Astrocyte-specific expression of tyrosine hydroxylase after intracerebral gene transfer induces behavioral recovery in experimental parkinsonism.
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Parkinson's disease is a neurodegenerative disorder characterized by the depletion of dopamine in the caudate putamen. Dopamine replacement with levodopa, a precursor of the neurotransmitter, is presently the most common treatment for this disease. However, in an effort to obtain better therapeutic results, tissue or cells that synthesize catecholamines have been grafted into experimental animals and human patients. In this paper, we present a novel technique to express tyrosine hydroxylase (TH) in the host's own astrocytes. This procedure uses a transgene in which the expression of a TH cDNA is under the control of a glial fibrillary acidic protein (GFAP) promoter, which confers astrocyte-specific expression and also increases its activity in response to brain injury. The method was tested in a rat model of Parkinson's disease produced by lesioning the striatum with 6-hydroxydopamine. Following microinjection of the transgene into the denervated striatum as a DNA-liposome complex, expression of the transgene was detected by RT-PCR and TH protein was observed specifically in astrocytes by using double-labeling immunofluorescence for GFAP and TH coupled with laser confocal microscopy. Efficacy was demonstrated by significant behavioral recovery, as assessed by a decrease in the pharmacologically induced turning behavior generated by the unilateral denervation of the rat striatum. These results suggest this is a valuable technique to express molecules of therapeutic interest in the brain. (+info)
Dietary control of triglyceride and phospholipid synthesis in rat liver slices.
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1. The effect of dietary manipulation on the synthesis of triglycerides and phospholipids was investigated by determining the incorporation of labeled long-chain fatty acid or glycerol into these lipids in liver slices derived from normally fed, fasted, and fat-free refed rats. 2. Triglyceride synthesis was affected markedly by the dietary regime of the animal; the lowest rates were measured with fasted rats, and the highest ones with fat-free refed rats. 3. In contrast to triglyceride synthesis, phospholipid synthesis occured at virtually constant rates regardless of the dietary conditions. 4. Addition of large amounts of fatty acid to the incubation mixture resulted in a marked stimulation of triglyceride synthesis, whereas phospholipid synthesis was affected to a much smaller extent. 5. These results indicate that the synthesis of triglycerides and that of phospholipids are controlled independently, and that the availability of fatty acid in the cell contributes to the control of triglyceride synthesis. (+info)
Kinetic and thermodynamic aspects of lipid translocation in biological membranes.
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A theoretical analysis of the lipid translocation in cellular bilayer membranes is presented. We focus on an integrative model of active and passive transport processes determining the asymmetrical distribution of the major lipid components between the monolayers. The active translocation of the aminophospholipids phosphatidylserine and phosphatidylethanolamine is mathematically described by kinetic equations resulting from a realistic ATP-dependent transport mechanism. Concerning the passive transport of the aminophospholipids as well as of phosphatidylcholine, sphingomyelin, and cholesterol, two different approaches are used. The first treatment makes use of thermodynamic flux-force relationships. Relevant forces are transversal concentration differences of the lipids as well as differences in the mechanical states of the monolayers due to lateral compressions. Both forces, originating primarily from the operation of an aminophospholipid translocase, are expressed as functions of the lipid compositions of the two monolayers. In the case of mechanical forces, lipid-specific parameters such as different molecular surface areas and compression force constants are taken into account. Using invariance principles, it is shown how the phenomenological coefficients depend on the total lipid amounts. In a second approach, passive transport is analyzed in terms of kinetic mechanisms of carrier-mediated translocation, where mechanical effects are incorporated into the translocation rate constants. The thermodynamic as well as the kinetic approach are applied to simulate the time-dependent redistribution of the lipid components in human red blood cells. In the thermodynamic model the steady-state asymmetrical lipid distribution of erythrocyte membranes is simulated well under certain parameter restrictions: 1) the time scales of uncoupled passive transbilayer movement must be different among the lipid species; 2) positive cross-couplings of the passive lipid fluxes are needed, which, however, may be chosen lipid-unspecifically. A comparison of the thermodynamic and the kinetic approaches reveals that antiport mechanisms for passive lipid movements may be excluded. Simulations with kinetic symport mechanisms are in qualitative agreement with experimental data but show discrepancies in the asymmetrical distribution for sphingomyelin. (+info)
Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids.
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The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins. (+info)
Antibodies against phospholipids and oxidized LDL in alcoholic patients.
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Antiphospholipid antibodies (APA) are a generic term describing antibodies that recognize various phospholipids. Hepatocyte damage is a cardinal event in the course of alcoholic liver injury and autoantibodies against phospholipids could play an important role in this process. APA in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression and they correlate significantly with disease severity. LDL oxidation is supposed to be one of the most important pathogenic mechanisms of atherosclerosis and antibodies against oxidized low-density lipoprotein (oxLDL) are some kind of an epiphenomenon of this process. The scope of our study was to determine some autoantibodies (IgG-oxLDL and antiphospholipid antibodies) and their possible changes in alcoholic patients. We studied IgG-oxLDL and four APA - anticardiolipin antibodies (ACA), antiphosphatidylserine antibodies (APSA) antiphosphatidylethanolamine antibodies (APE) and antiphosphatidylcholine antibodies (APCA) in 35 alcoholic patients with mildly affected liver function at the beginning of the abuse treatment. The control group consisted of 60 healthy blood donors. In the studied group, we obtained positive results concerning total ACA in 17.1 % of alcoholic patients (8.3 % in the control group), 11.4 % IgG-ACA (6.7 %), 8.6 % IgM-ACA (3.3 %), 14.3 % total APE (6.7 %), 14.3 % total APCA (8.3 %) and 20 % total APSA (8.3 % in the control group). The IgG-oxLDL (406.4+/-52.5 vs 499.9+/-52.5 mU/ml) was not affected in alcoholic patients. We conclude that the autoantibodies against oxLDL are present in sera of alcoholics and healthy blood donors. Based on our results which revealed a wide range of IgG-oxLDL titres in the healthy population, this parameter does not appear to be very promising for the evaluation of the risk of atherosclerosis. Alcoholics with only mild affection of liver functions did not exhibit a significantly higher prevalence of all studied antiphospholipid antibodies (ACA, APSA, APE, APCA) which could lead to membrane lesions in these patients. (+info)
Accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31p NMR study.
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Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been used to study accumulation of N-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl3-CH3OH-H2O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacyl-sn-glycero-3-phosphocholine (PCDIACYL) at -0.84 ppm). These signals were identified as originating from 1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (NAPEDIACYL) and 1-(1'-alkenyl)-2-acyl-sn -glycero-3-phospho-(N-acyl)-ethanolamine (NAPEPLAS), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N-acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N-acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with K-EDTA. The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPEDIACYL and NAPEPLAS) in rat brains by the use of 31P NMR spectroscopy. (+info)
Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium.
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A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme. (+info)
Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells.
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Formation of intracellular vesicles is initiated by membrane budding. Here we test the hypothesis that the plasma membrane surface area asymmetry could be a driving force for vesicle formation during endocytosis. The inner layer phospholipid number was therefore increased by adding exogenous aminophospholipids to living cells, which were then translocated from the outer to the inner layer of the membrane by the ubiquitous flippase. Addition of either phosphatidylserine or phosphatidylethanolamine led to an enhancement of endocytosis, showing that the observed acceleration does not depend on the lipid polar head group. Conversely, a closely related aminophospholipid that is not recognized by the flippase, lyso-alpha-phosphatidylserine, inhibited endocytosis, and similar results were obtained with a cholesterol derivative that also remains in the plasma membrane outer layer. Thus an increase of lipid concentration in the inner layer enhanced internalization, whereas an increase of the lipid concentration in the outer layer inhibited internalization. These experiments suggest that transient asymmetries in lipid concentration might contribute to the formation of endocytic vesicles. (+info)