Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellataroot extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Evaluation of total carotenoids, alpha- and beta-carotene in carrots (Daucus carota L.) during home processing

This study aims to analyze the influence of dehydration and different preparation methods during home processing related toalpha-carotene, beta-carotene and total carotenoids stability in carrots. Vitamin A values were evaluated after different treatments. Thus, carrots were submitted to steam cooking, water cooking with and without pressure, moist/dry cooking and conventional dehydration. Determination of alpha- and beta-carotenes was made by High-Performance Liquid Chromatography (HPLC) (conditions were developed by us) using spectrophotometric detection visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate (80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to 85.64%. Water cooking without pressure promoted higher retention levels of alpha- and beta-carotene and vitamin A values, while water cooking with pressure promoted higher retention levels of total carotenoids. Dehydration promoted the highest carotenoid losses. The results showed that, among the routinely utilized methods under domestic condition, cooking without pressure, if performed under controlled time and temperature, is the best method as it reduces losses in the amount of alpha- and beta-carotene, the main carotenoids present in the carrots. Despite the significant carotenoid losses, carrots prepared through domestic methods, remain a rich source of provitamin A.

Nutritional composition, fatty acid and tocopherol contents of buriti (Mauritia flexuosa) and patawa (Oenocarpus bataua) fruit pulp from the amazon region

Buriti and patawa are two endemic palm trees from the Amazon region. Their pulps are traditionally consumed by the local population, but are underused and lesser known worldwide. Nutritional composition, fatty acid and tocopherol contents of the two palm pulps were determined by modern analytical methods: Gas Chromatography (CG) and High Performance Liquid Chromatography (HPLC), based on the standards of AOCS (AMERICAN..., 2002) and AOAC (ASSOCIATION..., 1997), respectively. Buriti and patawa fruit pulps are highly nutritive, with respectively, high fat content (38.4% and 29.1% of dry matter (DM)), protein content (7.6% and 7.4% of DM) and dietary fibers (46% and 44.7% of DM). Buriti pulp can be considered healthy food due its high content of vitamin E (1169 µg.g-1 DM). Patawa pulp is highly oleaginous and its fatty acid composition is very similar to the ones of healthy oils, such as olive oil.

Incorporation of conjugated linoleic and alpha linolenic fatty acids into Pimedolus maculatus fillets

The objective of this study was to investigate the fatty acid composition of Pimedolus maculatus fillets of fish fed with different diets. The fish were submitted to an adaptation period (over 30 days) and fed only a control diet (diet I). After this period, the fish were divided into two groups: one group received a diet enriched with flaxseed oil (diet II), and the other received a diet enriched with conjugated linoleic acid (CLA) (diet III). Subsequent collections were undertaken after 25 and 50 days. Compared to the control samples, fish fed diet II showed an increase in concentrations of total fatty acid omega-3 (n-3) in the fillets, with an improvement ratio of n-6/n-3. The incorporation of CLA into fish fed diet III was successful. Supplementing the diet of Pimedolus maculatus with CLA and flaxseed oil led to improvements in nutritional quality and fatty acid profile of the fish fillets.

In-house method validation and occurrence of alpha-, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone and trifluralin residues in strawberry

A method for determination of organohalogen pesticides in strawberry by gas chromatography with electron capture detection was validated and applied in a monitoring program. Linearity, matrix effects, and day effect were evaluated for the analytes alpha-endosulfan, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone, and trifluralin. The linear range varied according to the chromatographic response of the analyte. Significant matrix effects were observed. The mean recoveries ranged from 74.6 to 115.4%, with repeatability standard deviations between 1.6 and 21.0% and intermediate precision between 5.9 and 21.0%. Detection, quantification and decision limit, and detection capacity ranged from 0.003 to 0.007 mg/kg, 0.005 to 0.013 mg/kg; 0.003 to 3.128 mg/kg; and 0.005 to 3.266 mg/kg, respectively. The method was fit for the purpose of monitoring organohalogen residues in strawberries. Residues of these pesticides were detected in 124 of the 186 samples analyzed between 2009 and 2011 in the state of Minas Gerais. Nine of them did not comply with the current legislation requirements; among them, seven (3.8%) had residues of unauthorized pesticide for the culture of strawberry, one (0.5%) had residues above the maximum residue limit, and another one (0.5%) exhibited both non-conformities.

Quality changes during frozen storage of blue shrimp (Litopenaeus stylirostris) with antioxidant, α-tocopherol, under different conditions

Fresh blue shrimp (Litopenaeus stylirostris) muscle was stored with antioxidants under different conditions: ANTIOX 2%, packed in bilayer film of polyamide-low density polyethylene film (PA-LDPE) with 2% α-tocopherol; ANTIOX 4%, packed in PA-LDPE film with 4% α-tocopherol; and ANTIOX-GLAZED, samples stored glazed with 2% α-tocopherol. Shrimps packed in PA-LDPE without α-tocopherol were used as CONTROL. All samples were stored at –20 °C for 120 days. As compared to the CONTROL, the shrimp stored with the antioxidant showed lower lipid oxidation (0.10-0.14 vs 1.58 mgMA/kg of muscle), lost less firmness and astaxanthin content. ANTIOX 2% and ANTIOX-GLAZED showed the lowest concentrations of formaldehyde (0.081-0.083 μM/g). There were no significant differences in color and sensory properties, but differences in the integrity of the muscle fibers were observed. The treatments with α-tocopherol maintained the shrimp muscle quality during frozen storage. However, no significant differences were found between these treatments.

Action of gibberellic acid (GA3) on dormancy and activity of alpha-amylase in rice seeds

To evaluate the effectiveness of gibberellic acid (GA3) in breaking rice seed dormancy and the use of alpha-amylase enzyme activity as an indicator of the dormancy level, seed from the intensively dormant irrigated cultivar Urucuia were used. The seeds were submitted to a pre-drying process in a forced air circulation chamber under 40ºC during 7 days and submersed in 30 mL of GA3 solution under 0, 10, 30 and 60 mg/L H2O concentrations, during 2, 24 and 36 hours. After the treatments, the alpha-amylase activity was determined by using the polyacrilamide electrophoresis and spectrophotometry. At the same time, the germination test was made. The results indicated a gain in germination and in alpha-amylase activity in higher concentrations and soaking time of seeds in GA3. These observations support the conclusion that soaking seed in 60 mg GA3/L during 36 hours can be used as a quick and efficient treatment in breaking rice seed dormancy and is equivalent to the forced air circulation chamber at 40ºC during 7 days. The alpha-amylase enzyme activity proved to be as an efficient marker of the seeds dormancy level.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Intramolecular carbenoid insertions : the reactions of [alpha]-diazoketones derived from furanyl, thienyl, benzofuranyl and benzothienyl acetic acids with rhodium (II) acetate /

A number of synthetically useful ring systems can be prepared via the intramolecular insertion of a metal-stabilized carbenoid into a heteroaromatic systems. The chemical outcome of these reactions are dependent not only on the nature of the heteroatom but also on the length of the aliphatic tether linking the carbenoid moiety with the aromatic fragment. Our work with furanyl and thienyl systems containing a single methylene tether have allowed for some rather atypical chemistry. For example, treatment of l-diazo-3-(2-thienyl)-2-propanone (6) with catalytic rhodium (II) acetate yields 5,6- dihydro-4^-cyclopenta[Z>]thiophen-5-one (3) while, the isomeric l-diazo-3-(3-thienyl)-2- propanone(15) gives a spiro-disulphide (20). Novel chemistry was also exhibited in the analogous furanyl systems. While treatment of l-diazo-3-(3-furanyl)-2-propanone (52) with Rh2(OAc)4 resulted in the expected 2-(4-Oxo-2-cyclopentenyliden)acetaldehyde (54), isomeric l-diazo-3-(2- furanyl)-2-propanone (8) undergoes vinylogous Wolff rearrangement to give a mixture of 6a-methyl-2,3,3a,6a-tetrahydrofuro[2,i-^>]furan-2-one (44) and 2-(2-methyl-3-furyl)acetic acid (43). Rhodium acetate catalyzed decomposition of l-diazo-3-(3-benzofuranyl)-2- propanone (84) and l-diazo-3-(2-benzofuranyl)-2-propanone (69)also allows for vinylogous Wolff rearrangement, a chemistry unseen in benzofuranyl systems with longer tethers. A number of interesting products were isolated from the trapping of intermediate ketenes. Decomposition of l-diazo-3-(3-benzothienyl)-2-propanone (100) resulted in the formation of 2,3-dihydro-l//-benzo[^]cyclopenta[^thiophen-2-one (102). However, in addition to (102), a dimer was also generated from the decomposition of l-diazo-3-(2- benzothienyl)-2-propanone (109). The insight into the mechanistic underpinnings of the above reactions are provided by molecular modeling at a PM3 level.

Investigation of the mechanism of transfer of a-tocopherol by the human a-tocopherol transfer protein (H-a-TTP) /

The human a-tocopherol transfer protein (h-a-TTP) is understood to be the entity responsible for the specific retention of a-tocopherol (a-toc) in human tissues over all other forms of vitamin E obtained from the diet. a-Tocopherol is the most biologically active form of vitamin E, and to date has been studied extensively with regard to its antioxidant properties and its role of terminating membrane lipid peroxidation chain reactions. However, information surrounding the distribution of a-tocopherol, specifically its delivery to intracellular membranes by a-TTP, is still unclear and the molecular factors influencing transfer remain elusive. To investigate the mechanism of ligand transfer by the h-a-TTP, a fluorescent analogue of a-toc has been used in the development of a fluorescence resonance energy transfer (FRET) assay. (/?)-2,5,7,8-tetramethyl-2-[9-(7-nitro-benzo[l,2,5]oxdiazol-4-ylamino)-nonyl]- chroman-6-ol (NBD-toc) has allowed for the development of the FRET-based ligand transfer assay. This ligand has been utilized in a series of experiments where changes were made to acceptor lipid membrane concentration and composition, as well as to the ionic strength and viscosity of the buffer medium. Such changes have yielded evidence supporting a collisional mechanism of ligand transfer by a-TTP, and have brought to light a new line of inquiry pertaining to the nature of the forces governing the collisional transfer interaction. Through elucidation of the transfer mechanism type, a deeper understanding of the transfer event and the in vivo fate of a-tocopherol have been obtained. Furthermore, the results presented here allow for a deeper investigation of the forces controlling the collisional protein-membrane interaction and their effect on the transfer of a-toc to membranes. Future investigation in this direction will raise the possibility of a complete understanding of the molecular events surrounding the distribution of a-toc within the cell and to the body's tissues.

Synthesis of fluorescent analogues of a-tocopherol as ligands for the human a-tocopherol transfer protein (a-TTP) /

To further understand in vivo localization and trafficking of a-tocopherol (a-Toe), the most biologically active form of vitamin E, between lipid environments, tocopherols are required that can be followed by teclu1iques such as confocal microscopy and fluorescence resonance energy transfer (FRET) assays. To this end, sixteen fluorescent analogues of a-tocopherol (la-d [(1)anthroy loxy -a-tocopherols, A O-a-Toes], 2a-d [w-nitro benzoxadiazole-a-tocopherols, NBD-aToes], 3a-d [w-dansyl-a-tocopherols, DAN-a-Toes], and 4a-d [w-N-methylanthranilamide-atocopherols, NMA-a-TocsD were prepared by substituting fluorescent labels at the terminus of w-functionalized alkyl chains extending from C-2 of the chroman ring while retaining key binding features of the natural ligand. These compounds were prepared starting from (S)-Trolox® acid VIa esterification, protection, and reduction producing the silyl-protected (S)-Trolox aldehyde that was coupled using Wittig chemistry to different w-hydroxyalkylphosphonium bromides. Reduction of the alkene generated the w-hydroxy functionalized 2-n-alkyl intermediates 9a-d having the necessary 2R stereochemistry. A series of functional group manipulations including mesylation, substitution with azide, and hydride reduction provided w-amino functionalized intermediates 12a-d as well. Coupling intermediates 9a-d and 12a-d with the selected fluorophores (9- anthracene carboxylic acid, 4-chloro-7-nitrobenz-2-oxa-l,3-diazole, 5- dimethylaminonapthalene-l-sulfonyl chloride, and I-methyl-2H-3,1-benzoxazine-2,4(1H)dione), followed by deprotection of the phenolic silyl group, gave the desired fluorescent ligands la-d, 2a-d, 3a-d and 4a-d in good yield. Assessment of their binding affinities with recombinant human a-tocopherol transfer protein (ha-TTP) utilizing fluorescent titration binding assays identified competent ligands for further use in protein studies. Compounds Id (C9-AO-a-Toc) and 2d (C9-NBD-a-Toc) both having nonyl alkyl chain extensions between the chromanol and fluorophore were shown to bind specifically to ha-TTP with dissociation constants (KdS) of approximately 280 nM and 55 nM respectively, as compared to 25 nM for the natural ligand 2R,4'R,^'R-a-tocophQxoL.

Intramolecular carbenoid insertion : reactions of [alpha] -diazoketones derived from benzothienyl, pyrolyl and indolyl alkanoic acids with rhodium (II) acetate

Recent studies have shown that the rhodium (II) acetate decomposition chemistry observed for a-diazoketones tethered to thienyl, furanyl, and benzofuranyl moieties is dependent not only on the nature of the heteroatom but also on the length of the aliphatic tether linking the diazoketone moiety with the aromatic fragment. The present thesis expands on these results and focuses on a-diazoketones tethered to benzothiophenes, pyrroles and indoles by a methylene linker. In the case of benzothiophenes, it was shown that the rhodium catalyst decomposition of I-diazo-4-(3-benzothienyl)-2-butanone (146) and 1-diazo-4-(3benzothienyl)- 2-butanone (152) allow for the isolation of 1,2,3a,3b-tetrahydro-3Hbenzo[ b]cyclopenta[1,3]cyclopropa- [1 ,2-d]thiophen-3-one (147) and 1,2,3a,3btetrahydro- 3H-benzo[b]cyclopenta[1,3]cyclopropa[1,2-d]thiophen-3-one (153). However treatment of 1-diazo-3-(3-Benzothienyl)-2-Propanone (165) with Rh(II) acetate results in the formation of 2,3-Dihydro-1H-benzo[b]cyclopenta[d]thiophen-2-one (159), while 1diazo- 3-(2-Benzothienyl)-2-Propanone with the same condition gives 5,5-bis( 1benzothiophen- 2-ylmethyl)-2(5H)-furanone (166) along with the tricycle 159. The chemistry of the pyrrolyl and the indolyl moieties linked to terminal adiazoketone systems was also investigated. The decomposition of I-diazo-(2-pyrrolyl)-2propanone (173) results in the formation of two products; the N-H insertion product IHpyrrolizin- 2(3H)-one (176) and the alkylation product 4,6-dihydrocyclopenta[b]pyrrol5( 1 H)-one (180). When 1-Diazo-3-(3-indoly)-3-propanone (194) is treated with catalytic amount of Rh (II) 3,4-dihydrocyclopenta[b]indol-2(1H)-one (193) is isolated quantitatively. The later reaction when monitored using IH NMR the intermediate 200 can be seen whose structure was confirmed by the comparison to series of model compounds. The mechanisms underlying these reactions as well as their synthetic utility is discussed.