In vitro study of platelet concentrate preparations from whole blood using N-(2-mercaptopropionyl)-glycine

The sulfhydryl group containing drug N-(2-mercaptopropionyl)-glycine (MPG) which inhibits platelet aggregation in a reversible manner permits to prepare platelet concentrates in non-siliconized glass containers at pH 7.4. Resuspension of platelets is possible immediately after centrifugation. In vitro platelets tests were carried out after washing out the MPG in MPG-free plasma. Thereafter, no inhibitory effects on platelet functions were found. Platelets concentrated in presence of MPG were significantly better with respect to yield, maintenance of discoid shape, aggregability, and hypotonic shock response compared with control platelets concentrated in absence of MPG.     

Effect of N-2-mercaptopropionyl glycine on exercise-induced cardiac adaptations

The purpose of this study was to test the hypothesis that exercise-induced cardiac adaptations would be attenuated by the free radical scavenger N-2-mercaptopropionyl glycine (MPG). Male Sprague-Dawley rats were divided into four groups (n = 9-13 per group) for 3-4 wk: sedentary (S), S+MPG (100 mg/kg ip daily), exercised on a treadmill (E) (60 min/day, 5 days/wk, at a speed of 20 m/min up a 6° grade in a 6°C room), or E+MPG given 10 min prior to exercise. Additional rats (n = 55) were used to determine acute exercise effects on myocardial redox state [nonprotein nonglutathione sulfhydryls (NPNGSH)] and PI3K/Akt signaling pathway activation. Compared with S, NPNGSH levels were 48% lower in E (P < 0.05) and unchanged in E+MPG (P > 0.05). MPG also attenuated exercise-induced activation of the signaling proteins Akt and S6. Hearts from the 4-wk groups were weighed, and cardiac function was evaluated using an isolated perfused working heart preparation. Similar increases (P < 0.05) in both exercised groups were observed for heart weight and heart weight-to-body weight ratio. Cardiac function improved in E vs. S, as indicated by greater (P < 0.05) external work performed (cardiac output × systolic pressure) and efficiency of external work (work/Vo(2)). MPG prevented these exercise-induced functional improvements. Skeletal muscle mitochondria content increased to similar levels in E and E+MPG. This study provides evidence that free radicals do not play an essential role in the development of exercise-induced cardiac hypertrophy; however, they appear to be involved in functional cardiac adaptations, which may be mediated through the PI3K/Akt pathway.     

The gastrointestinal absorption,tissue distribution,urinary excretion and metabolism of N-(2-aminoethyl)-glycine (AEG) in the rat

Radiolabeled N-(2-aminoethyl)-glycine (AEG) was synthesized and various aspects of its bioavailability were evaluated. AEG was rapidly and completely taken up by the small intestine of the rat. It was quickly absorbed into the portal vein. Most of the absorption took place during the first hour, with a peak at 30 min. Entry of this compound into the intestinal mucosal cell may be by a mechanism not involving active transport. Of many organs examined, only the liver took up significant amounts of AEG. The latter neither crossed the brain barrier nor was metabolized. Total urinary excretion (as intact AEG) averaged 80% of the administered dose within 4 hours and nearly 100% by 10 hours. Excluding the neutral-acidic amino acids and ammonia, AEG represented >99% of the ninhydrin positivity in the urine. AEG is thus an example of a substance which is rapidly and totally absorbed, as well as quickly and completely excreted.     

Attenuation of the acute adriamycin-induced cardiac and hepatic oxidative toxicity by N-(2-mercaptopropionyl) glycine in rats

The protective effect of the synthetic aminothiol, N-(2-mercaptopropionyl) glycine (MPG) on adriamycin (ADR) induced acute cardiac and hepatic oxidative toxicity was evaluated in rats. ADR toxicity, induced by a single intraperitoneal injection (15 mg/kg), was indicated by an elevation in the level of serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatine kinase isoenzyme (CK-MB), and lactic dehydrogenase (LDH). ADR produced significant elevation in thiobarbituric acid reactive substances (TBARS), indicating lipid peroxidation, and significantly inhibited the activity of superoxide dismutase (SOD) in heart and liver tissues. In contrast, a single injection of ADR did not affect the cardiac or hepatic glutathione (GSH) content and cardiac catalase (CAT) activity but elevated hepatic CAT. Pretreatment with MPG, (2.5 mg/kg) intragastrically, significantly reduced TBARS concentration in both heart and liver and ameliorated the inhibition of cardiac and hepatic SOD activity. In addition, MPG significantly decreased the serum level of GOT, GPT, CK-MB, and LDH of ADR treated rats. These results suggest that MPG exhibited antioxidative potentials that may protect heart and liver against ADR-induced acute oxidative toxicity. This protective effect might be mediated, at least in part, by the high redox potential of sulfhydryl groups that limit the activity of free radicals generated by ADR.     

Characterization of the binding properties of SIRT2 inhibitors with a N-(3-phenylpropenoyl)-glycine tryptamide backbone

SIRT2 inhibitors with a N-(3-phenylpropenoyl)-glycine tryptamide backbone were studied. This backbone has been developed in our group, and it is derived from a compound originally found by virtual screening. In addition, compounds with a smaller 3-phenylpropenoic acid tryptamide backbone were also included in the study. Binding modes for the new compounds and the previously reported compounds were analyzed with molecular modelling methods. The approach, which included a combination of molecular dynamics, molecular docking and cluster analysis, showed that certain docking poses were favourable despite the conformational variation in the target protein. The N-(3-phenylpropenoyl)-glycine tryptamide backbone is also a good backbone for SIRT2 inhibitors, and the series of compounds includes several potent SIRT2 inhibitors.     

Kinetic modelling of Amadori N-(1-deoxy-D-fructos-1-yl)-glycine degradation pathways. Part I--reaction mechanism

The fate of the Amadori compound N-(1-deoxy-D-fructos-1-yl)-glycine (DFG) was studied in aqueous model systems as a function of pH and temperature. The samples were heated at 100 and 120 degrees C with initial reaction pH of 5.5 and 6.8. Special attention was paid to the formation of the free amino acid, glycine; parent sugars, glucose and mannose; organic acids, formic and acetic acid and alpha-dicarbonyls, 1- and 3-deoxyosone together with methylglyoxal. For the studied conditions decreasing the initial reaction pH with 1.3 units or increasing the temperature with 20 degrees C has the same effect on the DFG degradation as well as on glycine formation. An increase in pH seems to favour the formation of 1-deoxyosone. The lower amount found comparatively to 3-deoxyosone, in all studied systems, seems to be related with the higher reactivity of 1-deoxyosone. Independently of the taken pathway, enolization or retro-aldolization, DFG degradation is accompanied by amino acid release. Together with glycine, acetic acid was the main end product formed. Values of 83 and 55 mol% were obtained, respectively. The rate of parent sugars formation increased with pH, but the type of sugar formed also changed with pH. Mannose was preferably formed at pH 5.5 whereas at pH 6.8 the opposite was observed, that is, glucose was formed in higher amounts than mannose. Also, independently of the temperature, at higher pH fructose was also detected. pH, more than temperature, had an influence on the reaction products formed. The initial steps for a complete multiresponse kinetic analysis have been discussed. Based on the established reaction network a kinetic model will be proposed and evaluated by multiresponse kinetic modelling in a subsequent paper.     

Kinetic modelling of Amadori N-(1-deoxy-D-fructos-1-yl)-glycine degradation pathways. Part II--kinetic analysis

A kinetic model for N-(1-deoxy-D-fructos-1-yl)-glycine (DFG) thermal decomposition was proposed. Two temperatures (100 and 120 degrees C) and two pHs (5.5 and 6.8) were studied. The measured responses were DFG, 3-deoxyosone, 1-deoxyosone, methylglyoxal, acetic acid, formic acid, glucose, fructose, mannose and melanoidins. For each system the model parameters, the rate constants, were estimated by non-linear regression, via multiresponse modelling. The determinant criterion was used as the statistical fit criterion. Model discrimination was performed by both chemical insight and statistical tests (Posterior Probability and Akaike criterion). Kinetic analysis showed that at lower pH DFG 1,2-enolization is favoured whereas with increasing pH 2,3-enolization becomes a more relevant degradation pathway. The lower amount observed of 1-DG is related with its high reactivity. It was shown that acetic acid, a main degradation product from DFG, was mainly formed through 1-DG degradation. Also from the estimated parameters 3-DG was found to be the main precursor in carbohydrate fragments formation, responsible for colour formation. Some indication was given that as the reaction proceeded other compounds besides DFG become reactants themselves with the formation among others of methylglyoxal. The multiresponse kinetic analysis was shown to be both helpful in deriving relevant kinetic parameters as well as in obtaining insight into the reaction mechanism.     

N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.     

Synthesis of N-(beta-D-glucopyranosyl)- and N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) amides as inhibitors of glycogen phosphorylase

2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl- and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl azides were transformed into the corresponding per-O-acetylated N-(beta-D-glycopyranosyl) amides via a PMe(3) mediated Staudinger protocol (generation of N-(beta-D-glycopyranosyl)imino-trimethylphosphoranes followed by acylation with carboxylic acids, acid chlorides or anhydrides). The deprotected compounds obtained by Zemplén deacetylation were evaluated as inhibitors of rabbit muscle glycogen phosphorylase b. The best inhibitor of this series has been N-(beta-D-glucopyranosyl) 3-(2-naphthyl)-propenoic amide (K(i)=3.5microM).     

Prevention by tiopronin (2-mercaptopropionyl glycine) of methylmercuric chloride-induced teratogenic and fetotoxic effects in mice

Previous investigations (Fuyuta et al., '76, '79) have shown that a single oral administration of 25 mg/kg methylmercuric chloride (MMC) to pregnant ICR mice on day 10 of pregnancy induced cleft palate in a remarkably high incidence in fetuses. Based on these findings, the present study dealing with the prevention of cleft palate by Tiopronin, (2-mercaptopropionyl glycine, Tp), was initiated. Twenty females in the positive control group were given 25 mg/kg MMC orally on day 10 of pregnancy and then given physiological saline intraperitoneally. Twenty females in the negative control group were given distilled water orally and then given saline intraperitoneally. Cleft palate was found in 98.1% of fetuses in the positive control group and none of them in the negative control group. Twenty females were pretreated with a single oral dose of 25 mg/kg MMC on day 10 of pregnancy and were posttreated with Tp intraperitoneally, immediately and at every 24, 48 and 72 hours after the MMC treatment. The doses of Tp were 320, 160 and 80 mg/kg/day. The incidences of cleft palate in fetuses were reduced to 1.49, 31.3 and 47.8% in the Tp-treated groups with the doses of 320, 160 and 80 mg/kg/day, respectively. Tiopronin could effectively prevent the expected incidence of cleft palate. Other types of abnormalities as well as fetotoxicity represented by reduced fetal body weight were also effectively prevented with the Tp-treatment.     

Conformations of N-(2-fluorophenyl)-N-phenylcarbamoyl-alpha-chymotrypsin

N-(2-Fluorophenyl)-N-phenylcarbamoyl chloride is shown to react with alpha-chymotrypsin to give a catalytically inactive material. A crystal structure determination shows that the chloride exists in the solid state in two conformations. In both of these the aromatic rings are tilted substantially relative to the plane through the atoms of the carbamoyl chloride group; the structures differ by a 180 degrees rotation of the 2-fluorophenyl ring. Fluorine NMR studies of alpha-chymotrypsin modified with this carbamoyl chloride show that, when bound to the enzyme, one aromatic ring of the diphenylcarbamoyl group likely rotates slowly while the other rotates much more rapidly or else is frozen in one dominant conformation. In the denatured enzyme (8 M urea) at room temperature and above, both aromatic rings of the diphenylcarbamoyl group appear to be rapidly rotating although differential linewidth changes observed at lower sample temperatures suggest that rotation of one ring becomes slow under these conditions. Rotation about the carbamoyl carbon-nitrogen bond is detected in fluorine NMR spectra of both the native and the denatured modified enzymes as the sample temperature is increased. Rates of carbamoyl rotation in the chloride, in the native modified enzyme, and in the denatured enzyme at 25 degrees C are approximately 66, 10, and 200 s-1, respectively.     

Synthesis of novel N-(2-hydroxy-2-p-tolylethyl)-amide and N-(2-oxo-2-p-tolylethyl)-amide derivatives and their antidyslipidemic and antioxidant activity

In continuation of our drug discovery program on metabolic diseases, we identified an alkaloidal amide, that is, Aegeline (V) from the plant Aegle marmelos leaves as a dual acting agent (antihyperlipidemic and antihyperglycemic). We therefore synthesized a series of alkaloidal amides [N-(2-hydroxy-2-p-tolylethyl)-amides and N-(2-oxo-2-p-tolylethyl)-amide derivatives] related to Aegeline and screened for their in vivo antihyperlipidemic activity in Triton induced hyperlipidemia model. The synthetic compounds 4, 17 and 20 showed equipotent activity to the natural product, that is, Aegeline (V). These compounds also showed strong antioxidant activity, which support their antihyperlipidemic activity. Compound 12 showed better antihyperlipidemic and antioxidant profile than the natural product V.     

The preparation and storage of cytochrome oxidase

The preparation of cytochrome oxidase by the method of Keilin and Hartree has been modified to give an increased yield and activity of the enzyme. The enzyme can be dried from the frozen state and stored for more than a year provided it is reconstituted in pH 9.5 buffer and treated with sound waves at 9,000 cycles/sec. This material is “soluble” in that it resists centrifugation at 11,000 × g for 2.5 hr. Its “solubility” depends in no way on autolysis of the heart muscle prior to extraction of the enzyme.     

Binary mixtures of (N-phosphonomethyl)-glycine with new aminophosphonates

The potential biological activity of binary mixtures of some new organophosphorous compounds, aminoalkane- and aminofluorenephosphonates, with (N-phosphonomethyl)-glycine (glyphosate, PMG) was studied. The inhibition of growth of wheat (Triticum aestivum) induced by individual compounds and their equimolar mixtures with PMG was a measure of that activity. The experiments were expected to show if the new compounds exhibited good biological activity to be used for agrochemical applications and if this activity can be improved when they are used in mixtures with glyphosate which is the active component of the well-known herbicide Roundup. The results obtained show that aminofluorenephosphonates inhibited wheat growth when used in micromolar concentrations. Thus, their efficiency can be compared to that of PMG. The efficiency of aminoalkanephosphonates was one order of magnitude weaker. The measure of the efficiency was the effective concentration inhibiting wheat growth by 50% (EC50). The most demanded interaction, i.e., a synergistic was observed for only one of binary mixtures of the compounds studied with PMG. Mostly they showed antagonistic or strong antagonistic interactions. Some of them were of the additive type. Such results exclude the possibility of potential use of all the compounds studied in binary mixtures with phosphonomethylglycine, especially as the mentioned synergistic interaction found was rather weak. The influence of structural features of anminophosphonates on the results obtained is discussed.     

Coriolus versicolor Laccase Catalyzes the Decarboxylation of 2-(4-Hydroxyphenyl)-glycine and 4-Hydroxymandelic Acid

Cyclodextrin glucanotransferase [1,4-α-D-glucan 4-α-D-(l,4-α-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from an alkalophilic Bacillus species A2–5a had a wider acceptor specificity than that from B. macerans, which was similar to those from B. stearo-thermophilus and B. circulans.

Glucosyl rhamnose produced by the CGTase was identified as glucopyranosyl-α-l,4-rhamnopyranose by α- and β-glucosidase treatments, and ¹H- and ^{1 3}C-NMR analyses.     

Facile synthesis of (R)N-2-hydroxyacyl-L-cysteine derivatives: (R)N-2-hydroxyacyl transfer from enzymatically-synthesized (R)S-2-hydroxyacylglutathione derivatives to L-cysteine

Summary N-(R)-2-Hydroxyacyl-L-cysteine derivatives were conveniently synthesized by the reaction of the corresponding S-(R)-2-hydroxyacyl-glutathione with cysteine. The (R)2-hydroxyacyl group was transferred from the S-glutathionyl moiety to S-cysteinyl, forming the corresponding (R)S-2-hydroxyacylcysteine; this rearranged to the (R)N-hydroxyacylcysteine. These compounds have anti-proliferative activity associated with the inhibition ofde novo pyrimidine synthesis.Abbreviations TRIS tris(hydroxymethyl) aminomethane - DTNB 5,5-dithiobis(2-nitrobenzoic acid)