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Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers.

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

Amino Acid Sequence↗

Effects of acylation on the structure, lipid binding, and transfer activity of wheat lipid transfer protein.

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.

Acylation↗

Low-density lipoprotein is the major carrier of lipid hydroperoxides in plasma. Relevance to determination of total plasma lipid hydroperoxide concentrations.

High-density lipoprotein (HDL) has been proposed as the principal carrier of hydroperoxides in plasma, based upon data gathered with an HPLC-chemiluminescence technique. To test this hypothesis we have measured total lipid hydroperoxides in native plasma using the ferrous oxidation in Xylenol Orange (FOX) assay and then fractionated plasma into very-low-density lipoprotein (LDL) and HDL fractions. Hydroperoxides were found to accumulate principally (more than 65%) in LDL, as judged by hydroperoxide content per amount of protein or cholesterol, or expressed as a proportion of total hydroperoxide in plasma. Plasma was also incubated at 37 degrees C in the presence and absence of 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), an azo-initiator of lipid peroxidation. The majority of hydroperoxides generated in plasma were recovered in the LDL fraction. Furthermore, when isolated lipoproteins were subject to oxidation initiated by AAPH, very-low-density lipoprotein and LDL showed the greatest propensity for hydroperoxide accumulation, whereas HDL seemed relatively resistant. Estimates for plasma and LDL peroxidation based upon techniques which measure total lipid hydroperoxides suggest that levels of hydroperoxides in plasma and LDL are far higher than that those estimates generated by ostensibly more selective techniques. Higher levels of hydroperoxides in LDL than those reported by HPLC-chemiluminescence also seem in greater accordance with other available data concerning LDL oxidation.

Amidines↗

Free radical initiation in proteins and amino acids by ionizing and ultraviolet radiations and lipid oxidation--part III: free radical transfer from oxidizing lipids.

Parallels and similarities in chemical and functional damage to proteins by ionizing and uv radiations and oxidizing lipids have been recognized for some time. However, only recently have oxidizing lipids been shown directly by electron spin resonance to be radiomimetic also in their capacity for protein free radical production. Free radicals play a key role in the transformation of energy to molecular and cellular damage. It is thus of critical importance to elucidate the general mechanisms of free radical formation and reactions in proteins in order to understand protein involvement in various pathological conditions and in food deterioration. Accordingly, this review is a detailed comparison of gamma-radiation, UV radiation, and lipid oxidation for what is presently known concerning (1) the specific modes of energy deposition and free radical formation, (2) the free radicals formed in proteins and amino acids, and (3) the typical damage correlating with these radicals.

Amino Acids↗

A soy protein diet alters hepatic lipid metabolism gene expression and reduces serum lipids and renal fibrogenic cytokines in rats with chronic nephrotic syndrome.

Nephrotic syndrome (NS) is characterized by the presence of proteinuria and hyperlipidemia. However, ingestion of soy protein has a hypolipidemic effect. The present study was designed to determine whether the ingestion of a 20% soy protein diet regulates the expression of hepatic sterol regulatory element binding protein (SREBP)-1, fatty acid synthase (FAS), malic enzyme, beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) reductase (r) and synthase (s), and LDL receptor (r), and to assess whether soy protein improves lipid and renal abnormalities in rats with chronic NS. Male Wistar rats were injected with vehicle or with puromycin aminonucleoside to induce NS and were fed either 20% casein or soy protein diets for 64 d. NS rats fed 20% soy protein had improved creatinine clearance and reduced proteinuria, hypercholesterolemia, hypertriglyceridemia, as well as VLDL-triglycerides and LDL cholesterol compared with NS rats fed the 20% casein diet. In addition, the soy protein diet decreased the incidence of glomerular sclerosis, and proinflammatory cytokines in kidney. Ingestion of the soy protein diet by control rats reduced the gene expression of SREBP-1, malic enzyme, FAS and increased HMG-CoAr, HMG-CoAs and LDLr. However, NS rats fed either casein or soy protein diets had low insulin concentrations with reductions in SREBP-1, FAS and malic enzyme expression compared with control rats fed the casein diet. NS rats fed the soy diet also had lower HMG-CoAr and LDLr mRNA levels than NS rats fed casein. In conclusion, the beneficial effects of soy protein on lipid metabolism are modulated in part by SREBP-1. However, in NS rats, the benefit may be through a direct effect of this protein on kidney rather than mediated by changes in expression of hepatic lipid metabolism genes.

Animals↗

Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells).

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.

Animals↗

Neutral lipid storage disease. Case report and lipid studies.

A 9-year-old boy of Greek-Cypriot origin had been diagnosed at the age of 3 years as suffering from non-bullous ichthyosiform erythroderma. However, he also had hepatomegaly and abnormal liver function tests, biochemical evidence of myopathy, early cataracts, and lipid vacuoles in white blood cells and basal keratinocytes. A diagnosis of neutral lipid (triglyceride) storage disease was confirmed by lipid studies on cultured fibroblasts.

Child↗

Effects of testosterone on lipid peroxidation, lipid profiles and some coagulation parameters in rabbits.

The purpose of this study was to investigate the effects of testosterone on some risk factors of atherosclerosis. Twenty-four male New Zealand white rabbits were randomly divided into three groups of eight. The first group was used as control. Second group was injected with 10 mg of testosterone propionate. Third group was castrated bilaterally. At the end of 6 weeks, lipid peroxidation (LPO), lipid profile, fibrinogen (FBN) level and coagulation parameters were evaluated. Testosterone administration decreased the level of high-density lipoprotein cholesterol (HDL-C), while castration increased this level (P < 0.05). Triglyceride (TG) and total cholesterol (TC) levels in the castration group were significantly higher (P < 0.05) than those in the testosterone group. The ratio of HDL-C:low-density lipoprotein cholesterol (LDL-C) decreased, while TC:HDL-C ratio increased (P < 0.05) in the testosterone group. No significant differences were found in the LDL-C and FBN levels among groups. However, there was a tendency for higher FBN level in the testosterone group. Testosterone administration resulted in an increase in the level of LPO (P < 0.05). Clotting time and prothrombin time prolonged in the castration group compared with testosterone group (P < 0.05). As a result, testosterone has exacerbating effect on atherosclerosis risk factors including lipid profile, LPO, FBN and coagulation system.

Animals↗

Lipid growth requirement and influence of lipid supplement on fatty acid and aldehyde composition of Syntrophococcus sucromutans.

Results concerning the ruminal fluid growth requirement of the ruminal acetogen, Syntrophococcus sucromutans, indicate that octadecenoic acid isomers satisfy this essential requirement. Complex lipids, such as triglycerides and phospholipids, can also support growth. The cellular fatty acid and aldehyde composition closely reflects that of the lipid supplement provided to the cells. Up to 98% of the fatty acids and 80% of the fatty aldehydes are identical in chain length and degree of unsaturation to the octadecenoic acid supplement provided in the medium. S. sucromutans shows a tendency to have a greater proportion of the aldehyde form among its 18 carbon chains than it does with the shorter-chain simple lipids, which may be interpreted as a strategy to maintain membrane fluidity. 14C labeling showed that most of the oleic acid taken up from the medium was incorporated into the membrane fraction of the cells.

Aldehydes↗

LIPIDS OF SARCINA LUTEA. II. HYDROCARBON CONTENT OF THE LIPID EXTRACTS.

Albro, Phillip W. (Ft. Detrick, Frederick, Md.), and Charles K. Huston. Lipids of Sarcina lutea. II. Hydrocarbon content of the lipid extracts. J. Bacteriol. 88:981-986. 1964.-The hydrocarbon fraction from Sarcina lutea lipid extracts was characterized by a combination of thin-layer and gas-liquid chromatography and infrared spectroscopy. A total of 37 components were observed by gas-liquid chromatography of this material. A breakdown of the components into classes indicated a composition consisting of 88.9% n-saturates, 1.2% monoenes, 2.1% dienes, 5.0% trienes, and 0.6% branched-saturates. Less than 0.1% of the hydrocarbon material was aromatic. No attempt was made in this study to relate the composition to either origin or function in the cell.

Chromatography↗

Body lipids of guinea pigs exposed to different dietary fats from mid-gestation to 3 months of age. II. The fatty acid composition of the lipids of liver, plasma, adipose tissue, muscle and red cell membranes at birth.

Pregnant guinea pigs were fed one of three diets: a commercial low-fat diet, or a high-fat diet containing maize oil or beef dripping. The young were killed at birth and the fatty acid composition of the lipids of the liver, plasma, adipose tissue, quadriceps muscle and red cell membranes was determined. Compared with those fed the commercial diet the tissue of the young of mothers fed maize oil had an increased percentage of linoleic acid whereas in those of the young of mothers fed beef dripping, the percentage of oleic acid was significantly higher. These changes occurred both in the triglycerides and in the membrane phospholipids (with the exceptin of sphingomyelins) of all tissues examined. These results demonstrate that the fatty acid composition of the tissue lipids of the newborn guinea pig can be influenced by the fatty acid composition of the maternal diet. Changes in the fatty acid composition of membrane phospholipids can be as great as those in storage lipids. This raises the question of long-term physiological significance for the animal.

Adipose Tissue↗

Hormonal contraceptive increases plasma lipid peroxides in female rats. Relationship to platelet aggregation and lipid biosynthesis.

We investigated whether changes in plasma oxidative properties could occur after oral (hormonal) contraceptive (OC) administration in female rats and whether such changes could be responsible for the platelet increase in aggregation and lipid biosynthesis observed with that treatment. Platelets and plasma (platelet-poor) from control and OC (ethinyl estradiol + lynestrenol)-treated rats were prepared separately. Thrombin-induced aggregation of control platelets was markedly enhanced after incubation for 4 (p less than 0.025) to 60 (p less than 0.001) minutes in OC as compared with control plasma. Under the same conditions, platelet lipid biosynthesis was increased also (p less than 0.05 to p less than 0.01), but after 3 hours incubation. The enhanced response of platelets to aggregation induced by OC plasma could be inhibited by adding either glutathione (p less than 0.025), vitamin E (p less than 0.025), catalase (p less than 0.05), or peroxidase + glutathione (p less than 0.005) to plasma or 2,6,di-bis(ter-butyl)p-cresol (p less than 0.05) to platelets before incubation. The peroxidized free fatty acids isolated from OC plasma added to normal platelets induced a 150% (p less than 0.001) increase in the response to thrombin as compared with the fatty acids from control plasma. In addition, the level of malondialdehyde and conjugated dienes was significantly (p less than 0.02 to p less than 0.001) increased in OC compared with control plasma. We conclude that the enhanced formation in plasma of lipid hydroperoxides seems to be the initial event stimulating platelets after OC treatment, at least in rats.

Adenosine Diphosphate↗

Dietary manipulation by perilla oil and fish oil of hepatic lipids and its influence on peroxisomal beta-oxidation and serum lipids in rat and mouse.

Rats and mice were fed a diet, prepared with soybean oil (SO), perilla oil (PO) or fish oil (FO), for 4 weeks. Compared with the groups of SO-feeding, FO-feeding increased content of eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) and PO-feeding elevated the content of alpha-linolenic acid (18:3 n-3), 20:5 n-3 and 22:5 n-3 or hepatic lipids of both rats and mice. FO-feeding increased the activity of peroxisomal beta-oxidation in the livers of both rats and mice. The activities of peroxisomal beta-oxidation in rats and mice that were fed FO diet at a high fat (40% of energy) concentration corresponded to 20% and 30%, respectively, of the maximum activities induced by peroxisome proliferators (4-chlorophenoxyisobutyric acid or perfluorooctanoic acid). To a lesser extent, PO-feeding elevated this activity too. There were significant correlations between peroxisomal beta-oxidation activity and the content of either 20:5 n-3, 22:5 n-3 or 22:6 n-3 in hepatic lipids. FO-feeding decreased effectively serum level of cholesterol of both rats and mice. The reduction in serum cholesterol by feeding PO was less pronounced than that observed with FO-feeding. A high correlation was found between 22:6 n-3 content in hepatic lipids and serum concentration of cholesterol. Although FO-feeding lowered the level of circulating triacylglycerol, PO-feeding produced no change. No substantial correlation was observed between the hepatic content of n-3 fatty acid and the concentration of serum triacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Phorbol myristate acetate stimulates degradation of a structural analogue of platelet-activating factor to a neutral lipid in human leukemic K562 cells: relevance to the release of lipids.

In our attempt to investigate the mechanism of the release of platelet-activating factor (PAF) from cells, the erythroleukemic cell line K562 was preloaded with a radiolabeled PAF analogue having an ethylcarbamyl residue, 1-O-octadecyl-2-O-ethylcarbamyl-sn-glycero-3-phosphocholine (ethylcarbamyl-PAF), that is resistant to the hydrolytic action of PAF acetylhydrolase. Its extracellular release was monitored using an albumin back-extraction method, and its metabolic degradation was analyzed by TLC. Phorbol myristate acetate (PMA) was found to stimulate the release of two radioactive lipids, ethylcarbamyl-PAF itself and its metabolite, 1-O-octadecyl-2-ethylcarbamyl-sn-glycerol, whereas only ethylcarbamyl-PAF was released from the resting cells. The increased release of radioactive lipids in PMA-stimulated cells was suggested to be due to stimulated degradation of intracellular ethylcarbamyl-PAF into the cell-permeable metabolite. Thus K562 cells have much less capacity to release intact PAF-like lipid in comparison with its high ability to uptake exogenously added PAF analogues previously described by us and others.

Biotransformation↗

Horseradish peroxidase degrades lipid hydroperoxides and suppresses lipid peroxidation of polyunsaturated fatty acids in the presence of phenolic antioxidants.

Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and alpha-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction.

Antioxidants↗

Selenium altered the levels of lipids, lipid peroxidation, and sulfhydryl groups in straitum and thalamus of rat.

The effect of sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, i.p.) on the lipid levels (total lipids, phospholipids, cholesterol, gangliosides), thiobarbituric acid reactive substance (TBARS), and sulfhydryl group (-SH) in the striatum and thalamus of a male Wistar rat was studied after 7 d of treatment. The level of total lipids and cholesterol was significantly and dose-dependently elevated in the striatum and thalamus with 0.1 and 0.2 mg/kg of sodium selenite. However, the cholesterol level was significantly increased only with 0.2 mg/kg of sodium selenite in the thalamus. The level of phospholipids and gangliosides was more significant with 0.1 mg/kg of sodium selenite as compared to 0.2 mg. No significant alteration on the gangliosides level was observed in the thalamus with various doses of sodium selenite although the elevation with 0.2 mg dose was 25.9%. The content of TBARS was elevated dose dependently in striatum, but its level was depleted significantly with 0.1-mg/kg dose of sodium selenite in the thalamus. The level of the -SH group was significantly depleted in the striatum with 0.1-mg/kg dose of sodium selenite; conversely, this dose has significantly elevated the levels of -SH group in the thalamus.

Animals↗

[Effect of five kinds of vegetable seed oil on serum lipid and lipid peroxidation in rats].

The effects of vegetable seed oil on hyperlipidemia induced by high lipid diet in rats. Male adult Wistar rats were fed on the test diet containing 94% high lipid diet and 6% lard pinon seed oil, perilla seed oil, blackcurrent seed oil, borage seed oil and evening primrose seed oil respectively for 3 weeks. The results showed that the vale of trilyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol (LDL-C), LDL-C/HDL-C(high density lipoprotein cholesterol) ratio increased and the vale of HDL-C/TC ratio and lecithin-cholesterol acyltransferase(LCAT) activity decreased in the groups with vegetable seed oil were less than that of the control group. The results suggested that all the five kinds of vegetable seed oil had the effect of regulating lipid metabolism of hyperlipidemia rats to some extent. Pinon seed oil and borage seed oil may be well suited for the prevention of atherosclerosis.

Animals↗

[Blood lipids and lipid peroxides in the families of patients with ischemic heart disease].

Levels of some fractions of blood neutral lipids and lipoperoxides were compared in the families of patients with coronary heart disease. The patients and their relatives (including healthy ones) were found to show a statistically significant increase in the atherogenic index, total cholesterol, low density lipoprotein cholesterol, triglyceride, total lipid, and lipoperoxide concentrations and a decrease in high density lipoprotein cholesterol levels as against controls. The changes in the values of lipids and lipoperoxides in the families of patients with coronary heart disease were homogenous and might be one of the pathogenetic links in the formation of hereditary predisposition to atherosclerotic cardiovascular diseases which might be detected in a prehospital period.

Adolescent↗