Convenient syntheses of symmetrical and unsymmetrical glycosyl carbodiimides and N,N-bis(glycosyl)cyanamides

Reaction of glycosyl trimethylphosphinimides with carbon disulfide under mild conditions (room temperature, short reaction time) leads to symmetrical glycosyl carbodiimides. Addition of bis(trimethylsilyl)carbodiimide to peracetylated aldoses under the influence of SnCl(4) afforded N,N-bis(glycosyl)cyanamides for the first time. Readily accessible unsymmetrical N,N'-bis(glycosyl)thioureas can be desulfurated and transformed into the corresponding carbodiimides using HgO in CHCl(3)/water at room temperature.     

山羊小腔卵泡DNA含量的荧光分析

用显微手术法剥离山羊3 mm以下完整小腔卵泡,用胶原酶和Triton X-100进行处理,选用荧光试剂4,6-二脒基-2-苯吲哚(4,6-diamidino-2-phenylindol-2HCl,DAPI),对其DNA含量进行分析.结果表明,山羊3 mm以下卵泡的DNA含量与卵泡直径相关性极显著(r=0.9824＞0.7800=r_0.01,n=12).该法可测定5 ng的DNA.提示该法在卵泡生长发育的研究中具有重要应用价值.     

Fluorometric determination of alpha-ketosuccinamic acid in rat tissues

A method for the fluorometric determination of alpha-ketosuccinamic acid, the alpha-keto acid analog of asparagine, is described. The procedure involves the hydrolysis of alpha-ketosuccinamate to oxaloacetate by omega-amidase followed by NADH-dependent reduction of oxaloacetate to malate by malate dehydrogenase. A correction for endogenous oxaloacetate is made by using control samples lacking omega-amidase. Of the rat tissues investigated, liver contained the highest concentration, followed by kidney (53 +/- 6 (n = 11) and 18 +/- 3 (n = 3) mumol/kg wet wt, respectively). alpha-Ketosuccinamate was not detected in brain (less than 8 mumol/kg wet wt). Some chemical properties of alpha-ketosuccinamate were investigated. Concentrated solutions of sodium alpha-ketosuccinamate frozen for extended periods and the solid sodium salt of alpha-ketosuccinamate dimer heated to 130 degrees C are converted to at least 10 products by processes involving dimerization, dehydration, and decarboxylation. Isobutane chemical ionization mass spectral analysis (170-230 degrees C) of the free acid monomer yielded similar products. Many of the breakdown products were identified as di- and monoheterocyclic compounds, some of which are known to be of biological importance.     

Evaluation of antileishmanial activity of trans-aconitic acid

TAA, an inhibitor of the enzyme aconitase, inhibits the growth of L. donovani promastigotes. Morphogenic transformation of the amastigote to the promastigote (table; see text) form in vitro was also inhibited by 2 mM TAA. TAA also reduced multiplication of the parasite in macrophage culture. In the hamster model of leishmania, TAA significantly reduced the parasitic burden of liver. In acute toxicity tests with BALB/c mice no deaths were recorded even at a dose level of 2 g/kg body wt/day.     

Fluorometric determination of inulin using 5-quinolineboronic acid and inulinase

Inulin is a polysaccharide composed mainly of D-fructose units and is the most reliable indicator of the glomerular filtration rate. We have proposed an inulin detection method that involves the hydrolysis of inulin to D-fructose using inulinase and the selective binding of D-fructose from inulin using 5-quinolineboronic acid. In this method, the fluorescence of 5-quinolineboronic acid increases, depending on inulin concentration. For inulin in plasma, the detection and quantitation limits were calculated to be 3.7 and 11 μg/ml, respectively.     

Fluorometric determination of carbohydrate with 2-aminothiophenol

The 2-aminothiophenol-based fluorometric assay of Nakano et al. (1973, J. Pharm. Soc. Jpn. 93, 350-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 50 ng of carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates.     

Fluorometric determination of trioses with o-phenylphenol.

A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2′-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2′-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 50W-X12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 μg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.     

Fluorometric determination of carbohydrates

Fluorometric methods are described for the determination of the enzymes hexokinase and β-glucosidase, and the carbohydrates glucose, fructose, maltose, cellobiose, lactose, glycogen, and salicin Glucose and fructose are determined fluorometrically using hexokinase and the resazurin-resorufin indicator reaction. Maltose, cellobiose, lactose, glycogen, and salicin are enzymically hydrolyzed to glucose, which is then determined fluorometrically using glucose oxidase, p-hydroxyphenylacetic acid, and peroxidase. All the carbohydrates were assayed in the range 0.01–50 μg/ml and the enzymes hexokinase and β-glucosidase in the range 10^?3 to 10^?1 unit/ml with an accuracy and precision of about 1.5%.     

Fluorometric determination of deoxyribonuclease I activity with PicoGreen

A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.     

Chemical modification of hyaluronic acid by carbodiimides.

Hyaluronic acid (HA) is a linear polysaccharide with repeating disaccharide units of glucuronic acid and N-acetylglucosamine and is found in the extracellular matrix of connective tissues. Reaction of high molecular weight sodium hyaluronate (NaHA, MW approximately 2 x 10(6] with EDC at pH 4.75, either in the presence or absence of a primary diamine, gave the N-acylurea and O-acylisourea as NaHA-carbodiimide adducts. None of the expected intermolecular coupling with the amine component was observed. On the basis of this new observation, this method for chemical modification of HA was used in conjunction with new synthetic carbodiimides to prepare HA derivatives bearing lipophilic, aromatic, cross-linked, and tethered functional groups. The degree of conversion to NaHA-acylurea products appears to depend upon both the characteristics of various carbodiimides and the conformational structure of NaHA.     

Fluorometric determination of urinary kynurenic acid by flow injection analysis equipped with a "bypass line"

A flow injection analysis involving a photochemical reaction and fluorometric detection has been developed for the determination of urinary kynurenic acid. Kynurenic acid was found to fluoresce on irradiation with ultraviolet light at pH 7.2 in the presence of hydrogen peroxide. This method was applied to flow injection analysis using a new procedure involving a "bypass line" for the simultaneous determination of urinary kynurenic acid and background fluorescence. The calibration graph showed linearity over the range of 0.20 to 120 pmol. For pretreatment of urinary kynurenic acid, a PRE-SEP C18 cartridge was used. The mean recovery of kynurenic acid from urine was 94.5%. The content of urinary kynurenic acid was 13.0 +/- 2.68 mumol/day. There was good correlation (r = 0.9729) between values determined by flow injection analysis and high-performance liquid chromatography.     

党参类多糖比色法含量测定

党参的主要成分是糖类,传统经验认为,党参“味甜者佳”,其品质好坏,多以含糖量来衡量。药理研究表明,党参的升血糖作用是其所含糖分所致。刘中煜等曾对5种党参中单糖含量进行了测定,但其多糖含量测定尚无报道。本文采用苯酚一硫酸比色法对党参类多糖含量进行了测定。