Anti-obesity and lipid lowering effects of Cymodocea nodosa sulphated polysaccharide on high cholesterol-fed-rats.

This study aims to evaluate for the first time the effects of Cymodocea nodosa sulphated polysaccharide (CNSP) on lipase activity in vitro and in vivo to high fat diet (HFD)-rats on body weight, lipid profile and liver-kidney functions. The administration of CNSP decreases the body weight and inhibits lipase activity of obese rats in serum and intestine as compared with untreated HDF-rats. This decrease in lipase activity leads to lipid regulation shown by the decrease of total cholesterol (T-Ch), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) and an increase in high density lipoprotein cholesterol (HDL-C) levels in HFD-rats. Additionally, CNSP administration to HFD-rats induces anti-oxidant activity observed by the increase of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities and the decrease in Thiobarbituric acid reactive substances (TBARS) levels and protects liver-kidney functions proven by a decrease in the levels of toxicity parameters in blood.

Inhibitory effects of Cymodocea nodosa sulphated polysaccharide on α-amylase activity, liver-kidney toxicities and lipid profile disorders in diabetic rats.

This study aimed to evaluate for the first time the effects of Cymodocea nodosa sulphated polysaccharide (CNSP) on the α-amylase activity, hyperglycaemia, liver-kidney functions, and pancreatic architecture of alloxan-induced diabetic rats. Animals were allocated into four groups of seven rats each, the body weight and blood glucose levels were estimated periodically for 2 months of treatment by gastric gavages route. The CNSP effect was confirmed by biochemical procedures and histological study. The inhibition of α-amylase activity and protection of pancreatic ß-cells induced a decrease in the blood glucose levels and regulated the lipid profile in the plasma of the treated diabetic rats, which helped to maintain the homeostasis of blood lipid. Moreover, CNSP administration induced a significant decrease in the levels of lipid peroxidation in the pancreas, liver and kidney of diabetic rats and protects their functions attested by a decrease in the levels of toxicity parameters in blood.

Role of disulphide linkages between protein-coated lipid droplets and the protein matrix in the rheological properties of porcine myofibrillar protein-peanut oil emulsion composite gels.

The objective of the study was to establish disulphide interaction between protein-coated oil droplets and the surrounding protein matrix in myofibrillar protein (MP)-emulsion composite gels. An MP-stabilized peanut oil emulsion was treated with 0, 1, 3, 5 and 10 mM N-ethylmaleimide (NEM, a sulphydryl-blocking agent) and subsequently incorporated into a bulk MP sol to produce 5%-lipid, 2%-protein composites at pH 6.2. About 69% of sulphydryls in the emulsion (1% protein) were blocked by 1 mM NEM, and almost all were bound at ≥3 mM NEM. The loss of free sulphydryls resulted in a significant drop in the storage modulus (G') and rupture force of the composite gels. Microstructural examination revealed pores and oil leakage from emulsion droplets by NEM treatments, corresponding to declining rheological properties of the MP-emulsion composites. The results supported the hypothesis that disulphide cross-linking between MP-coated oil droplets and protein matrix contributed to the stabilization and reinforcement of protein-emulsion composite gels formed in comminuted muscle foods.

Measurement of ligand binding to tubulin by sulfhydryl reactivity.

Ligand binding can induce shifts in protein conformation. In the case of tubulin, these drug-induced confirmational changes can prevent or stabilize microtubule polymerization. 5',5'-Dithiobis(2-nitrobenzoate) (DTNB) reacts with free and accessible sulfhydryls and stoichiometrically produces a detectable product, which allows an exact measurement of reacted thiols. Since binding of small ligands may alter conformational dynamics, it may also affect the reactivity of thiols on tubulin. Differences in DTNB reactivity with thiols upon ligand binding can therefore be used to deduce binding characteristics. We will describe two methods that use tubulin cysteine reactivity with DTNB in the presence of drug to define ligand-binding characteristics.

Functional activity of a monocarboxylate transporter, MCT1, in the human retinal pigmented epithelium cell line, ARPE-19.

The purpose of this study was to identify and characterize the functional activity of monocarboxylic acid transporter 1 (MCT1) on the human retinal pigmented epithelium (RPE) cell line, ARPE-19, and to evaluate whether the cell line can function as an in vitro screening tool for intravitreally administered drugs/prodrugs targeted to the MCT1 expressed in RPE. Uptake studies were carried out at 37 degrees C, for 30 s, with ARPE-19 cells. [(14)C]l-Lactic acid was selected as a substrate for this transporter. Uptake of [(14)C]L-lactic acid by ARPE-19 cells was found to exhibit saturable kinetics (K(m) = 3.1 +/- 0.6 mM and V(max) = 63.1 +/- 4.1 pmol/min/mg of protein). Monocarboxylic acids, such as benzoic acid, salicylic acid, and pyruvic acid, inhibited the uptake of [(14)C]L-lactic acid whereas di- and tricarboxylic acids, such as phthalic, succinic, and citric acids, did not demonstrate any inhibitory effect. Uptake was stereospecific where D-lactic acid was less effective in inhibiting [(14)C]L-lactic acid uptake than unlabeled L-lactic acid. ELISA indicated the expression of only MCT1, MCT4, and MCT8 isoforms by ARPE-19 cells. Increase in [(14)C]L-lactic acid uptake was observed as the uptake medium pH was lowered from 7.4 to 5.0. Moreover, inhibition of [(14)C]L-lactic acid uptake was observed in the presence of the protonophore 2,4-dinitrophenol. Uptake was significantly decreased in the presence of sodium azide, ouabain, p-chloromercuribenzoic acid (pCMBA), N-ethylmaleamide, dithiothreitol, and p-chloromercuribenzene sulfonate (pCMBS). However, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and L-thyroxine did not inhibit [(14)C]L-lactic acid. RT-PCR studies and sequence analysis of the PCR product confirmed the expression of MCT1 by ARPE-19 cells. Our results indicate that MCT1 is functionally active and is the only MCT isoform involved in the apical uptake of monocarboxylates by ARPE-19 cells. This cell line may thus be used as an effective screening tool for intravitreally administered drugs/prodrugs targeted toward MCT1 expressed on the RPE.

Phosphorylation-dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component.

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A, and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation.

Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds.

Mass spectrometry, proteomics, and protein chemistry methods are used to characterize the cleavage products of 79 kDa transferrin proteins induced by iron-catalyzed oxidation, including a novel C-terminal polypeptide released upon disulfide reduction. Top-down electrospray ionization tandem mass spectrometry (ESI-MS/MS) of intact multiply-charged transferrin from a variety of species (human, bovine, rabbit, chicken) performed on a quadrupole time-of-flight mass spectrometer yields multiply-charged b(n)-products originating near residues 56-69 from the N-terminal region, in addition to their complementary y(n)-products. Incubation of transferrin with reductants, such as dithiothreitol (DTT) or tris(2-carboxyethyl)-phosphine (TCEP), yields an increase in multiple charging observed by ESI-MS and an increase in molecular weight consistent with disulfide reduction. However, mammalian transferrins release a 6-8 kDa fragment upon disulfide reduction. Protein acetylation and MS/MS sequencing demonstrate that the fragment originates from the C-terminus of the protein, and that it is a separate polypeptide linked via three disulfide bonds to the main transferrin chain. The existence of a separate C-terminal chain is not annotated in protein sequence databases and, to date, has not been reported in the literature. Iron-catalyzed cleavage induces fragments originating from both the N- and C-terminus of transferrin.

RPR112378 and RPR115781: two representatives of a new family of microtubule assembly inhibitors.

A screening program aimed at the discovery of new antimicrotubule agents yielded RPR112378 and RPR115781, two natural compounds extracted from the Indian plant Ottelia alismoides. We report their isolation, structural determination, and mechanisms of action. RPR112378 is an efficient inhibitor of tubulin polymerization (IC(50) = 1.2 microM) and is able to disassemble preformed microtubules. Regarding tubulin activity, RPR115781 is 5-fold less active than RPR112378. Tubulin-RPR112378 complexes, when isolated by gel filtration, were able to block further tubulin addition to growing microtubules, a mechanism that accounts for the substoichiometric effect of the drug. RPR112378 was found to prevent colchicine binding but not vinblastine binding to tubulin. Although colchicine binding is known to induce an increase of tubulin GTPase activity, no such increase was observed with RPR112378. We show that RPR112378 is a highly cytotoxic compound and that RPR115781 is 10, 000-fold less active as an inhibitor of KB cell growth. Part of the cytotoxicity of RPR112378 is probably caused by a reaction of addition with sulfhydryl groups, an observation that has not been made with RPR115781. In conclusion, these molecules represent a new class of inhibitors of microtubule assembly with potential therapeutic value.

MALDI mass spectrometry combined with avidin-biotin chemistry for analysis of protein modifications.

A general mass spectrometric method that combines purification and analysis in one step is described for the rapid and sensitive determination of protein modification that involves covalent attachment of a modifying group. In this method, the modifying group is first labeled with a biotin moiety, and the covalent interaction of this group with the targeted protein results in a biotinylated product. The modified protein can then be subjected to enzymatic digestion, followed by the isolation of the biotinylated peptide based on a previously described MALDI method incorporating the avidin-biotin interaction (Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387). To illustrate the validity of the method, a study of a model system was undertaken, involving the interaction between avian skeletal muscle troponin C and a sulfhydryl-specific biotinylation reagent. It is shown that isolation of a modified peptide with an immobilized avidin product could be achieved, even in the presence of an excess of contaminating protein. Exoproteases could be added to the crude tryptic digest to generate peptide ladders, each containing biotin, which could be analyzed by the avidin-biotin/MALDI method for sequence information. Complementary sequence information could be obtained from the application of this technique in a tandem sector/time-of-flight mass spectrometer for MALDI MS/MS analysis, which allowed for the identification of the modification site.

Compound A: solubility in saline and olive oil; destruction by blood.

Compound A is a degradation product of sevoflurane. Knowledge of the solubility of Compound A, CH2F-O-C(=CF2)(CF3), in blood and other solvents would aid in the definition of its kinetics. Accordingly, we determined solvent/gas partition coefficients of Compound A for saline (0.166 +/- 0.002 [mean +/- SD; n = 4]) and olive oil (20.1 +/- 1.1 [n = 4]). Measurement of solubility in blood was confounded by degradation of Compound A in blood and blood components. If a mixture of 99.3% saline and 0.7% oil provides the solubility equivalent to that possessed by blood (as it does for the parent compound, sevoflurane), then blood solubility and solubility in plasma, albumin, red blood cells, or pure hemoglobin is approximately 0.31. The order of Compound A degradation was human plasma = rat blood > whole human blood >5% human serum albumin = washed human red blood cells (hematocrit 50%) = 5% pure hemoglobin. Presuming a solvent/gas partition coefficient of 0.31, respective approximate times for 50% degradation equaled 2.7, 2.8, 4.6, 9.9, 11.0, and 12 min. The accuracy of these approximations was limited by the need to estimate, rather than determine, the solubility of Compound A in such solvents. Pasteurization (heating to 60 degrees C for 12 h) or pretreatment with N-ethylmaleimide (a compound that reversibly binds to sulfhydryl groups) decreased the degradation rate in plasma. These results suggest that degradation arises, at least in part, from reaction of Compound A with proteins in blood, possibly from covalent reaction of Compound A with protein and/or from an enzymatically mediated reaction. The products of degradation, the binding sites, and the clinical implications of such binding and degradation remain to be determined.