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1.
The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1-14C]arachidonic acid into synaptosomal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration-dependent manner, with 50% inhibition occurring at less than 25 microM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non-competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.  相似文献   

2.
Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.  相似文献   

3.
Fatty acid beta-oxidation is a key process in mammalian lipid catabolism. Disturbance of this process results in severe clinical symptoms, including dysfunction of the liver, a major beta-oxidizing tissue. For a thorough understanding of this process, a comprehensive analysis of involved fatty acid and acyl-carnitine intermediates is desired, but capable methods are lacking. Here, we introduce oxaalkyne and alkyne fatty acids as novel tracers to study the beta-oxidation of long- and medium-chain fatty acids in liver lysates and primary hepatocytes. Combining these new tracer tools with highly sensitive chromatography and mass spectrometry analyses, this study confirms differences in metabolic handling of fatty acids of different chain length. Unlike longer chains, we found that medium-chain fatty acids that were activated inside or outside of mitochondria by different acyl-CoA synthetases could enter mitochondria in the form of free fatty acids or as carnitine esters. Upon mitochondrial beta-oxidation, shortened acyl-carnitine metabolites were then produced and released from mitochondria. In addition, we show that hepatocytes ultimately also secreted these shortened acyl chains into their surroundings. Furthermore, when mitochondrial beta-oxidation was hindered, we show that peroxisomal beta-oxidation likely acts as a salvage pathway, thereby maintaining the levels of shortened fatty acid secretion. Taken together, we conclude that this new method based on oxaalkyne and alkyne fatty acids allows for metabolic tracing of the beta-oxidation pathway in tissue lysate and in living cells with unique coverage of metabolic intermediates and at unprecedented detail.  相似文献   

4.
Weaning mice were fed a diet supplemented with beef tallow (BT) or BT plus docosahexaenoic acid (DHA) containing 100 mg alpha-tocopherol/kg (alpha-Toc100) or 500 mg alpha-tocopherol/kg (alpha-Toc500) for 4 wk to modify membrane fatty acid unsaturation, and then were administered ferric nitrilotriacetate (Fe-NTA). The mortality caused by Fe-NTA was higher in the group fed the DHA (alpha-Toc100) diet than in the BT diet groups but the DHA (alpha-Toc500) diet suppressed this increase. Serum and kidney alpha-tocopherol contents were slightly influenced by the dietary fatty acids but not significantly. These results indicate that the increased unsaturation of tissue lipids enhances oxidative damage induced by Fe-NTA in mice fed DHA (alpha-Toc100) but not when additional alpha-tocopherol is supplemented. The apparent discrepancy between the observed enhancement by dietary DHA of oxidative damage and the beneficial effects of dietary DHA on the so-called free radical diseases is discussed in terms of strong bolus oxidative stress and moderate chronic oxidative stress.  相似文献   

5.
The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.  相似文献   

6.
The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.  相似文献   

7.
Rat thymocytes, spleen lymphocytes and isolated nuclei were incubated with fatty acids and then labelled with 5-doxylstearic acid and 12-doxylstearic acid. The ESR spectra only in the case of 5-doxylstearic acid showed changes which were demonstrable only under those conditions which resulted in cytolysis. Thymocytes in medium with 10% serum showed the effect at 10 microM, splenic lymphocytes at 100 microM. The effect was maximal at 2 min and was not enhanced by higher concentrations. The uptake of fatty acid by spleen cells required to cause this change was determined using 14C-oleic acid, to be 0.6 mumol/g tissue. This quantity is less than that required (label:lipid ratio less than 1:10) to produce major perturbations in membranes. Free fatty acids of C-8 to C-18 produced the effect, but not esters or amides. It was concluded that free fatty acids induce changes proximal to the polar region of membrane lipids which, if not progressive and essential to the ultimate process of lysis, are at least indicative of impending cell death at an early time.  相似文献   

8.
Neurocalcin is a member of a novel family of neuronal calcium sensors that belongs to the superfamily of EF-hand Ca(2+)-binding proteins. Neurocalcin is myristoylated on its N-terminus in vivo and can associate with biological membranes in a calcium and myristoyl-dependent manner. This process known as "Ca(2+)-myristoyl switch" has been best described for the photoreceptor specific protein, recoverin, as well as for several other neuronal calcium sensors. Here, we used reversed micelles to chemically acylate nonmyristoylated neurocalcin at its N-terminus with fatty acids of different lengths (from C12 to C16). This approach allowed us to prepare neurocalcin derivatives in which a single fatty acid is selectively linked to the N-terminal glycine of the polypeptide chain through an amide bond. The membrane binding properties of the monoacylated neurocalcins were then examined by cosedimentation with phospholipid vesicles and direct binding to lipid monolayers by surface plasmon resonance spectroscopy (Biacore). Our results show that neurocalcins monoacylated with lauric, myristic, or palmitic acid were able to associate with membrane in a calcium-dependent manner. This indicates that the Ca(2+)-myristoyl switch can function with different lipid moieties and is not strictly restricted to myristate. The ability to modify at will the fatty acid linked to the N-terminal glycine should be useful to analyze the contribution of the fatty acid moiety to the biological function of this family of neuronal calcium sensors.  相似文献   

9.
Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.  相似文献   

10.
Leaf Fatty-Acid Content in Relation to Hardening and Chilling Injury   总被引:4,自引:0,他引:4  
At 25?C the leaves of chill-resistant plants did not containmore unsaturated fatty acid than chill-sensitive plants. Furthermorewhen chill-sensitive plants were hardened at 12?C there wasno increase in the amount of unsaturated fatty acid or totalweight of fatty acid in the leaf. These results cast furtherdoubt on the importance of the degree of unsaturation of thefatty acids in determining the critical temperature at whichthe phase change occurs in the lipid portion of the membrane. When chill-sensitive plants are chilled (5?C) the percentageof linolenic acid and total weight of fatty acid decreases rapidlybut remains constant in chill-resistant species. Hardening mayreduce the degree of damage to the plant by slowing down detrimentalprocesses such as the decrease in the percentage of linolenicacid which occurs on chilling.  相似文献   

11.
Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.  相似文献   

12.
In the course of liver injury induced by CCl4 in rats the change of the endoplasmic reticulum takes 5 hours and that of the lysosomal membrane, 18 hours to develop. The latter change precedes hepatocellular necrosis. Elevation of plasma free fatty acids and fatty infiltration of the liver can be observed at 3 hours after CCl4 administration. The maximum of fatty infiltration, hepatocellular necrosis and the highest degree of lysosomal damage develop at the same time. Since CCl4 is eliminated in a few hours, it must initiate a cellular process which then leads to lysosomal membrane damage and hepatocellular necrosis.  相似文献   

13.
Encapsulation of polyuridylic acid in phospholipid vesicles.   总被引:2,自引:0,他引:2  
Entrapment of polyuridylic acid by neutral, positive and negatively charged phospholipid multilamellar vesicles was studied. The polyuridylic acid was found to be involved with the liposomes in two ways. Liposome-associated polyuridylic acid was readily degraded by bovine pancreatic RNase, while entrapped polynucleotide was found to be RNase-resistant. Sepharose 4B column chromatography showed the presence of liposome-associated and liposome entrapped polynucleotide. Approximately 14–26% of the polynucleotide became entrapped in the liposomes. Multilamellar vesicles prepared with dipalmitoylphosphatidylcholine or purified egg lecithin did not differ in the amount of polynucleotide entrapped nor in Sepharose 4B column chromatography behavior. Entrapment in liposomes protected the polynucleotide from degradation by serum nucleases.  相似文献   

14.
细胞液中乙酰辅酶A的持续供应是脂肪酸高效积累的必要条件。考虑到甲羟戊酸和脂肪酸合成途径共用相同的前体乙酰辅酶A,抑制甲羟戊酸途径可能促使更多的乙酰辅酶A流向脂肪酸合成。通过添加前体物质或/和甲羟戊酸途径酶的抑制剂以强化乙酰辅酶A的供应,即在裂殖壶菌发酵起始或/和后期添加乙酸、发酵起始添加甲羟戊酸途径酶的抑制剂辛伐他汀或柠檬酸、发酵起始同时添加乙酸和辛伐他汀或柠檬酸并考察其对裂殖壶菌合成二十二碳六烯酸 (DHA)的影响,结果发现发酵起始同时添加6mmol/L的乙酸和1μmol/L的辛伐他汀时,DHA产量最高,达到13.21g/L,比对照提高了46.61%。  相似文献   

15.
The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.  相似文献   

16.
One approach to studying the importance of membranes in freeze-thaw damage is to modify their composition and study the effect of this modification on survival after freeze-thaw damage. Fatty acid desaturase auxotrophs of yeast cells were enriched with two fatty acids having substantially different physical properties thus resulting in cells whose membranes had very different physical properties. The fatty acids were stearolic acid (mp = +45 °C) and linolenic acid (mp = ?10 °C). Electron-spin resonance studies showed that membranes containing the latter fatty acid were more fluid than those containing stearolic acid. The yeast were grown under either anaerobic or aerobic conditions. In the former case, the mitochondria appear as membraneous shells with little, if any, internal membrane structure; thus, the plasma and tonoplast membranes are the primary membranes. Yeast cells grown under these conditions survived freezethaw damage (?196 °C) significantly better when the fatty acid composition was mainly stearolic acid rather than linolenic acid. The absolute survival depended on the freezing rate and the differences in survival became small at fast rates. With yeast cells grown under aerobic conditions, when functional mitochondria are formed, the pattern in freeze-thaw survival reversed; cells with γ-linolenic acid in their membranes survived significantly better than cells containing stearolic acid.  相似文献   

17.
Binding of the recA gene product from Escherichia coli to single-stranded polynucleotides has been investigated using poly(dA) that have been modified by chloroacetaldehyde to yield fluorescent 1,N6-ethenoadenine (epsilon A) bases. A strong enhancement of the fluorescent quantum yield of poly(d epsilon A) is induced upon RecA protein binding. A 4-fold increase is observed in the absence of ATP or ATP gamma S and a 7-fold increase in the presence of either nucleoside triphosphate. RecA protein can bind to poly(d epsilon A) in the absence of both Mg2+ ions and ATP (or ATP gamma S) but Mg2+ ions are required to observe RecA protein binding in the presence of ATP (or ATP gamma S) at pH 7.5. ATP binding to the RecA-poly(d epsilon A) complex induces a dissociation of RecA from the polynucleotide followed by re-binding of [RecA-ATP-Mg2+] ternary complex. Whereas ATP-induced dissociation of RecA-poly(d epsilon A) complexes is a fast process, the subsequent binding reaction of [RecA-ATP-Mg2+] is slow. A model is proposed whereby [RecA-ATP-Mg2+] binding to poly(d epsilon A) involves slow nucleation and elongation processes along the polynucleotide backbone. The nucleation reaction is shown to involve at least a trimer or a tetramer. Polymerization of the [RecA-ATP-Mg2+] ternary complex stops when the polynucleotide is entirely covered with 6 +/- 1 nucleotides per RecA monomer. ATP hydrolysis then induces a release of RecA-ADP complexes from the polynucleotide template.  相似文献   

18.
Young adult rats, either control or essential fatty acid deficient, were administered either [3-H] oleic acid or [3-H] arachidonic acid by stomach tube. In addition, a group of control rats was given [3-H] palmitic acid. The rats were killed at various times therafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, wheras the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.  相似文献   

19.
The 3-thia fatty acid tetradecylthioacetic acid (TTA) is a synthetic modified fatty acid, which, similar with dietary fish oil (FO), influences the regulation of lipid metabolism, the inflammatory response and redox status. This study was aimed to penetrate the difference in TTA's mode of action compared to FO in a long-term experiment (50 weeks of feeding). Male Wistar rats were fed a control, high-fat (25% w/v) diet or a high-fat diet supplemented with either TTA (0.375% w/v) or FO (10% w/v) or their combination. Plasma fatty acid composition, hepatic lipids and expression of relevant genes in the liver and biomarkers of oxidative damage to protein were assessed at the end point of the experiment. Both supplements given in combination demonstrated an additive effect on the decrease in plasma cholesterol levels. The FO diet alone led to removal of plasma cholesterol and a concurrent cholesterol accumulation in liver; however, with TTA cotreatment, the hepatic cholesterol level was significantly reduced. Dietary FO supplementation led to an increased oxidative damage, as seen by biomarkers of protein oxidation and lipoxidation. Tetradecylthioacetic acid administration reduced the levels of these biomarkers confirming its protective role against lipoxidation and protein oxidative damage. Our findings explore the lipid reducing effects of TTA and FO and demonstrate that these bioactive dietary compounds might act in a different manner. The experiment confirms the antioxidant capacity of TTA, showing an improvement in FO-induced oxidative stress.  相似文献   

20.
Mutants of Saccharomyces cerevisiae, deficient in cytosolic superoxide dismutase and catalase activities were used to study the role of various oxygen species in the process of lipid peroxidation in yeast cells. Lipid peroxidation does not occur normally in yeast, because this organism is unable to form fatty acids with more than one double bond, whereas under physiological conditions, only fatty acids with at least two double bonds undergo this process. The fatty acid content of cellular lipids was modified by growing the cells in anoxia in the presence of oleic or linolenic acid. Toxic effects of oxygen were observed almost exclusively in those cells of yeast mutants deficient in superoxide dismutase, which contain linolenic acid in cellular lipids. Hypersensitivity of the mutant cells, however, results mainly from toxic effects of the products of autooxidation of extracellular fatty acids. These facts suggest that superoxide dismutases are in some way involved in preventing toxic effects of the products of lipid peroxidation and to some extent prevent the process of lipid peroxidation.  相似文献   

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