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21.
Paola Ceccaroli Paola Cardoni Michele Buffalini RobertaDe Bellis Giovanni Piccoli Vilberto Stocchi 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,702(1-2)
Hydrophobic interaction chromatography (HIC) has been used extensively for the separation of proteins and peptides by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this paper we compare different hydrophobic interaction chromatographic media for the separation of multiple forms of hexokinase from rabbit reticulocytes. Among the different hydrophobic chromatographic media tested (Toyopearl Phenyl 650S, Ether 650S and Butyl 650S) Toyopearl Phenyl 650S offered the best separation of multiple forms of hexokinase, probably due to its intermediate hydrophobicity. In order to establish the optimal experimental conditions, we evaluated the effects of different salts, and the results obtained demonstrated that among the antichaotropic salts, ammonium sulphate is the most suitable for the separation of hexokinase sub-types. The sample loading capacity of the three Toyopearl supports was investigated and the recovery of enzymatic activity obtained ranged from 60% to 90%, depending on the different salts and hydrophobic media used. The chromatographic profiles of hexokinase activity from various mammalian and fungal tissues also demonstrate that Toyopearl Phenyl 650S can be successfully employed for the separation of multiple forms of enzymes from different biological sources. 相似文献
22.
23.
Sarichelou I Cappuccio I Ferranti F Mosillo P Ciceroni C Sale P Stocchi F Battaglia G Nicoletti F Melchiorri D 《Cell death and differentiation》2008,15(4):700-707
Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells. 相似文献
24.
25.
Rossi I. Bartolacci B. Potenza L. Bertini L. Barbieri E. Stocchi V. 《Plant and Soil》2000,219(1-2):127-133
Morphologically very similar species of white truffle were analyzed by the random amplified polymorphic DNA (RAPD) technique. Species-specific RAPD fragments were selected and pairs of primers were designed on their sequences. Sequence-characterized amplified regions were developed and applied to identify these species throughout their entire life cycle: fruit body, mycelium, ectomycorrhiza. This procedure provides an unambiguous and rapid tool for typing species whose morphology is very similar. 相似文献
26.
Anatomical and morphological characterization of mycorrhizas of five strains of Tuber borchii Vittad
G. Giomaro A. Zambonelli D. Sisti M. Cecchini V. Evangelista V. Stocchi 《Mycorrhiza》2000,10(3):107-114
Tilia platyphyllos Scop. plantlets were inoculated in vitro with five Tuber borchii Vittad. strains (1BO, 17BO, 43BO, 71BO and 10RA) to test their intraspecific variability. The ability of the strains to form
mycorrhizas varied, with the mean degree of ectomycorrhizal infection ranging from 50.6% (for 1BO) to 82.1% (for 10RA). The
anatomical/morphological characteristics of the resulting mycorrhizas were determined. Although the morphological features
of the mycorrhizas and the characteristics of the cystidia were similar for all strains tested, differences were found in
the anatomical features of the mantle. The form of the mantle cells was examined in the surface and inner layers (6 and 12 μm
deep, respectively) by both conventional and confocal microscopy. These cells were polygonal in 1BO, and 71BO, epidermoid
in 43BO and intermediate in 17BO and 10RA. The structure of the mantle also varied and thus provided little information with
which to identify T. borchii mycorrhizas.
Accepted: 7 July 2000 相似文献
27.
V. Stocchi F. Palma G. Piccoli B. Biagiarelli M. Magnani L. Masat L. Cucchiarini 《Amino acids》1992,3(3):303-309
Summary In this paper we report the complete separation of amino acids as DABS-derivatives using a 3µm Supelcosil LC-18 (25 cm × 2.1 mm I.D.) narrowbore column. The system described makes it possible to perform the analysis of DABS-amino acids with a sensitivity to the femtomole level. We have also studied the conditions necessary for using the narrow-bore columns for routine analysis, paying particular attention to the problem of providing adequate protection for the analytical column. We have found it very suitable to use a (2 cm × 2.1 mm I.D.) guard column filled with a 40µm Pelliguard LC-18, pellicular packing resin, without affecting the complete resolution of the DABS-amino acids. Comparing the results obtained using conventional HPLC columns (3–5µm Supelcosil LC-18) of different lengths (15 and 25 cm × 4.6 mm I.D.) with those obtainable with the narrow-bore columns used in this work, it is possible to achieve a much greater sensitivity using the narrow-bore columns. In short, using the appropriate guard column and the standard HPLC apparatus used, the narrow-bore columns are very useful for routine analyses of DABS-amino acids with a sensitivity at the femtomole level. 相似文献
28.
Magnani Mauro Serafini Giordano Stocchi Vilberto 《Molecular and cellular biochemistry》1989,85(1):3-7
In rabbit reticulocytes more than half of the total hexokinase activity is mitochondrial bound and shows a fast decay during reticulocyte maturation. During in vitro incubation of rabbit reticulocytes, Ca2+ increases the decay of hexokinase while salicylhydroxamate (SHAM), an inhibitor of lipoxygenase, reduces the decay. Swelling of mitochondria, by incubation of the cells in hypotonic solutions, greatly enhances hexokinase decay, but both the Ca2+ and SHAM are still appreciable suggesting that Ca2+ and the swelling act by additive mechanisms, both able to influence hexokinase decay. This was confirmed by incubation of rabbit brain mitochondria in hypotonic solutions which does not promote any hexokinase decay, while the presence of Ca2+ does. Analyses of hexokinase isozymic pattern after incubation of reticulocytes in hypotonic solution both with and without Ca2+ and SHAM showed that the decay of hexokinase mainly involves the mitochrondrial bound isozymic forms.Abbreviations SHAM
Salicylhydroxamate
- HPLC
High-Performance Liquid Chromatography 相似文献
29.
Giordano Serafini Mauro Magnani Vilberto Stocchi Marina Dachà Giorgio Forniani 《Molecular and cellular biochemistry》1986,69(2):179-185
Summary Rat erythrocytes, in contrast to red blood cells from other mammals, have been shown to contain only one hexokinase isozymic form identified as type I by chromatographic and kinetic properties. Rat reticulocytes contain 3.6-times the hexokinase activity found in mature erythrocytes but exactly the same isozyme. By a combination of ion-exchange chromatography, dye-ligand chromatography and high-pressure liquid chromatography the rat erythrocyte hexokinase was purified more than 84 000-fold to a specific activity of 143 units/mg protein and shown to be homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native protein showed a molecular weight of 100 000 by gel-filtration and an apparent molecular weight of 98 000 under denaturating conditions in sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was shown to be 6.3 pH units. This data provides evidence of only one form of hexokinase in the erythrocytes of a mammal. 相似文献
30.
Red blood cell galactokinase activity was measured in 70 patients with cataracts to assess a possible correlation between galactokinase activity levels and risk of cataract development. Among all, 15 patients developed cataracts during the first year of life, 25 patients under the age of 50 and 30 later in life. No cases of total or partial galactokinase deficiency were found. These results, taken together with the absence of cataracts in 9 patients with partial galactokinase deficiency render less certain the cause and effect relationship between partial galactokinase deficiency and the appearance of cataracts. 相似文献