of the membrane are discussed.

Animals↗
PubMed1986

Studies on calcium binding to brush-border membranes from rabbit small intestine.

A study was made of the uptake of Ca2+ by brush-border membrane vesicles prepared from rabbit small intestine. The process was found to be time, temperature and substrate concentration dependent, displayed saturability, did not depend on added energy sources and occurred optimally in a pH range of 7.5-8.0. Although the transport of D-glucose by these membrane vesicles responded to changes in osmotic pressure as modified by adding cellobiose to the medium, the uptake of Ca2+ was found not to be osmotically-sensitive. Moreover, the equilibrium uptake value obtained when vesicles were exposed to 0.36 mM Ca2+ was some 60-fold higher than the amount that could have been accommodated by the intravesicular space, calculated from the equilibrium uptake of D-glucose. It was concluded from these results that the uptake involved complete binding of the Ca2+ to the membrane. The ionophore A23187 enhanced the rates of uptake and efflux of Ca2+ without affecting equilibrium values, which suggests that the binding of Ca2+ measured under our conditions was to interior sites of the membrane. The binding capacity was decreased in the presence of 10 mM lidocaine as indicated by a diminution of the equilibrium binding values. Generating an electrochemical potential (negative inside) by addition of valinomycin to vesicles pre-equilibrated with K2SO4, enhanced the rate of uptake of Ca2+. Addition of metal ions, on the other hand, inhibited the uptake, La3+ and Tb3+ being most effective followed by Mn2+, Ba2+ and Mg2+. Na+ and K+ were the least inhibitory. The properties of the Ca2+ uptake process found in rabbit brush-border membranes were compared to those of similar processes occurring in other species.

Adenosine Triphosphate↗
PubMed1986

A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation.

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.

Animals↗
PubMed1986

Effects of exogenous fatty acids on calcium uptake by brush-border membrane vesicles from rabbit small intestine.

The uptake of a variety of fatty acids by isolated brush-border membranes from rabbit small intestine was studied. This uptake increased with acyl chain-length and was not diminished by washing of the lipid-treated membranes with 0.25 M CsBr. The binding of fatty acid was not accompanied by a decrease in endogenous acyl groups or of cholesterol and therefore corresponded to a net uptake accountable qualitatively and quantitatively by the fatty acid added to the membranes. The uptake of Ca2+ was stimulated by treatment of the membranes with low concentrations of unsaturated fatty acids (0.05 mM) as well as with various concentrations of caprylic acid (0.10-3.00 mM) and inhibited by treatment with higher concentrations of unsaturated fatty acids (0.20-0.60 mM). Saturated fatty acids had no marked effects on Ca2+ uptake. The stimulatory concentrations of unsaturated fatty acids did not change the Ca2+-binding characteristics of the membranes, whereas the higher concentrations decreased equilibrium binding of Ca2+ and very probably the number of high-affinity binding sites. The results of this study are assessed in terms of the effects of normal fatty acids found in the diet on the absorptive properties of the brush-border membranes.

Animals↗
PubMed1986

Factors influencing the uptake of cholesterol by isolated brush border membranes from rabbit small intestine.

The uptake of [3H] cholesterol by isolated brush border membranes from rabbit small intestine was studied under different conditions. In initial experiments the uptake from taurocholate-micellized suspensions was found to be greatly enhanced by Ca2+. Maximum stimulation was obtained with 10 mM Ca2+ and this condition was adopted for some of the subsequent experiments. In the presence of this cation, the uptake was too rapid to allow kinetic studies and only equilibrium values could be measured. Addition of lecithins to the micelles had an inhibitory effect on the equilibrium uptake in the presence of Ca2+ and on the rate of uptake in the absence of this cation which increased with acyl chain length. Admixture of the sterol with dipalmitoylphosphatidylethanolamine stimulated the uptake whereas admixture with dipalmitoylphosphatidylglycerol as well as with 1-monoolein alone or together with oleic acid had little or no effect on uptake in the presence of Ca2+. Again with this cation present, preincubation of membranes with the dipalmitoyl analogue of lecithin increased the equilibrium uptake of the sterol and abolished the inhibitory effect of the choline lipid. This inhibitory effect could be seen only with untreated membranes when lecithin and cholesterol were micellized together. In the absence of Ca2+, preincubation with dipalmitoylphosphatidylcholine had no marked effect on the rate of cholesterol uptake and the suppressive effect of lecithin on this rate could be seen even with lipid-treated membranes provided the lecithin and sterol were micellized together.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗