Biomedical subjects

F Schroeder

Publications and source records attributed to F Schroeder.

At least 73 records · Page 4Linked to original sources

PubMed1997

In vivo measurement of lung preservation solution efficacy: comparison of LPD, UW, EC and low K+-EC following short and extended ischemia.

OBJECTIVE: The impact of storage solution composition on graft performance was evaluated following perfusion with either Euro-Collins (EC), low potassium Euro-Collins (rEC), low potassium dextran (LPD) or University of Wisconsin solution (UW) after brief (2 h) and extended ischemia (16 h) in an acute double lung transplantation model in the rat. METHODS: Following flush perfusion and ischemia the lungs were implanted in recipient rats allowing serial assessment of graft pulmonary vascular resistance (PVR) and alveolar arterial oxygen difference (AaDO2) during 120 min of reperfusion. Graft dynamic lung compliance (DLC) was determined by separate ventilation. Final evaluation included weight gain and histology. RESULTS: After extended ischemia LPD provided superior graft function in respect to DLC (repeated measures ANOVA; LPD versus rEC P < 0.05; versus EC P < 0.03; versus UW P < 0.05) and AaDO2 (LPD versus rEC P < 0.04; versus EC P < 0.006). The PVR was significantly lower in LPD versus UW (P < 0.05). At the end of reperfusion the weight increase amounted to 229 +/- 49% in rEC, 207 +/- 22% in EC, 115 +/- 22% in UW and 87 + 17% in LPD (LPD versus rEC P < 0.01, LPD versus EC P < 0.001). The type of preservation solution used had little impact on graft function after 2 h ischemia. CONCLUSIONS: Low potassium dextran provides superior graft function after extended ischemia. After short ischemia the type of preservation solution used in this study had little impact on global lung function.

Adenosine↗

PubMed1997

Fatty acid uptake in diabetic rat adipocytes.

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structure, lipid composition, and cytosolic fatty acid binding protein.

Adipocytes↗

PubMed1997

Amyloid beta-peptides increase annular and bulk fluidity and induce lipid peroxidation in brain synaptic plasma membranes.

Amyloid beta-peptides (A beta) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of A beta(1-40) and A beta(25-35) on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p < or = 0.02) by A beta(1-40). Similar effects on fluidity were observed for A beta(25-35) (P < or = 0.002). Increased fluidity was associated with lipid peroxidation. Both A beta peptides significantly increased (p < or = 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of A beta on lipid peroxidation and fluidity in SPM. The fluidizing action of A beta peptides on SPM may be due to the induction of lipid peroxidation by those peptides. A beta-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.

Amyloid beta-Peptides↗

PubMed1996

Sterol carrier protein-2, a new fatty acyl coenzyme A-binding protein.

The ability of sterol carrier protein-2 (SCP-2) to interact with long chain fatty acyl-CoAs was examined. SCP-2 bound fluorescent fatty acyl-CoAs at a single site with high affinity. Kd values for cis- and trans-parinaroyl-CoA were 4.5 and 2.8 nM, respectively. Saturated 10-18-carbon and unsaturated 14-20-carbon fatty acyl-CoAs displaced SCP-2-bound fluorescent ligand. Oleoyl-CoA and oleic acid (but not coenzyme A) significantly altered SCP-2 Trp50 emission and anisotropy decay, thereby increasing SCP-2 rotational correlation time, SCP-2 hydrodynamic radius, and SCP-2 Trp50 remaining anisotropy up to 1.7-, 1.2-, and 1.3-fold, respectively. These changes were not accompanied by significant alterations in protein secondary structure as determined by circular dichroism. Finally, SCP-2 differentially altered the fluorescence emission and anisotropy decays of bound cis- and trans-parinaroyl-CoA. Both fluorescent fatty acyl-CoAs were located within a very ordered (limited cone angle of rotation) environment within SCP-2, as shown by a remaining anisotropy of 0.365 and 0.361 and a wobbling cone angle of 12 and 13 degrees , respectively. These anisotropy values were very close to those of such ligands in a propylene glass. However, the rotational relaxation times exhibited by SCP-2-bound cis- and trans-parinaroyl-CoA, 8.4-8.8 ns, were longer than those for the corresponding free fatty acid, 7.5-6.6 ns. These data show for the first time that SCP-2 is a fatty acyl-CoA-binding protein.

Acyl Coenzyme A↗

PubMed1996

Effect of insulin on fatty acid uptake and esterification in L-cell fibroblasts.

We examined the effects of insulin on fatty acid uptake in L-cell fibroblasts, using cis-parinaric acid to measure uptake rates in the absence of esterification and [3H]oleic acid to measure uptake rates in the presence of esterification. L-cells exhibited both high and low affinity insulin binding sites with Kd of 23 nM and 220 nM and a cellular density of 1.4 and 6.8 x 10(5) sites/cell, respectively. Insulin in the range 10(-9) to 10(-7) M significantly decreased both the initial rate and maximal extent of cis-parinaric acid uptake by 24 to 30%. Insulin also reduced [3H]oleic acid uptake up to 35%, depending on insulin concentration and decreased the amount of fatty acid esterified into the phospholipids and neutral lipids by 28 and 70%, respectively. In contrast, glucagon or epinephrine stimulated both the initial rate and extent of cis-parinaric acid uptake 18 and 25%, respectively. Because L-cells lack P-adrenergic receptors, the epinephrine effect was not the result of P-receptor stimulation. Hence, insulin altered not only fatty acid uptake, as determined by cis-parinaric and oleic acid uptake, but also altered the intracellular oleic acid esterification.

Adenylyl Cyclases↗

PubMed1996

Intestinal fatty acid-binding protein expression stimulates fibroblast fatty acid esterification.

The effect of intestinal fatty acid binding protein (I-FABP) expression on cell growth and cell lipid content is not known. Therefore, mouse L-cell fibroblasts were transfected with the cDNA encoding for I-FABP. The high expression clones expressed 0.35% of the total cytosolic proteins as I-FABP. Mock transfected L-cells did not differ from control L-cells in any properties tested. Neither the growth rate, maximal cell density, nor [3H]oleic acid uptake differed in I-FABP expressing as compared to control cells. In contrast, I-FABP expression increased triacylglycerol and cholesteryl ester mass (nmol/mg protein) by 63% and 25%, respectively. Phospholipid mass was unchanged in I-FABP expressing cells. The initial [3H]oleic acid esterification into triacylglycerols and cholesteryl esters was increased 3.9- and 2.5-fold in I-FABP expressing cells. Although, the initial [3H]oleic acid esterification into total phospholipids was unchanged, within the phospholipid fraction the initial [3H]oleic acid esterification into phosphatidylethanolamine was increased 70% and decreased 50% in phosphatidylcholine in I-FABP expressing cells. These observed differences suggest a distinct role for I-FABP in stimulating net formation, and not just turnover, of triacylglycerides and cholesteryl esters in transfected L-cell fibroblasts.

Animals↗

PubMed1996

Fluorescence studies of lipid association-induced conformational adaptations of an exchangeable amphipathic apolipoprotein.

The conformational adaptability of Manduca sexta apolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145 is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145 and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKa of Tyr145 in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the (lambda)max of absorbance of Tyr145 was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145 fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145 in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145 exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145 remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145 which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145 is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145 distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.

Animals↗

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 and intestinal fatty acid binding proteins in control and TGF beta 1 gene targeted deficient mice.

The effect of transforming growth factor beta-1 (TGF beta 1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF beta 1-deficient mice. Homozygous TGF beta 1-deficient 129 x CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF beta 1-deficient/immunodeficient C3H mice on a SCID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (I-FABP) which decreased 3-fold in the TGF beta 1-deficient/immunodeficient C3H mice only. Thus, TGF beta 1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.

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↗

PubMed1996

Direct binding of ethanol to bovine serum albumin: a fluorescent and 13C NMR multiplet relaxation study.

Molecular mechanisms of ethanol interaction with proteins are not well-understood. In the present study, direct interaction of ethanol with hydrophobic binding sites on fatty acid free bovine serum albumin (BSA) was determined using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), cis-parinaric acid, and 13C NMR. The affinity of ethanol for BSA (Kd) was (5.21 +/- 0.31) x 10(-2) mol. Ethanol (25-200 mmol) competitively inhibited 1,8-ANS binding to BSA in a concentration-dependent manner with a Ki (concentration of ethanol that decreased 1,8-ANS binding by 50%) of 658 mmol. Preincubation of BSA with ethanol significantly decreased cis-parinaric acid binding to BSA, indicating interaction of ethanol with hydrophobic fatty acid-binding site(s) on BSA. Furthermore, ethanol was found to act on three of the five fatty acid-binding sites on BSA. These data indicated selectivity in the interaction of ethanol with hydrophobic sites on BSA. 13C NMR multiplet relaxation was used to characterize the interaction of ethanol with binding sites on BSA. Detailed analysis of [13C]ethanol relaxation data obtained in the presence of increasing BSA concentrations (25-200 mg/mL) led to the conclusion that the ethanol methyl group, as opposed to its hydroxyl group, binds in a hydrophobic pocket(s) on the protein. Ethanol-induced changes in activity of certain proteins may result from direct binding of ethanol to specific hydrophobic binding sites and/or displacement of endogenous ligands from those sites.

Anilino Naphthalenesulfonates↗

PubMed1996

Acyl-CoA binding proteins: multiplicity and function.

The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

Animals↗

PubMed1996

Cycloheximide-induced apoptosis in melanoma cells derived from regressing cutaneous tumours of Sinclair swine.

The role of apoptosis in the spontaneous regression of Sinclair swine melanoma was investigated in vitro with swine melanoma cell lines. Growth characteristics and sensitivity to cycloheximide-induced apoptosis were determined in melanoma cell lines derived from tumours that were progressing or undergoing regression in vivo. In contrast to cell lines derived from progressing tumours, those derived from regressing tumours showed induction of apoptosis; this phenomenon was dependent on dose but independent of cell growth stage in culture. Chromatin condensation, cell shrinkage, and fragmentation into apoptotic bodies, as well as DNA fragmentation into large kilobase fragments, occurred in cell lines from regressing tumours but not from progressing tumours. These findings suggest that swine melanoma cells are inherently more sensitive to cell death during tumour regression. The apoptosis-sensitive and resistant cell lines will be important for further studies of the roles of cell signalling pathways and gene expression in tumour regression.

Animals↗

PubMed1996

Effects of chronic ethanol consumption on sterol transfer proteins in mouse brain.

Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross-reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein-2 (SCP-2) and the liver form of fatty acid-binding protein (L-FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP-2 and L-FABP were used to identify the lipid transfer proteins in the brains of alcohol-treated and control mice. L-FABP was not detectable in brain of either control or chronic ethanol-treated mice. In contrast, SCP-2 not only was present, but its level was significantly (p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP-2 reflected true levels of SCP-2 in brain, the cDNA sequence for brain SCP-2 was isolated from a brain cDNA library. The mouse brain SCP-2 sequence was 99.99% identical to the mouse liver SCP-2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP-2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl-CoA binding protein, a lipid-binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP-2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP-2 may contribute to membrane tolerance to ethanol.

Alcoholism↗

PubMed1996

Isolation and characterization of two fatty acid binding proteins from mouse brain.

Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.

Animals↗

PubMed1996

Increasing age alters transbilayer fluidity and cholesterol asymmetry in synaptic plasma membranes of mice.

Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3-4-, 14-15-, and 24-25-month old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.

Aging↗