60 research outputs found
Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae.
No full textA phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid
Effects of elicitors and Ca2+ deprivation on the levels of sterols and 1alpha,25-dihydroxy vitamin D3 in cell cultures of Solanum malacoxylon
No full text1alpha,25-dihydroxy vitamin D-3[1alpha,25(OH)(2)D-3], the hormonal form of vitamin D-3 that is essential for the maintenance of calcium-phosphorus homeostasis in birds and mammals, has been found in several plants. In order to study the metabolic role of 1alpha,25(OH)(2)D-3 in plant cells, we monitored the changes of cellular levels of 1alpha,25(OH)(2)D-3 in cell suspension cultures of Solanum malacoxylon Sendt. under different experimental conditions. We also measured the levels of cholesterol, the assumed precursor of vitamin D-3, and other end-product sterols such as beta-sitosterol, stigmasterol and campesterol. It was found that when cells are incubated in a calcium-deprived medium, the level of 1alpha,25(OH)(2)D-3 increases markedly within a few hours. Treatment with methyl jasmonate (MeJ), a linolenic acid-derived compound that accumulates during wounding stress, was found to reduce the cellular level of 1alpha,25(OH)(2)D-3 and to promote its secretion into the culture medium. After treatment with cellulysin, a cellulase of fungal origin, the metabolite was no longer detectable, either in the medium or in the cells. Calcium deprivation in the medium and treatment with the elicitors MeJ and cellulase lowered the cellular level of all the sterols tested. The possible role of 1alpha,25(OH)(2)D-3 is discussed in light of these results
Phytosterols in grapes and wine, and effects of agrochemicals on their levels
No full textTo improve the knowledge on the chemical diversity and complexity of grapevine, we investigated the plant sterol content of berry and seed tissues at pre-véraison and véraison stages in 2009 and 2010. We also assessed the effects of benzothiadiazole and chitosan elicitors on content of sterols in grapes and their levels in the corresponding experimental wines. β-Sitosterol was the most abundant component in berry tissues, in both growth stages and years, with the highest amounts in the flesh and skin at pre-véraison and véraison, respectively. Stigmasterol and campesterol were present in lower concentrations in both phenological stages and vintages. During the transition from pre-véraison to véraison, phytosterols decreased in all tissues, in both years, apart from stigmasterol in seeds. In addition, the results showed that the plant activators were more effective than conventional fungicides in rising the levels of sterols, particularly β-sitosterol, both in grapes and in microvinificates
Permeability of lepidopteran larval midgut to the potential insecticidal peptides proctolin and TMOF
No full textGreat efforts have been recently devoted to the identification of innovative gene products to be developed as biopesticides. A large number of studies constantly detect new naturally derived peptides and proteins with haemocoelic receptors that could be used as potential insecticides. However, it is still to be defined their efficacy by oral delivery, because very limited information is available on how the epithelial barrier of the insect gut handles these kind of molecules.
We have considered two small peptides, both active as internal regulative factors in insects, that have lately been considered potential bioinsecticides, the pentapeptide proctolin and the decapeptide Trypsin Modulating Oostatic Factor (TMOF). We have performed a functional study of proctolin and TMOF permeability across the two barriers of Bombyx mori larval midgut, the peritrophic membrane (PM) and the epithelium, separately isolated and perfused in vitro in Ussing chambers. We observed that both peptides crossed easily the peritrophic membrane, but their permeability coefficient was highly affected by the molecular weight and steric conformation. Once crossed the PM, peptides present in vivo in the ectoperitrophic space have to pass through the second and more selective barrier represented by the intestinal epithelial cell monolayer. While proteins can cross B. mori epithelium from the lumen by transcytosis, no data are available for small peptides: here we tested the hypothesis that peptides with few amino acid residues could reach the insect haemocoel through the paracellular pathway, lined by the smooth septate junctions.
First, we characterised the selectivity properties of midgut paracellular pathway by measuring the lumen to haemolymph transepithelial fluxes of a number of organic molecules with different size and electric charge. The results obtained indicated that septate junction is highly selective to both the dimension and the charge of the permeant compounds. Then, we determined the route followed by proctolin and TMOF. The confocal images of whole-mount midguts incubated for two hours with rhodamine(rh)-proctolin or FITC-TMOF revealed that rh-proctolin was localized exclusively in the intercellular spaces, while labelled TMOF was present only inside the enterocytes’ cytoplasm. Therefore, proctolin does not enter midgut cells but crosses the epithelium through the paracellular pathway, whereas TMOF follows the transcellular route by a mechanisms to be clarified
The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides
No full textEndogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited
information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx
mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic
membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway.Wecharacterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images ofwhole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of
the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through
the transcellular route
Identification of enzyme activities involved in vitamin D3 metabolism in Solanum malacoxylon
No full textIn animals vitamin D3 is made from 7-deydrocholesterol through the action of UV light in the skin; the activation process of vitamin D3 involves a 25-hydroxylation in the liver, followed by a 1a-hydroxylation in the kidney, leading to 1a,25(OH)2D3, the hormone active on the regulation of calcium homeostasis. Vitamin D3 and the related hydroxylated metabolites, 25-hydroxy vitamin D3 [25-OH D3] and 1a,25-dihydroxy vitamin D3 or calcitriol [1a,25-(OH)2D3], have been found also in different plants[1]. The physiological role of these substances in plants has not been clarified yet but different hypothesis have been considered [2].
Aburjai et al. [3] observed that when calli of Solanum malacoxylon were grown in the light, vitamin D3 and the related hydroxylated metabolites were identified; if the calli were grown in the dark, only 7-dehydrocholesterol was found: these findings suggest that in plant cells may be present a biosynthetic pathway for 1a,25-(OH)2D3 similar to the one acting in animals.
To confirm this hypothesis we attempted to identify the enzymes involved in vitamin D3 metabolism in plant. We detected a 25(OH) vitamin D3 1α-hydroxylase activity in mitochondrial membranes purified both from leaves and from cell cultures of Solanum malacoxylon.
An activity vitamin D3 25-hydroxylase has also been measured in a reconstituted system containing: a solubilized enzyme fraction obtained from a cell cultures microsomal preparation, NADPH, ferredoxin-NADP+ reductase and ferredoxin I, both recombinant proteins from spinach. An attempt to identify also a 24-hydroxylase, the enzyme responsible for 25-OH D3 and 1a,25-(OH)2D3 catabolism in animals, is in progress
Effetto di una chitinasi ricombinante (AcMNPV-ChiA) sulla permeabilità della membrana peritrofica e sulla fisiologia intestinale di un lepidottero
No full textMetal compounds of calixarenes, detergent compositions and lubricant compositions
No full textThe invention relates to a metal compd. of calixarene not contg. sulfur and dispersible in oil, which can be partially salified, or neutral, or basic or overbased, wherein the calixarene has the general formula (I), where R1, R2, R3 and R4 are independently selected from hydrogen, or a group contg. carbon and hydrogen, or a group contg. heteroatoms in addn. to carbon and hydrogen, provided that the heteroatoms are not sulfur; one of the two substituents R5 and R6 is hydrogen, while the other may be hydrogen, or a linear or branched C1-C6-alkyl, preferably Me, Et, 1-Pr, 2-Pr, Bu, sec-Bu, more preferably Me or ethyl; n is the no. of units of the calixarene ring and is in the range between 4 and 16, preferably between 5 and 12, wherein in at least one of the n units of the calixarene ring, at least one of the substituents R1, R2, R3 and R4 contains at least one carboxylic acid group available for the reaction with a metal base, with the proviso that the carboxylic acid group is not contained in a unit of the calixarene ring derived from salicylic acid. [on SciFinder(R)
Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures
No full textBenzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D-3 metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 mu M BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3'-diaminobenzidine (DAB) for hydrogen peroxide (H2O2) detection and phloroglucinol for lignin staining showed that BTH causes H2O2 accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (beta-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D-3, and the hydroxylated metabolites 25-hydroxyvitamin D-3 [25(OH)D-3] and 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D3, though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D-3 and 1 alpha,25(OH)(2)D-3 metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol
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