Thiol Components Research Articles

Protein Adsorption on Mixed Self-Assembled Monolayers: Influence of Chain Length and Terminal Group.

Mixed self-assembled monolayers (SAMs) are often used as highly tunable substrates for biomedical and biosensing applications. It is well documented, however, that mixed SAMs can be highly disordered at the molecular level and do not pack as closely or homogeneously as single-component SAMs, particularly when the chain lengths and head groups of the SAM thiol components are significantly different. In this study, we explore the impact of SAM structure and mixing ratio (-OH and -CH3 termini) on the weak physisorption behavior of bovine serum albumin (BSA), which adsorbs more readily to hydrophobic, methyl-terminated SAMs. Our results suggest that once the mixture includes 50% or more of the methyl terminus, the mixing ratio alone is a relatively good predictor of adsorption, regardless of the relative chain lengths of the thiols used in the mixture. This trend persists at any mixing ratio for SAMs where methyl- and hydroxyl-terminated groups are the same length or where the hydroxyl-terminated thiol is longer. The only variance observed is at low mixing ratios (<50% methyl-terminated) for a mixed SAM where the methyl-terminated component has a longer chain length. Relative protein adsorption increases on these mixtures, perhaps due to the disordered exposure of the excess alkane backbone. Taken together, however, we do not find significant evidence that varying chain lengths for mixed SAMs prepared on polycrystalline substrates and analyzed in air have an outsized influence on nanoscopic adsorption behavior, despite molecular-level disorder in the SAM itself.

Monitoring physiological processes of fast-growing broilers during the whole life cycle: Changes of redox-homeostasis effected to trassulfuration pathway predicting the development of non-alcoholic fatty liver disease.

In the broiler industry, the average daily gain and feed conversion ratio are extremely favorable, but the birds are beginning to approach the maximum of their genetic capacity. However, as a consequence of strong genetic selection, the occurrence of certain metabolic diseases, such as myopathies, ascites, sudden cardiac death and tibial dyschondroplasia, is increasing. These metabolic diseases can greatly affect the health status and welfare of birds, as well as the quality of meat. The main goal of this study was to investigate the changes in the main parameters of redox homeostasis during the rearing (1-42 days of age) of broilers with high genetic capacity, such as the concentrations of malondialdehyde, vitamin C, vitamin E, and reduced glutathione, the activities of glutathione peroxidase and glutathione reductase, and the inhibition rate of superoxide dismutase. Damage to the transsulfuration pathway during growth and the reason for changes in the level of homocysteine were investigated. Further, the parameters that can characterize the biochemical changes occurring in the birds were examined. Our study is the first characterize plasma albumin saturation. A method was developed to measure the levels of other small molecule thiol components of plasma. Changes in redox homeostasis induce increases in the concentrations of tumor necrosis factor alpha and inflammatory interleukins interleukin 2, interleukin 6 and interleukin 8 in broilers reared according to current large-scale husbandry technology and feeding protocols. A significant difference in all parameters tested was observed on the 21st day. The concentrations of cytokines and homocysteine increased, while the concentrations of glutathione and cysteine in the plasma decreased. Our findings suggest that observed changes in the abovementioned biochemical indices have a negative effect on poultry health.

Effect of methylmercury on fetal neurobehavioral development: an overview of the possible mechanisms of toxicity and the neuroprotective effect of phytochemicals.

Methylmercury (MeHg) is a global environmental pollutant with neurotoxic effects. Exposure to MeHg via consumption of seafood and fish can severely impact fetal neurobehavioral development even when MeHg levels in maternal blood are as low as about 5μg/L, which the mother tolerates well. Persistent motor dysfunctions and cognitive deficits may result from trans-placental exposure. The present review summarizes current knowledge on the mechanisms of MeHg toxicity during the period of nervous system development. Although cerebellar Purkinje cells are MeHg targets, the actions of MeHg on thiol components in the neuronal cytoskeleton as well as on mitochondrial enzymes and induction of disturbances of glutamate signaling can impair extra-cerebellar functions, also at levels well tolerated by adult individuals. Numerous herbal substances possess neuroprotective effects, predominantly represented by natural polyphenolic molecules that might be utilized to develop natural drugs to alleviate neurotoxicity symptoms caused by MeHg or other Hg compounds.

Inhibition of an organophosphate-detoxifying bacterial phosphotriesterase by albumin and plasma thiol components

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.

Exogenous melatonin regulates endogenous phytohormone homeostasis and thiol-mediated detoxification in two indica rice cultivars under arsenic stress.

Melatonin enhanced arsenic (As) tolerance by inhibiting As bioaccumulation, modulating the expression of As transporters and phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid. The present study was aimed at investigating the influence of exogenous melatonin on the regulation of endogenous plant growth regulators and their cumulative effects on metal(loid)-binding ligands in two contrasting indica rice cultivars, viz., Khitish (arsenic sensitive) and Muktashri (arsenic tolerant) under arsenic stress. Melatonin supplementation ameliorated arsenic-induced perturbations by triggering endogenous levels of gibberellic acid and melatonin, via up-regulating the expression of key biosynthetic genes like GA3ox, TDC, SNAT and ASMT. The endogenous abscisic acid content was also enhanced upon melatonin treatment by induced expression of the key anabolic gene, NCED3 and concomitant suppression of ABA8ox1. Enhanced melatonin content induced accumulation of higher polyamines (spermidine and spermine), together with up-regulation of SPDS and SPMS in Khitish, thereby modulating stress condition. On the contrary, melatonin escalated putrescine and spermidine levels in Muktashri, via enhanced expression of ADC and SAMDC. The role of melatonin appeared to be more prominent in Khitish, as evident from better utilization of thiol components like cysteine, GSH, non-protein thiols and phytochelatins, with higher GSH/GSSG ratio, despite down-regulated expression of corresponding thiol-metabolic genes (OsMT2 and OsPCS1) to deal with arsenic toxicity. The extent of arsenic bioaccumulation, which was magnified several folds, particularly in Khitish, was decreased upon melatonin application. Overall, our observation highlighted the fact that melatonin enhanced arsenic tolerance by inhibiting arsenic bioaccumulation, via modulating the expression levels of selected arsenic transporters (OsNramp1, OsPT2, OsPT8, OsLsi1) and controlling endogenous phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid.

Studies on the Thiol Components of Isolated Nuclei

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.

Protective mechanisms of melatonin against selenium toxicity in Brassica napus: insights into physiological traits, thiol biosynthesis and antioxidant machinery

BackgroundThe ubiquitous signaling molecule melatonin (N-acetyl-5-methoxytryptamine) (MT) plays vital roles in plant development and stress tolerance. Selenium (Se) may be phytotoxic at high concentrations. Interactions between MT and Se (IV) stress in higher plants are poorly understood. The aim of this study was to evaluate the defensive roles of exogenous MT (0 μM, 50 μM, and 100 μM) against Se (IV) (0 μM, 50 μM, 100 μM, and 200 μM) stress based on the physiological and biochemical properties, thiol biosynthesis, and antioxidant system of Brassica napus plants subjected to these treatments.ResultsSe (IV) stress inhibited B. napus growth and biomass accumulation, reduced pigment content, and lowered net photosynthetic rate (Pn) and PSII photochemical efficiency (Fv/Fm) in a dose-dependent manner. All of the aforementioned responses were effectively alleviated by exogenous MT treatment. Exogenous MT mitigated oxidative damage and lipid peroxidation and protected the plasma membranes from Se toxicity by reducing Se-induced reactive oxygen species (ROS) accumulation. MT also alleviated osmotic stress by restoring foliar water and sugar levels. Relative to standalone Se treatment, the combination of MT and Se upregulated the ROS-detoxifying enzymes SOD, APX, GR, and CAT, increased proline, free amino acids, and the thiol components GSH, GSSG, GSH/GSSG, NPTs, PCs, and cys and upregulated the metabolic enzymes γ-ECS, GST, and PCS. Therefore, MT application attenuates Se-induce oxidative damage in plants. MT promotes the accumulation of chelating agents in the roots, detoxifies Se there, and impedes its further translocation to the leaves.ConclusionsExogenous MT improves the physiological traits, antioxidant system, and thiol ligand biosynthesis in B. napus subjected to Se stress primarily by enhancing Se detoxification and sequestration especially at the root level. Our results reveal better understanding of Se-phytotoxicity and Se-stress alleviation by the adequate supply of MT. The mechanisms of MT-induced plant tolerance to Se stress have potential implications in developing novel strategies for safe crop production in Se-rich soils.Graphical abstract

Intracellular GSH of Streptococcus thermophilus shows anti-oxidative activity against low-density lipoprotein oxidation in vitro and in a hyperlipidaemic hamster model.

Streptococcus thermophilus YIT 2001 (ST-1), a lactic acid bacterial strain, was shown to have inhibitory effects on the oxidation of low-density lipoprotein (LDL) and the development of aortic fatty lesions in an animal model, and lower the serum levels of malondialdehyde-modified LDL, an oxidative modification product of LDL, in a clinical trial. This study aimed to identify the intracellular active component of ST-1 associated with anti-oxidative activity against LDL oxidation. High-performance liquid chromatography-electrospray ionisation mass spectrometry analysis after fractionation of the cellular extract by reversed-phase chromatography demonstrated that the active fraction contained reduced glutathione (GSH). GSH showed anti-oxidative activity in a dose-dependent manner, while this activity disappeared following thiol derivatisation. ST-1 had the strongest anti-oxidative activity against LDL oxidation and the highest level of intracellular GSH among five strains of S. thermophilus. In addition, the anti-oxidative activity of ST-1 after thiol derivatisation decreased by about half, which was similar to that of three other strains containing poor or no intracellular GSH or thiol components. Moreover, anti-oxidative activity against LDL oxidation was observed in hyperlipidaemic hamsters fed with high GSH ST-1 cells but not in those given low GSH cells. These findings suggest that intracellular GSH in ST-1 may provide beneficial effects via anti-oxidative activity against LDL oxidation and excess oxidative stress in the blood.

Design and Optimization of a Bacterial Sensor Based on the Innate Immune System

The rapid and accurate detection of bacteria is of great concern in both clinical and defence settings. However, to date, the gold standard in the detection of bacteria remains the method of culturing bacteria in a laboratory setting. This process is slow and laborious, often taking multiple days before confirmation is given. This delay between presentation and identification is detrimental in both settings, as patients could go untreated or, in the case of biological terrorism or warfare, many potential victims could be exposed to a pathogen. As such, there is a need for the development of a rapid and sensitive detection system capable of identifying the presence of bacteria outside of a clinical laboratory. In the human body, this function is fulfilled by the innate immune system, which responds to pathogen-associated molecular patterns (PAMPs) that are present on broad classifications of biological threats. For example, Toll-Like Receptor-4 (TLR-4), a pattern recognition receptor in the innate immune system, responds to the presence of lipopolysaccharide, the PAMP displayed on Gram-negative bacteria. As Gram-negative and Gram-positive bacteria can be treated with different antibiotics, even this broad distinction can be medically relevant. TLR-4 and the other proteins in the Toll-Like Receptor family have recently been studied for their use in biosensors1–5, and have shown great promise in being able to rapidly differentiate classes of bacteria and other biological threat agents. One major advantage over aptamer or antibody based sensors is that the user does not need to know what the biological agent is. Instead, the sensor can be used as a broad screening tool. This is especially true for the use of electrochemically based Toll-Like Receptor sensors, where the protein is attached to a surface via a linker, such as a self-assembled monolayer (SAM). However, these sensors have relied almost exclusively on the presence of relatively high resistance (> 1 kΩ) systems that provide small signals that are ill-suited to implementation outside of a laboratory1. To address this deficiency, we sought to optimize a TLR-4/SAM-based biosensor. Towards this aim, we have built upon previous work in which TLR-4 is oriented in a bio-mimicking fashion on a Au electrode surface1. The protein was tethered to a Ni2+-nitrilitriacetic acid (NTA) functional group, covalently attached to the end of a carboxyl-terminated thiol-based SAM. Various SAMs were explored in this work, ranging from long-chain single-component monolayers to tripartite SAMs containing multiple functional groups. An optimal ratio of thiol components was determined, based on the TLR-4 sensor response to the presence of Gram-negative bacteria, while remaining insensitive to the presence of Gram-positive bacteria or viral particles, with all samples presenting ≤1 kΩ of interfacial resistance to the rectification reaction. This represents a marked decrease in resistance compared to the current state-of-the-art for TLR-based sensors, providing a convenient avenue for future studies. 1. Mayall, R. M., Renaud-Young, M., Chan, N. W. C. & Birss, V. I. An electrochemical lipopolysaccharide sensor based on an immobilized Toll-Like Receptor-4. Biosens. Bioelectron. 87, 794–801 (2017). 2. Amini, K., Chan, N. W. C. & Kraatz, H.-B. Toll-like receptor 3 modified Au electrodes: an investigation into the interaction of TLR3 immobilized on Au surfaces with poly(I:C). Anal. Methods 6, 3322 (2014). 3. She, Z. et al. Investigation of the utility of complementary electrochemical detection techniques to examine the in vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor. Anal. Chem. 150222112048003 (2015). doi:10.1021/ac5042439 4. Amini, K. & Kraatz, H.-B. Toll-like receptors for pathogen detection in water: challenges and benefits. Int. J. Environ. Anal. Chem. 7319, 1–9 (2016). 5. Yeo, T. Y. et al. Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes. Biosens. Bioelectron. 28, 139–145 (2011).

Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor

It has now been shown that amines and thiols can be tethered together through a Meldrum's acid-derived conjugate acceptor for applications ranging from dynamic combinatorial chemistry to biochemistry to polymer chemistry. Furthering the utility of this chemistry, the ability to then chemically ‘declick' back to the unaltered amine and thiol components is also demonstrated.

Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins.

Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures.

Selenite Protection of Tellurite Toxicity Toward Escherichia coli.

In this work the influence of selenite on metal resistance in Escherichia coli was examined. Both synergistic and antagonistic resistance and toxicities were found upon co exposure with selenite. In wild type cells co-exposure to selenite had little effect on arsenic resistance, decreased resistance to cadmium and mercury but led to a dramatically increased resistance to tellurite of 32-fold. Due to the potential importance of thiol chemistry in metal biochemistry, deletion strains in γ-glutamylcysteine synthetase (key step in glutathione biosynthesis, encoded by gshA), thioredoxin (trxA), glutaredoxin (grxA), glutathione oxidoreductase (gor), and the periplasmic glutathione transporter (cydD) were also evaluated for resistance to various metals in the presence of selenite. The protective effect of selenite on tellurite toxicity was seen in several of the mutants and was pronounced in the gshA mutant were resistance to tellurite was increased up to 1000-fold relative to growth in the absence of selenite. Thiol oxidation studies revealed a faster rate of loss of reduced thiol content in the cell with selenite than with tellurite, indicating differential thiol reactivity. Selenite addition resulted in reactive oxygen species (ROS) production equivalent to levels associated with H2O2 addition. Tellurite addition resulted in considerably lower ROS generation while vanadate and chromate treatment did not increase ROS production above that of background. This work shows increased resistance toward most oxyanions in mutants of thiol redox suggesting that metalloid reaction with thiol components such as glutathione actually enhances toxicity of some metalloids.

Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry.

We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines.

Functional interplay between glutathione and hydrogen sulfide in regulation of thiol cascade during arsenate tolerance of common bean (Phaseolus vulgaris L.) genotypes.

Changes in expressions of up- and downstream thiol cascade were studied in leaves of Phaseolus vulgaris L. cv. VL-63 and its mutant, pvsod1 (deficient in superoxide dismutase activity) under 50 μM sodium arsenate (As), As + l-buthionine-sulfoximine (BSO) and As + BSO + Sodium hydrosulfide (NaHS)-treatments for 10 days. Main objective was to investigate the functional relationship between hydrogen sulfide (H2S) and glutathione (GSH) in regulation of sulfate transporters and cysteine metabolisms as up-stream thiol components and GSH, phytochelatins (PCs) and antioxidant defense response as downstream cascade under As-exposure. As treatment alone initiated coordinated inductions of sulfate transport, biosynthesis of cysteine, GSH, and PCs, and GSH-mediated antioxidant defense in the pvsod1 mutant. At As + BSO, GSH synthesis was blocked, resulting in significantly low GSH redox pool and steep decline in GSH-dependent antioxidant capacity of both the genotypes. However, unlike VL-63, cysteine-degradation pathway was induced in pvsod1 mutant, resulting in significant accumulation of endogenous H2S. The H2S-surge in the pvsod1 mutant stimulated ascorbate-dependent antioxidant defense and catalases and regulated O-acetylserine (thiol)lyase activity, preventing overaccumulation of H2O2 and free cysteine, respectively. No As-induced oxidative stress symptom was observed in the mutant. This trend was maintained at As + BSO + NaHS treatment, also. In contrast, failure to induce entire cascade from sulfate transport to downstream antioxidant defense led to onset of As-induced oxidative damage in VL-63 plant. Results revealed dual roles of H2S as (a) stimulator of GSH-independent antioxidant defense and (b) regulator of cysteine homeostasis through its metabolic diversion during As-exposure and blockage of GSH biosynthesis.

Diverse topologies in dynamic combinatorial libraries from tri- and mono-thiols in water: sensitivity to weak supramolecular interactions

We describe here the use of disulfide exchange reactions to generate distinct supramolecular topologies within a dynamic combinatorial library (DCL) derived from tri- and mono-functionalised thiol components in water. This approach allows for the effective expansion of structural diversity within a DCL, and also provides a sensitive probe for weak supramolecular interactions.

Cooperative functions of manganese and thiol redox system against oxidative stress in human spermatozoa

AIMS:In this study, the effects of 0.1 mM Mn2+ on thiol components (total thiols [TSH], glutathione reduced [GSH], glutathione oxidized [GSSG] and redox ratio [GSH/ GSSG]) have been determined in human spermatozoa.SETTINGS AND DESIGN:The subjects of the study were healthy males having more than 75% motility and 80 × 106 sperms/mL.MATERIALS AND METHODS:Fresh semen was suspended in phosphate-buffered saline (PBS) (pH 7.2) and this suspension was divided into eight equal fractions. All fractions, control (containing PBS) and experimental (treated/untreated with [ferrous ascorbate, FeAA - 200 FeSO4 μM, 1000 μM ascorbic acid, nicotine (0.5 mM) and FeAA + nicotine], supplemented/unsupplemented with Mn2+ [0.1 mM]), were incubated for 2 h at 37°C. These fractions were assessed for determining the thiol components.STATISTICAL ANALYSIS:The data were statistically analyzed by Students “t” test.RESULTS AND CONCLUSIONS:Ferrous ascorbate, nicotine and ferrous ascorbate + nicotine induced oxidative stress and decreased GSH and redox ratio (GSH/GSSG ratio) but increased the TSH and GSSG levels. Mn2+ supplementation improved TSH, GSH and redox ratio (GSH/GSSG) but decreased the GSSG level under normal and oxidative stress conditions. Thiol groups serve as defense mechanisms of sperm cells to fight against oxidative stress induced by stress inducers such as ferrous ascorbate, nicotine and their combination (ferrous ascorbate + nicotine). In addition, Mn2+ supplementation maintains the thiol level by reducing oxidative stress.

Quartz crystal microbalance bioaffinity sensor for rapid identification of glycosyldisulfide lectin inhibitors from a dynamic combinatorial library

Carbohydrate-lectin interactions were probed with dynamic combinatorial libraries, using the plant lectin Concanavalin A as target species. The dynamic combinatorial libraries were generated from a pool of thiol components through reversible thiol-disulfide interchange, and screened using a simple and efficient method based on a quartz crystal microbalance setup. It was found that dimers based on 1-thio- and 6-thio-mannose analogues were the most active inhibitors. Furthermore, the results clearly show that the 6-thio-mannose possess unique characteristics compared to its oxygen-containing counterpart.

Comparative study on the use of ortho-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde and anthracene-2,3-dicarboxaldehyde reagents for α-amino acids followed by the enantiomer separation of the formed isoindolin-1-one derivatives using quinine-type chiral stationary phases

Cinchona alkaloid based chiral stationary phases (CSPs) were evaluated and compared for the enantiomer separation of a set of α-amino acid derivatives as selectands (SA), using ortho-phthalaldehyde (OPA), naphthalene-2,3-dicarboxaldehyde (NDA) and anthracene-2,3-dicarboxaldehyde (ADA) as reagents in the presence of acetonitrile. Protocols have been developed for the derivatization of most common amino acids in the absence of the usual thiol components (2-mercaptoethanol, mercaptosulphonic acid, sodium sulfite) under acidic and neutral conditions providing the corresponding isoindolin-1-one (phthalimidine) derivatives. They are stable for hours at various reaction conditions compared to thiol or sulfide modified isoindoles resulted by the OPA-thiol reaction type. Among the derivatizing agents, ADA afforded the highest retention factors ( k) and for the majority of the analytes also resolution ( R S) and enantioselectivity ( α) values (i.e. for tryptophan k 1 = 23, R S = 4.93 and α = 1.43). Structure variation of the CSPs and selector (SO), respectively indicates that steric arrangement around the binding cleft plays a major role in the enantiodiscriminating events. To provide more detailed information about the derivatization reaction itself, the proposed mechanism for the formation of the OPA derivative (isoindolin-1-one) was further evaluated by deuterium labeling and LC–MS analysis.

Some physiological studies on the effect of onion and garlic juices on the fish, Clarias lazera

Fish, Clarias Lazera were orally administered onion (Allium cepa) or garlic (Allium sativum) juices. Contents of glucose, free amino acids, total lipids, triglycerides, cholesterol, albumin, total protein, urea, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum as well as glycogen, free amino acids, total lipids, triglycerides and cholesterol contents in liver, white and red muscle were determined 5 h and 24 h after a single dose or after repeated doses (1 dose every 24 h for 5 days). Both onion and garlic treatments caused a temporary hypoglycemia as well as liver glycogen muscle depletion accompanied with a significant elevation in white muscle glycogen suggesting that dietary onion or garlic may enhance the process of glycogenesis in white muscle. Onion feeding caused hypolipidemia accompanied with significant decreases in liver content of total lipids in response to the repeated doses for 5 days and a severe rise in the lipid content of red muscle at all periods. Such results suggest that dietary onion may enhance the process of lipogenesis in red muscle of Clarias lazera. Probably, the main precursor of such lipogenesis in the red muscle was the serum fatty acids. Moreover, dietary onion led to hypocholesterolemia accompanied with a significant rise in cholesterol level of both liver and red muscle. On the other hand, dietary garlic significantly decreased serum free amino acids after 5 h and 5 days. Furthermore, significant elevations of liver free amino acids were dedected in the onion-treated fish group (24 h and 5 days) and in the garlic-treated fish group (5 h), probably due to oxidation of thiol components (sulfur attached to the proteins). This result was accompanied with either a marked increase in white muscle free amino acids at all periods of garlic fish group, or a noticeable drop in the onion group (repeated doses). Also, dietary onion and garlic treatment led to an elevation in hepatic ureagenesis in spite of the unchanged serum urea level. Significant decreases of both serum AST and ALT in garlic groups (all periods) and in onion groups (repeated doses) were observed. In liver, AST and ALT showed temporary rises after onion or garlic supplementation. In general, it could be concluded that both dietary onion and garlic have a transitory hypoglycemic action as well as a promoting effect on the process of glycogenesis in white muscle whereas dietary onion promotes lipogenesis in red muscle of fish. Also, dietary garlic may enhance the white muscle uptake of free amino acids which, in turn, may promote protein synthesis.

The Redox State of Glutathione, Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.