Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the effect of ionol. II. Physico- chemical properties of the sarcoplasmic reticulum membrane

Physico-chemical parameters of membranes of skeletal muscles' sarcoplasmic reticulum in antioxidant insufficiency, which was modelled by excluding alpha-tocopherol from the animals ration, and after treatment with phenol antioxidant ionol were studied. It was shown that activation of lipid peroxidation in vitamin E insufficiency results in a significant lowering of microviscosity of lipid bilayer membranes of sarcoplasmic reticulum. Using polarography significant changes in membrane protein conformation were revealed, which were characterized by lowering of integrity and by disorganization of protein globules. Treatment of animals with antioxidant insufficiency with ionol led to certain normalization of changes of physico-chemical characteristics of the learned membrane structures caused by lipid peroxidation.     

Polychlorinated biphenyls-induced lipid peroxidation as measured by thiobarbituric acid-reactive substances in liver subcellular fractions of rats

Rats were given a 0.05% polychlorinated biphenyls (PCB) diet supplemented with adequate nutrients for 10 days and not only PCB-induced lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances but also variations of lipid peroxides scavengers in liver and its subcellular fractions (nuclei and cell debris, mitochondrial, microsomal and cytosolic fractions) were investigated. The lipid peroxidation in liver and subcellular fractions in the PCB-treated group increased significantly except in the nuclei and cell debris fraction. The increase in lipid peroxidation in the microsomal fraction appeared to be associated in part with the decrease in vitamin E (alpha-tocopherol) content and induction of drug-metabolizing enzymes. In the cytosolic fraction, the total lipid content increased, glutathione peroxidase (GSHPx) activity decreased and the quantity of free radical-reactive substances suppressing lipid peroxidation was low as measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) value. From these results, the increase in lipid peroxidation in the cytosolic fraction in the PCB-treated group was ascribed to the abundance and availability of oxidizable substrate attended with fatty liver, to the decline in GSHPx activity, and to the insufficiency in antioxygenic activity as observed by the decrease in the DPPH value.     

Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation

Prescott, B. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), O. Sobeslavsky, G. Caldes, and R. M. Chanock. Isolation and characterization of fractions of Mycoplasma pneumoniae. I. Chemical and chromatographic separation. J. Bacteriol. 91:2117-2125. 1966.-Fractionation of Mycoplasma pneumoniae, cultured on a beef heart infusion-horse serum-yeast extract medium, was carried out by chemical and chromatographic procedures. The chemical method yielded eight fractions consisting of lipid, carbohydrates, and proteins. Four protein-rich fractions were isolated by chromatographing a supernatant fluid of sonically treated organisms on Sephadex G-25. The 12 fractions were tested for serological and antigenic activity in vitro and in vivo. The lipid fraction was serologically active and the relative order of activity of the protein fractions appeared to depend on the amount of lipid present in the molecule. The highly serologically active Sephadex G-25 protein fraction 1 prepared chromatographically contained 15% lipid in the molecule, whereas the less serologically active protein fraction 2 prepared by chemical means contained 2% lipid. The acetone-extracted lipid fraction was chromatographed on thin-layer chromatography plates and found to consist of nine fractions. Serological activity was associated with only the first three spots above the origin. Lipid extracted from the protein fractions seemed to be similar to the acetone-extracted lipid from the sediment of the sonically treated organisms.     

Note: Antioxidant properties of lipid fractions from Deinococcus radiophilus strain UWO1055

Lipids of the radio-resistant bacterium Deinococcus radiophilus were tested for their antioxidant properties. The crude lipid extract showed a significant antioxidant effect in linoleic acid emulsion. The crude extract was separated to polar and non-polar lipid fractions. The non-polar fraction showed an antioxidant effect in both suspensions and emulsions of linoleic acid, and inhibition of oxidation in a β-carotene emulsion. Lipids of the non-polar fraction were separated and their antioxidant activity was determined in a β-carotene emulsion; the lipid that was marked NP9 showed the highest antioxidant effect. Lipid NP9 inhibited oxidation in a β-carotene emulsion in the concentration range of 5–51 ppm. It is suggested that the antioxidant activity of lipids of D. radiophilus contribute to its radio-resistance.     

Effects of Chlorpromazine on the Activities of Antioxidant Enzymes and Lipid Peroxidation in the Various Regions of Aging Rat Brain

In this work, the effect of chronic intraperitoneal administration of chlorpromazine (5 and 10 mg/kg) on the antioxidant enzymes superoxide dismutase (SOD), catalase (CA), glutathione reductase (GR), and glutathione peroxidase (GP); lipid peroxidation; and lipofuscin accumulation in the brains of rats ages 6, 9, and 12 months was studied. Chlorpromazine increased the activities of SOD, GR, and GP in particulate fraction from cerebrum, cerebellum, and brain stem in a dose-dependent manner. While GR and SOD associated with soluble fraction increased, GP associated with soluble fraction was not affected. CA did not change after chlorpromazine administration in any regions of the brain of rats from all age groups. Chlorpromazine, thus, had a somewhat different action on antioxidant enzymes in different subcellular fractions. Chlorpromazine inhibited lipid peroxidation, both in vivo and in vitro, and it also inhibited accumulation of lipid peroxidation fluorescent products (lipofuscin), which was studied histochemically and biochemically as well. The data indicate that chlorpromazine inhibition of lipid peroxidation and of accumulation of lipofuscin can result from elevation of the activity of brain antioxidant enzymes.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Effects of hypothyroidism induced by 6-n-propylthiouracil and its reversal by T3 on rat heart superoxide dismutase, catalase and lipid peroxidation

The present study critically evaluates the effects of hypothyroid and hyperthyroid states on lipid peroxidation and two enzymes of active oxygen metabolism, namely superoxide dismutase (SOD) and catalase (CAT) in the rat heart mitochondrial and post-mitochondrial fractions. Lipid peroxidation, an index of oxidative stress, was elevated in the heart tissue in hypothyroid state but reduced upon T3 supplementation. Hyperthyroidism registered increased SOD activity in post-mitochondrial fraction. Mitochondrial SOD activity was reduced in hypothyroid state, which was further reduced by T3 administration. In contrast, different thyroid states had no effect on catalase activity in the mitochondrial fraction. The hypothyroid state however, significantly augmented catalase activity in post-mitochondrial fraction. The results suggest that the antioxidant defence status of cardiac tissue is well modulated by thyroid hormone.     

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.     

Cyclopentenosine, major trifunctional crosslinking amino acid isolated from acid hydrolysate of elastin

Young male rats were sacrificed either at rest or immediately after a single bout of swimming lasting either 5 or 8 h. Mitochondrial population, obtained by centrifugation (10,000g for 10 min) from liver homogenates freed from debris and nuclei, was resolved by differential centrifugation into three fractions. Homogenates and mitochondrial preparations were examined for their protein content, oxidative capacity (by cytochrome oxidase activity), peroxidative processes (by thiobarbituric acid reactive substance and hydroperoxide levels), antioxidant status (by reduced glutathione and vitamin E levels and whole antioxidant capacity), and susceptibility to in vitro oxidative stress. In all groups, the antioxidant level was smaller and oxidative capacity, lipid peroxidation, and susceptibility to oxidants were greater in the heavy mitochondrial fraction. Exercise of shorter duration did not significantly affect most of the parameters; only the resulting homogenate glutathione level and susceptibility to oxidative stress decreased and increased, respectively, compared with control values. In contrast, more prolonged exercise was associated with increased lipid peroxidation and susceptibility to oxidative stress and decreased antioxidant levels in all preparations. The contribution of each fraction to the whole mitochondrial population was also modified in that the heavy fraction decreased and light fractions increased. These results suggest that liver antioxidant defence systems are able to withstand oxidative challenge due to low-intensity exercise of moderate duration. In contrast, the free radical production associated with long-lasting exercise causes oxidative injury in cellular components and in particular induces protein degradation in the heavy mitochondrial fraction characterized by higher susceptibility to oxidative stress.     

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.     

Effect of C27-steroids on lipid peroxidation in rat liver mitochondria

The influence of C27-steroids--vitamin D3 and 20-hydroxyecdysone in free-radical reactions on lipid peroxidation in the mitochondrial fraction of rat's liver has been studied. Antioxidant activity of superoxide dismutase and catalase and the content of lipid components--cholesterol and phospholipids has been determined. The assumption was put forward that antioxidant action of steroids was possible as a result of to the modification of mitochondrial membrane through its lipid content and changing of its cholesterol: phospholipids content.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Structural organization of kinetoplast DNA and its compactization in a model system in vitro

Studies on compactization and decompactization of the genome are of great importance for elucidation of structural mechanisms taking part in the regulation of gene activity. Kinetoplast DNA (kpDNA) is a convenient model for studies of compactization processes. KpDNA represents unique structure ("network"), consisting of catenated circular molecules of two types: minicircles (900 b.p.) and maxicircles (40 000 b.p.). The compactization process of kpDNA in vitro caused by interaction with synthetic peptide-dansylhydraside trivaline was studied. It was shown that at the initial stages the hairpins are observed on minicircles as if triple rings are being organized. The formation of hairpin is probably favoured by the presence in the minicircles of bent DNA, a specific nucleotide sequence causing rigid bending of the DNA helix. The hairpin does not make contact with the neighbouring DNA segment to form a triple ring, because the sizes of minicircles are too small. The minicircles compactization is finished with a complete collapse of the minicircles with the formation of rod-like structures. The catenation causes branching of rod-like structures. As a result of their intermolecular interaction, the branched rod-like structures become thicker. The process is completed with formation of the compact network, its diameter being 3-6 times smaller compared to the initial one.     

EXISTENCE OF ASCORBIC ACID AS AN ACTIVATOR OF THIAMINE DlPHOSPHATASE IN RAT BRAIN

The effect of the supernatant fraction (105,000 g for 60 min) of rat brain on the microsomal thiamine diphosphatase activity was examined. The thiamine diphosphatase activity was increased by addition of the supernatant fraction. The factor activating the enzyme was a heat-stable and dialyzable substance. It caused lipid peroxidation in the microsomes and the increase of the enzyme activity was mediated through lipid peroxidation of the preparation. When the supernatant fraction was chromatographed on columns of Sephadex G-25 and Dowex 1 × 2, the activator was eluted in fractions containing ascorbic acid. The inhibitory factor of ATPase present in the supernatant fraction was also eluted with the activator. The u.v.-spectrum of the active fraction obtained by these chromatographies was the same as that of ascorbic acid. These findings indicate the existence of ascorbic acid as an activator of thiamine diphosphatase in rat brain and confirm the previous finding that the soluble factor inhibiting ATPase activity is ascorbic acid.     

Dietary oils high in oleic acid, but with different non-glyceride contents, have different effects on lipid profiles and peroxidation in rabbit hepatic mitochondria

The influence on the lipid profile and lipid peroxidation in rabbit-liver mitochondria exerted by different edible oils high in oleic acid but different non-glyceride phenolic fractions was studied. High-phenolic virgin olive oil from the variety "Picual", the same oil submitted to an exhaustive process of washing to eliminate the phenolic fraction without altering the lipid profile and high-oleic sunflower oil (poor in phenolic compounds) were added to rabbit diets. The results reveal the importance of the different oleic: linoleic ratio of the lipid sources on the lipid profile of mitochondrial membranes. This is highlighted by the greater proportion of saturated fatty acids and the lower content in oleic acid (p < 0.05) shown by the rabbits fed on high-oleic sunflower oil. The group fed on the fat rich in phenolics exhibited the highest level of antioxidants (alpha-tocopherol, ubiquinone 10) and the highest activity of glutathione peroxidase as well as the lowest content in hydroperoxides and TBARS. The study provides evidences in vivo about the considerable antioxidant capacity of the phenolic fraction of virgin olive oil in rabbit-liver mitochondria and the important role that this non-glyceride fraction can play in the overall antioxidant benefits attributed to this oil.     

Separation of Antioxidant-Rich Alternanthera Sessilis Red Extracts by Sephadex LH-20 and Identification of Polyphenols Using HPLC-QToF-MS/MS

This study aimed to fractionate Alternanthera sessilis Red (ASR) crude extracts and determine their antioxidant activities as well as the related active components in the whole plant. ASR was extracted with water and ethanol, and further separated using a Sephadex LH-20 column. Following the assessments of the polyphenolic contents and antioxidant activities of crude extracts (H₂O_ASR and EtOH_ASR) and fractions, a HPLC-QToF analysis was performed on the crude extracts and selected fractions (H₂O_ASR FII and EtOH_ASR FII). Three water fractions (H₂O_ASR FI, FII and FIII) and four ethanolic fractions (EtOH_ASR FI, FII, FIII and FIV) were derived from their crude extracts, respectively. EtOH_ASR FII exhibited the greatest total phenolic content (120.41 mg GAE/g fraction), total flavonoid content (223.07 mg RE/g fraction), and antioxidant activities (DPPH IC₅₀=159.43 μg/mL; FRAP=1.93 mmol Fe²⁺/g fraction; TEAC=0.90 mmol TE/g fraction). Correlation analysis showed significant (p<0.01) positive correlations between both TPC (r=0.748–0.970) and TFC (r=0.686–0.949) with antioxidant activities in the crude extracts and fractions. Flavonoids were the major compounds in the four selected samples tentatively identified using HPLC-QToF-MS/MS, with the highest number of 30 polyphenol compounds detected in the most active fraction, EtOH_ASR FII.     

Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries

The antioxidant potential of crude extracts and fractions from leaves of Ouratea parviflora, a Brazilian medicinal plant used for the treatment of inflammatory diseases, was investigated in vitro through the scavenging of radicals 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydroxyl radical (HO*), superoxide anion (O2*-), and lipid peroxidation in rat liver homogenate. The crude extract (CEOP) and hydro-alcoholic fraction (OP4) showed strong inhibitory activity toward lipid peroxidation induced by tert-butyl peroxide (IC50 = 2.3 +/- 0.2 and 1.9 +/- 0.1 microg/ml, respectively). The same products exhibited a strong concentration-dependent inhibition of deoxyribose oxidation (14.9 +/- 0.2 and 0.2 +/- 0.1 microg/ml, respectively), and also showed a considerable antioxidant activity against O2*- (87.3 +/- 0.1 and 73.1 +/- 0.4 microg/ml, respectively) and DPPH radicals (55.4 +/- 0.3 and 38.3 +/- 0.4 microg/ml, respectively). The protective effects of CEOP and OP4 were also studied in mouse liver. CCl4 significantly increased (by 90%) levels of lipid hydroperoxides, carbonyl protein content (64%), DNA damage index (133%), aspartate aminotransferase (261%), alanine aminotransferase (212%), catalase activity (23%), and also caused a decrease of 60% in GSH content. The results showed that CEOP and OP4 exerted cytoprotective effects against oxidative injury caused by CCl4 in rat liver, probably related to the antioxidant activity showed by the in vitro free radical scavenging property.