P. Proulx

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

Abstract 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.

Vitamin E suppresses diacylglycerol (DAG) level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity

The present study has examined the role of vitamin E, a natural lipid antioxidant, in the production of diacylglycerol (DAG) and phosphatidic acid (PA) in thrombin-stimulated human endothelial cells. Cells were labelled with [3H]myristate and the incorporation and distribution of [3H]myristate into cellular lipids was not affected by vitamin E. However, in response to thrombin stimulation, considerably more PA and less DAG were formed in cells enriched with vitamin E. The time-course of thrombin stimulation indicated that vitamin E attenuated the accumulation of sustained DAG levels with a concomitant increase in PA. Direct determination of DAG mass further confirmed that vitamin E suppresses the accumulation of DAG induced by thrombin. In the presence of ethanol, the formation of [3H]phosphatidylethanol (PEt) in [3H]myristate-labelled cells stimulated by thrombin was unaffected by vitamin E enrichment. DL-Propranolol, a PA phosphohydrolase inhibitor, caused an accumulation of PA, without affecting DAG formation in either vitamin E-treated and untreated cells. This indicated that the increase in PA and decrease in DAG in vitamin E-treated cells was not due to a stimulation of phospholipase D or an inhibition of PA phosphohydrolase. Determination of inositol phosphates formation in response to thrombin showed that the change of DAG levels elicited by vitamin E was independent of phospholipase C-induced hydrolysis of inositol phospholipids. In contrast, analysis of DAG kinase activity revealed that vitamin E enrichment enhanced the activity of the enzyme in both basal and thrombin-stimulated cells. Taken together, these data indicated that vitamin E caused an increased conversion of DAG to PA by activating DAG kinase activity without causing any change in the activities of phospholipase D, PA phosphohydrolase or phospholipase C.

Phospholipase a activity in growing Eacherichia coli cells

The turnover of phosphoglycerides in Escherichia coli B, B fad and K 19 was investigated under normal growth conditions. E. coli B fad displayed appreciable turnover of phosphatidylethanolamine and polyglycerophosphatides. During growth, this strain released phospholipids, lipopolysaccharide material and proteins including phospholipase A to the medium and produced sizeable amounts of free fatty acids due mainly to phosphatidylethanolamine hydrolysis. The two other strains displayed little or no turnover of phosphatidylethanolamine and a lower production of free fatty acid: however polyglycerophosphatide breakdown in this case was similar to that of strain B fad. Also E. coli B and K 19 had a more rigid cell envelope structure than B fad as could be assessed by the tendency to release outer membrane materials to the medium and susceptibility to lysis. The results suggested that the levels of phospholipase A activity in various strains of growing E. coli is related to the integrity of the cell envelope.

Studies on the mechanism of hemolysis by acyl carnitines, lysolecithins and acyl cholines

Abstract 1. 1. The effect of isolated membrane components and normal cytosol constituents on lysis of rat erythrocytes by synthetic, palmitoyl- d,l -carnitine, palmitoyl choline and (1-palmitoyl)-lysolecithin were studied. 2. 2. A number of these constituents were found to act as modifiers of hemolytic activity. Their action as inhibitors or accelerators were highly dependent on the order with which erythrocytes, lysin and modifier substance were added to constitute the lysis test. 3. 3. Preincubation of the lysin with any one membrane component prior to erythrocyte addition resulted in an inhibition greater than in the corresponding case with a similar addition sequence and no preincubation. On the other hand, preincubation of erythrocytes with various membrane constituents prior to lysin addition did not increase inhibition except in the case of membrane protein. It was concluded that the inhibitory effects obtained resulted from a non-specific interaction of the lysins tested with the various membrane constituents added. In the case of added membrane protein, protection against lysis would also result from its interaction with the cell surface. 4. 4. When membrane and cytosol constituents were each added to erythrocytes immediately after cell exposure to a weakly -hemolytic concentration of lysin, this resulted in an increase rather than a decrease in the extent of hemolysis. 5. 5. These results are discussed in terms of the mechanism of lysis by quaternary ammonium amphipathic derivatives.

Metabolism of phosphoglycerides in Escherichia coli during growth at 37 °C and during a cold-induced lag phase

Abstract 1. 1. The turnover of phosphoglyceride classes in Escherichia coli cells growing at 37 °C or kept stationary at 10 °C was studied. At 37 °C, the levels of phosphatidyl ethanolamine, doubly labelled with 32P and 14C increased slightly during a 3 h chase. This change was accompanied by a moderate and parallel turnover of both these labels in phosphatidyl glycerol and cardiolipin. In cells lagging at 10 °C there was little or no turnover in the phospholipid and neutral lipid classes. Analysis of the turnover and synthesis rates of the acyl groups of phosphatidyl ethanolamine and phosphatidyl glycerol revealed that a phospholipase A1:lysophosphoglyceride acyltransferase cycle was not operative at 10 °C. At either temperature no evidence indicating a partial turnover pathway sparing either diglycerides or acyl groups could be obtained. 2. 2. Turnover and synthesis of phosphatide species were examined simultaneously at either 37 or 10 °C. In cells chased at 37 °C the metabolism of 3H-labelled acyl groups of phosphatidyl ethanolamine and phosphatidyl glycerol was typified by a conversion of disaturated species to cyclopropane fatty acid-containing analogues whereas this conversion was impaired and little change occurred in cells chased at 10 °C. During the chase at 37 °C, synthesis of 14C-labelled phosphoglycerides favoured saturated species whereas at 10 °C, unsaturated species formation was predominant. 3. 3. Total lipids from cells growing, or starved at 37 °C had a fatty acid composition quite different than that of total lipids from cells lagging at 10 °C. The latter condition was characterized by a much higher degree of unsaturation and a correspondingly lower content of cyclopropane fatty acid. 4. 4. The increase in unsaturation accompanying cold exposure is discussed in terms of possible factors influencing cyclopropane fatty acid synthesis as well as other phenomena.

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.

A role for PKCε and MAP kinase in bradykinin-induced arachidonic acid release in rabbit CCD cells

Arachidonic acid (AA) release is the rate-limiting step in the production of prostaglandins, an important class of autocrine/paracrine factors that modulate collecting duct function. Previous results from this laboratory have established cytosolic phospholipase A2(cPLA2) as the enzyme responsible for bradykinin (BK)-stimulated AA mobilization in rabbit cortical collecting duct (RCCD) cells, and the present study pursues the intracellular signaling mechanisms responsible for its activation. Pretreatment of cells with Ro-31-8220, an inhibitor of protein kinase C (PKC), or PD-98059, an inhibitor of the mitogen-activated protein kinase (MAPK) cascade, resulted in a 50-60% reduction in BK-stimulated AA release. Incubation of RCCD cells with a combination of both Ro-31-8220 and PD-98059 did not achieve a greater inhibition of either BK-stimulated AA release or cPLA2 activity, possibly indicating that MAPK activation was dependent upon prior activation of PKC. This was supported by the observation that BK-induced MAPK activation could be reversed by either inhibitor. Additional experiments dealing with immunoblots for PKC isozymes revealed that RCCD cells express PKC species α, γ, ε, and ζ. Following BK stimulation, only PKCε translocated to the particulate fraction. Based on these results, it appears that PKC is activated and involved in the sequential activation of MAPK and cPLA2 following BK treatment. The results also suggest that PKCε may be the isozyme implicated in the process.

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.

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.

Cardiolipin specific phospholipase D activity in Escherichia coli extracts

Abstract Escherichia coli homogenates were found to specifically hydrolyse 32P-labelled cardiolipin to yield phosphatidic acid and phosphatidyl glycerol as labelled products. This phospholipase D activity occurred in unfrozen preparations provided Mg2+ was added. In preparations that had been frozen and thawed the activity required the presence of ATP as well as Mg2+. The significance of these findings is discussed in terms of the metabolism of polyglycerophosphatides in normal and energy-depleted E. coli cells.