Biomedical subjects

E J Murphy

Publications and source records attributed to E J Murphy.

At least 19 recordsLinked to original sources

PubMed1996

Sterol carrier protein-2 expression in mouse L-cell fibroblasts alters cholesterol uptake.

Despite the progress made on the possible functions of sterol carrier protein (SCP-2) using assays in vitro, very little is known regarding the role of SCP-2 in intact cells. To further elucidate this role, mouse L-cell fibroblasts were transfected with cDNA encoding for mouse 15 kDa or 13.2 kDa SCP-2. The data show for the first time, that SCP-2 expression increases cholesterol uptake into transfected L-cell fibroblasts. Untransfected L-cells expressed SCP-2 at levels near or below the lower limit of detectability. SCP-2 immunoreactive protein levels were 0.030 +/- 0.004% and 0.036 +/- 0.002% of total cytosolic proteins in the 15 and 13.2 kDa stable transfectants, respectively. Both the 15 and 13.2 kDa SCP-2 expressions products were found as 13.2 kDa proteins, consistent with rapid post-translational cleavage of the putative amino terminal mitochondrial targeting sequence from the 15 kDa SCP-2. The effect of expressing either form of SCP-2 on [3H]cholesterol uptake was determined. Expression of the 15 kDa form, but not the 13.2 kDa form of SCP-2, enhanced the rate and extent of [3H]cholesterol uptake compared to control or mock-transfected L-cells. The [3H]cholesterol uptake rate in 15 kDa SCP-2 expressing cells was increased 1.3-fold, while the extent of [3H]cholesterol uptake was increased 1.4-fold after 12 h of uptake compared to control L-cells. The differences in cholesterol uptake between the cells expressing the 13.2 versus the 15 kDa protein, suggest that the 15 kDa form of SCP-2 is functionally localized within the cell, while the 13.2 kDa product is not.

Animals↗

PubMed1996

Spontaneous and protein-mediated sterol transfer between intracellular membranes.

Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4-5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell.

Animals↗

PubMed1996

Liver fatty acid-binding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism.

The role of cytosolic liver fatty acid binding protein (L-FABP) in fatty acid uptake and metabolism was examined using cultured L-cell fibroblasts transfected with the cDNA encoding for L-FABP. [3H]Oleic acid was used to determine the effects of intracellular esterification on fatty acid uptake and to determine esterified fatty acid localization to specific lipid classes. cis-Parinaric acid, a poorly esterified fatty acid, was used to determine uptake in the absence of any appreciable esterification. High-expression L-cells had a 80% and 50% greater initial uptake rate for both [3H]oleic acid and cis-parinaric acid, respectively compared to low-expression L-cells. Maximal uptake of [3H]oleic acid did not plateau because of intracellular esterification. In high-expressing cells, maximal cis-parinaric acid uptake rapidly plateaued at a level 34% higher than in low-expression cells. After 1 min of incubation, the majority of cellular [3H]oleic acid was unesterified, with the bulk of the esterified portion preferentially localized to phospholipids. After 5 and 30 min, cells expressing L-FABP esterified a significantly greater amount of [3H]oleic acid into both the neutral lipid and phospholipid fractions than did low-expression cells. L-FABP expression also selectively stimulated [3H]oleic acid incorporation into choline glycerophospholipids. Thus, L-FABP expression not only stimulated fatty acid uptake at all time points, but also stimulated intracellular esterification into specific lipid pools. These results show in detail for the first time using an intact cell culture system that L-FABP expression not only stimulated fatty acid uptake, but also increased intracellular esterification of exogenously supplied fatty acids.

Animals↗

PubMed1996

Fatty acid double bond orientation alters interaction with L-cell fibroblasts.

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while 3H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (Km = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (Km = 4.17 uM). Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.

Animals↗

PubMed1995

Altered lipid metabolism in the presence and absence of extracellular Ca2+ during combined oxygen-glucose deprivation in primary astrocyte cultures.

The effect of combined oxygen-glucose deprivation (COGD) on lipid metabolism in primary rat cortical astrocyte cultures was studied in both the presence and absence of extracellular Ca2+. In this study, increases in intracellular Ca2+ from internal Ca2+ stores were not inhibited nor were internal Ca2+ levels buffered. Combined oxygen-glucose deprivation resulted in a quantitative reduction in phospholipid levels and an increase in free fatty acid and lysophospholipid levels. Four hours after the onset of COGD, ethanolamine- and choline glycerophospholipid levels were decreased by 40 and 46% from control levels in the presence of Ca2+, respectively. A similar decrease was found 6 hr after onset of COGD in the absence of Ca2+. These changes were accompanied by elevated levels of the corresponding lysophospholipids. However, the increases in lysophospholipid content did not account for the entire loss of ethanolamine- or choline glycerophospholipid. Phosphatidylserine was reduced in both the presence and absence of extracellular Ca2+ but phosphatidylinositol was only decreased in the absence of Ca2+. Statistically significant increases in total fatty acid (FA) and polyunsaturated fatty acid (PUFA) levels occurred at 30 min and 3 hr after the onset of COGD in the absence and presence of Ca2+, respectively. Arachidonic acid levels were increased in both groups by 1 hr. These increases in FA, PUFA, and specifically arachidonic acid were time-dependent and increased over the 12 hr of COGD. Collectively, these results indicate the activation of an acylhydrolase mechanism in the possible presence of an inhibited reacylation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells.

Differential effects of intestinal (I-FABP) or liver (L-FABP) fatty acid binding proteins on fatty acid uptake and esterification were examined using transfected mouse L-cell fibroblasts. L-FABP, but not I-FABP, expression increased the initial rate and extent of cis-parinaric acid uptake by 50 and 29%, respectively, compared to control cells. I-FABP and L-FABP expression preferentially increased [3H]-oleic acid incorporation into triacylglycerols by 5.5-fold and 3.8-fold, respectively. While both L-FABP and I-FABP increased esterification of [3H]-oleic acid into ethanolamine glycerophospholipids, these proteins had opposite effect on esterification into choline glycerophospholipids. These data show for the first time that distinct FABP differentially affect both fatty acid uptake and intracellular esterification.

Animals↗

PubMed1994

Lipid alterations following impact spinal cord injury in the rat.

A computer-controlled impactor was used to produce a severe spinal cord injury in the rat thoracic spinal cord. Cords were rapidly frozen in situ at 5, 15, 30, and 60 min and 6, 12, and 24 h postinjury. Control cords were noninjured cords from animals having undergone a laminectomy and allowed to recover for 90 min postlaminectomy. The cords were assayed for alterations in lipid metabolism. Specifically, there were rapid increases in prostaglandin F2 alpha and thromboxane, with a peak increase in thromboxane levels at 30 min. Prostaglandin F2 alpha levels peaked at 15 min with levels remaining nearly constant for 12 h. There were no detectable changes in phospholipid levels, although diacylglycerol levels and free fatty acid levels were increased. Total free fatty acids were increased at 12 and 24 h postinjury by 2.3- and 3.2-fold over control levels, respectively. Arachidonic acid levels were not significantly elevated at early time points, however, these early time points correspond to elevated eicosanoid synthesis and this may account for the lack of early detectable increases in arachidonic acid. After 6 h postinjury, arachidonic acid levels were 20-fold greater than control levels and remained elevated at 24 h. There were minimal decreases in cholesterol and no decrease in either choline or ethanolamine plasmalogen levels. These results suggest a rapid turnover of arachidonic acid following spinal cord injury with a concomitant increase in vasoconstrictive eicosanoid synthesis. The lack of changes in major membrane constituents suggests the mechanisms may not involve general membrane degradation, but an over-stimulation of phospholipase A2-linked membrane receptors.

Animals↗

PubMed1993

Effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells.

The effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells were determined. The cellular lipids were extracted on days 0, 3 and 7, following the addition of 1.2% dimethylsulfoxide to induce cellular differentiation. Proportions of ethanolamine glycerophospholipids (EtnGpl), phosphatidylinositol (PtdIns) and sphingomyelin (CerPCho) were significantly elevated following differentiation. The mole percentage of choline glycerophospholipids (ChoGpl) decreased with differentiation. The plasmalogens, both choline and ethanolamine, increased by 1.3- and 2.3-fold, respectively, during differentiation. The fatty acid composition of the phospholipid classes was also altered. PtdIns and ChoGpl had decreased proportions of polyenoic fatty acids, while these proportions were increased in EtnGpl. Both ChoGpl and EtnGpl had increased n-3/n-6 series fatty acid ratios, but this ratio was decreased in PtdIns. The mole percentage of arachidonic acid was significantly decreased in both PtdIns and ChoGpl, but elevated in EtnGpl and may be a result of the increase in ethanolamine plasmalogen. Thus, differentiation did not increase the overall mole percentage of polyenoic FA in the cells nor increase the n-6 series fatty acid proportions. We speculate plasmalogens may have a role in the differentiation process or in maintaining the cell in the differentiated state.

Cell Differentiation↗

PubMed1993

Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures.

The phospholipid and phospholipid fatty acid compositions of mixed murine spinal cord neuronal cultures are reported. The phospholipid composition was primarily comprised of ethanolamine glycerophospholipids (44.8%) and choline glycerophospholipids (43.5%). Plasmalogens made up 29.1% of the ethanolamine glycerophospholipids (13.0% of the total phospholipids) and 4.5% of the choline glycerophospholipids (1.9% of the total phospholipids). Other phospholipids ranged from 2.9% for sphingomyelin to 1.0% for phosphatidylinositol 4-phosphate. The fatty acid compositions of the ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylserine, and phosphatidylinositol were also determined. The choline glycerophospholipids were the most saturated and contained the smallest amount of polyunsaturated fatty acids. The ethanolamine glycerophospholipids were the most unsaturated and contained the highest amount of polyunsaturated fatty acids. The phospholipids contained minimal amounts of 20:3 n-9 (Mead acid) and are not considered polyunsaturated fatty acid deficient. Thus, for the mixed neuronal spinal cord cultures, the phospholipid fatty acid compositions were not polyunsaturated fatty acid deficient and contained a large amount of polyenoic fatty acids of both the n-3 and n-6 series.

Animals↗

PubMed1993

High-performance liquid chromatography separation and quantitation of methylprednisolone from rat brain.

A sensitive, reliable method for the extraction, separation, and quantitation of methylprednisolone from rat brain is reported. The method can accurately quantitate methylprednisolone levels between 9.8 and 2500 ng/injection using a two-step HPLC separation and monitoring absorbance at 254 nm. A 90% extraction recovery of methylprednisolone (interday variation of 9.0% and an intraday variation of 0.0 to 7.7%) from rat cortex was obtained with a double extraction method using low toxicity solvents. These solvents are known to quantitatively extract the neutral lipids and phospholipids from brain. Combined with the ability to separate the neutral lipid and methylprednisolone fractions for further separation, and the ability to separate all phospholipid classes in the first run, this method offers great utility combined with the reliable, high extraction recovery and sensitive quantitation of methylprednisolone.

Animals↗

PubMed1993

Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography.

A simple, quantitative method for determining the plasmalogen content of small samples is reported here. The method uses the different susceptibility to acid-catalyzed hydrolysis of the alkyl, alkenyl and acyl linkages to separate the plasmalogen subclass from the other two non-labile subclasses. Hydrolysis of plasmenylethanolamine and plasmenylcholine was complete after 4 and 1 min of acid treatment, respectively. The acid-catalyzed hydrolysis did not alter the phospholipid fatty acid composition, making this method useful for fatty acid compositional analysis of the plasmalogen subclass. High-performance liquid chromatography was used for separations, and phospholipids were quantitated by assay of lipid phosphorus or by direct quantitation of peak area. Using this method, small amounts (10 nmol) of ethanolamine glycerophospholipid and choline glycerophospholipid are subjected to acid-catalyzed hydrolysis and subsequent separation of the resulting lysocompounds obtained from plasmalogens from the more acid-stable alkylacyl and diacyl glycerophospholipid fractions. Our values for plasmalogens from commercial preparations of choline and ethanolamine glycerophospholipids agree with literature values. The usefulness of the method is demonstrated for small glycerophospholipid samples that are equivalent to samples from cultured neural cells.

Acids↗

PubMed1993

Composition of the phospholipids and their fatty acids in the ROC-1 oligodendroglial cell line.

ROC-1 cells are a hybrid of C-6 rat glioma and rat oligodendroglia cells. Biochemically these cells resemble the oligodendroglia parent, but their lipid composition is unknown. The phospholipid composition in mole % was: cardiolipin, 1.0; phosphatidylglycerol, 1.2; ethanolamine glycerophospholipids, 27.6; phosphatidylinositol, 5.8; lysophosphatidylethanolamine, 0.8; phosphatidylserine, 5.6; choline glycerophospholipids, 43.7; sphingomyelin, 13.7; phosphatidylinositol-4-monophosphate, 0.8; and lysophosphatidylcholine, 0.6. The choline and ethanolamine plasmalogens made up 7.2 and 18.4% of the total phospholipids, respectively. The phospholipid composition reflects that of both parental cells. The cells had moderate to high levels of 20:3n-9 indicating n-6 series fatty acid deficiency. The phosphatidylinositol had very high 20:3n-9 levels with a 20:3n-9/20:4n-6 ratio of 2.1 compared to 0.44 and 0.58 for ethanolamine glycerophospholipids (EtnGpl) and choline glycerophospholipids (ChoGpl) respectively. The saturated/polyenoic fatty acid ratios were 0.40 for EtnGpl, 3.38 for ChoGpl and 1.48 for phosphatidylinositol.

Animals↗

PubMed1993

Cytotoxicity of aluminum silicates in primary neuronal cultures.

To study their cytotoxicity, clays containing aluminum silicates were added to cultures of primary murine spinal cord neurons and differentiated N1E-115 neuroblastoma cells. Bentonite (0.1 mg/ml) and montmorillonite (0.1 mg/ml) rapidly associated with the outer membrane of both N1E-115 and neuronal cells. Erionite (0.1 mg/ml) was randomly distributed throughout the culture. Both bentonite and montmorillonite caused complete cell lysis in the neuronal cultures within 60 min following addition. Erionite had no effect. None of the clays appeared to be cytotoxic to the differentiated N1E-115 cells even though bentonite and montmorillonite were closely associated with the cell membrane. N1E-115 cell lysis did not occur up to 18 h after addition of the clay. Aluminum silicate-containing clays caused a rapid lysis of primary neuronal cells. Differentiated N1E-115 neuroblastoma cells were not susceptible to clay-induced lysis, suggesting that the lytic mechanism is not a general phenomenon that affects all cell types equally.

Aluminum Silicates↗

PubMed1993

Aluminum silicate toxicity in cell cultures.

To assess the cytotoxicity of four clays containing an aluminum silicate--montmorillonite, bentonite, kaolinite and erionite--we used human umbilical vein endothelial, N1E-115 neuroblastoma, and ROC-1 oligodendroglial cells. Morphological examination, lactate dehydrogenase release and fatty acid release were used as indices of trauma. The clays were added in suspension to the cell cultures at concentrations of 0.1, 0.03 and 0.01 mg/ml of medium and the cells were incubated for 1, 6 and 24 h. The clays did not lyse ROC-1 and N1E-115 cells and did not cause a dose-dependent increase in fatty acid levels at 24 h. There were no significant increases in lactate dehydrogenase activity in N1E-115 neuroblastoma or ROC-1 oligodendroglial cells. In human umbilical vein endothelial cells, montmorillonite, kaolinite and bentonite caused a dose-dependent increase in fatty acids at 24 h. All three clays caused cell lysis. We postulate that the cytotoxicity of the clays containing an aluminum silicate towards endothelial cells may disrupt the blood-brain barrier in the affected areas, allowing the entry of the clay particle into the brain. Aluminum silicate clays caused a dose-dependent release of fatty acids in human umbilical vein endothelial cells. The clays also caused lysis of these cells. ROC-1 oligodendroglia and N1E-115 neuroblastoma cells were not lysed by the clays, suggesting that this is not a general phenomenon.

Aluminum Silicates↗

PubMed1993

A model for compression trauma: pressure-induced injury in cell cultures.

An increase in pressure up to 15 atm was used to condense the cellular membrane of cells in culture thereby eliciting a mechanical-like trauma. This trauma is similar to a compression-like spinal cord injury or brain injury. The cells used in this study were ROC-1 oligodendroglia, N1E-115 neuroblastoma, and human umbilical vein endothelial (HUVE) cells. Total fatty acid (FA) release and release of lactate dehydrogenase (LDH) into the extracellular medium were used as indices of cellular trauma. Pressure-induced FA release, dependent on pressure and pressure duration, occurred with all cell types. The level of pressure needed to cause the greatest increase in FA levels was 10 atm for ROC-1 cells (3 min duration), 15 atm for N1E-115 cells (3 min duration), and 15 atm for HUVE cells (10 min duration). With each cell type, the released FA were reacylated or metabolized between 10 and 30 min of recovery. Following a 12- to 24-h recovery period, N1E-115 and HUVE cells release more FA, indicating that the initial perturbation of the membrane was not fully reversible. LDH levels were significantly increased in both the N1E-115 and HUVE cultures following 24 h of recovery. This efflux of LDH indicates irreversible membrane damage, suggesting that the trauma may be irreversible at longer recovery times.

Atmospheric Pressure↗

PubMed1992

Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths.

31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.

Animals↗

PubMed1992

Extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation.

We tested the hypothesis that extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation. Both differentiated and undifferentiated astroglial cultures were exposed to combined glucose-oxygen deprivation in the presence and absence of extracellular calcium. Lactate dehydrogenase efflux was used as an index of cellular injury. Both types of cultures exhibited significantly less cellular injury when exposed to combined glucose-oxygen deprivation in the absence of extracellular calcium (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: presence of calcium, 65.2 +/- 2.5% vs. absence of calcium, 21.4 +/- 1.3%). To further elucidate the mechanism by which extracellular calcium produces injury, we studied the effect of nimodipine, an L-type calcium channel blocker, on astroglial injury resulting from combined glucose-oxygen deprivation. Nimodipine decreased cellular injury in both types of cultures (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: untreated, 65.4 +/- 2.2% vs. 10 nM nimodipine, 44.6 +/- 4.2%). Extracellular calcium appears to be a mediator of astroglial injury during combined glucose-oxygen deprivation. These results suggest that influx of extracellular calcium via L-type voltage-gated calcium channels may contribute to astroglial injury during cerebral ischemia.

Animals↗

PubMed1992

Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR.

31P-NMR spectra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) region and decreases in the phosphodiester (PDE) region as early as 12 h post partial hepatectomy, which return to normal by 8 days. The compounds primarily responsible for these changes have been identified in perchloric acid extracts as the phosphomonoester phosphoethanolamine and the phosphodiester glycerophosphoethanolamine (GPE), indicating altered phosphatidylethanolamine metabolism. A corresponding increase in diacylglycerol (DAG) levels during regeneration indicates a possible role for a phosphatidylethanolamine-specific phospholipase C in cellular proliferation. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid cellular proliferation. The spectral changes observed in the regenerating rat liver are similar to changes seen in spectra from the livers of human patients in several disease states, indicating that 31P-NMR may allow non-invasive study of cell turnover in liver disease.

Adenosine Triphosphate↗