Nedda Burlini

Nutritional Traits of Bean (Phaseolus vulgaris) Seeds from Plants Chronically Exposed to Ozone Pollution

The effect of chronic exposure to ozone pollution on nutritional traits of bean ( Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) seeds from plants grown in filtered and nonfiltered open-top chambers (OTCs) has been investigated. Results showed that, among seed macronutrients, ozone significantly raised total lipids, crude proteins, and dietary fiber and slightly decreased total free amino acid content, although with a significant reduction of asparagine, lysine, valine, methionine, and glycine, compensated by a conspicuous augmentation of ornithine and tryptophan. Phytosterol analysis showed a marked increase of beta-sitosterol, stigmasterol, and campesterol in seeds collected from nonfiltered OTCs. With regard to secondary metabolites, ozone exposure induced a slight increase of total polyphenol content, although causing a significant reduction of some flavonols (aglycone kaempferol and its 3-glucoside derivative) and hydroxycinnamates (caffeic, p-coumaric, and sinapic acids). Total anthocyanins decreased significantly, too. Nevertheless, ozone-exposed seeds showed higher antioxidant activity, with higher Trolox equivalent antioxidant capacity (TEAC) values than those measured in seeds collected from filtered air.

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Phytosterols in grapes and wine, and effects of agrochemicals on their levels

To 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.

Dependence on cyclic AMP of glucose-induced inactivation of yeast gluconeogenetic enzymes

1. INTRODUCTION In some yeasts, addition of glucose or metabol- ically related sugars to cells adapted to a sugar- lacking medium causes a time-dependent disap- pearance of some enzymes. The proposed physio- logical role of this ‘catabolite inactivation’ is the regulation of glucose formation, since nearly all of the involved enzymes take part in metabolic reac- tions that provide the cells with glucose

Extracellular space determination in rat small intestine by using markers of different molecular weights.

The apparent extracellular space (ECS) of rat jejunum, everted and cannulated “in vitro”, has been measured by using extracellular markers of different molecular weights. The markers used were two polyethyleneglycols,14C and3H labelled (14C-PEG MW 4000 and3H-PEG MW 900) and3H-sucrose. The ECSs for the mucosal and serosal sides have been separately determined throughout the time course, and it has been found that the two spaces are identical when PEG 4000 was used but the serosal ECS is almost the double when using PEG 900. The serosal ECS determined with sucrose is four times as big as the mucosal ECS. It seems reasonable to conclude that the best marker for the measure of total apparent ECS is sucrose, placed in the serosal compartment, taking into account that the mucosal ECS is four times smaller than the serosal one. All the markers used reach equilibrium with ECS, more rapidly in the mucosal than in the serosal ECS.Finally, by comparing cell water and cell Na concentrations, one observes that there is a statistical difference between the results obtained by using PEG 4000 as an extracellular marker and those obtained with sucrose.

Intracellular proteases from the extremely thermophilic archaebacteriumSulfolobus solfataricus

Proteolytic activities from the extremely thermoacidophilic archaebacteriumSulfolobus solfataricus were detected with the aid of synthetic substrates in a cell extract fractionated by gel filtration. Two aminopeptidases (aminopeptidase I and II), three endopeptidases (proteinase I, II and III) and one carboxypeptidase could be identified. Experiments carried out with protease inhibitors led to the identification of the exopeptidases as metalloproteases. Proteinases I and II behaved as chymotrypsin-like serine proteases, and proteinase III as a cysteine protease with a trypsin-like specificity. Molecular weight values assessed with the aid of marker proteins were as follows: aminopeptidase I, >450 kDa; aminopeptidase II, 170 kDa; carboxypeptidase, 160 kDa; proteinase I, 115 kDa; proteinase II, 32 kDa; proteinase III, 27 kDa. On incubation for 15 min they retained most of their activity up to a temperature of 90°C, with the sole exception of proteinase II, which was rapidly inactivated at 60°C. Protease content was also determined in crude extracts from cells grown in a mineral medium both to the stationary and to the exponential phase, with glucose or with yeast extract as carbon sources. No dramatic change was detected depending on the growth phase; however, carboxypeptidase level was three- to four-fold higher when yeast extract was present in the medium instead of glucose; this might suggest an involvement of this enzyme in the digestion of extracellularly available peptides.

The intestinal barrier in lepidopteran larvae : Permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous 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. We characterized 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 of whole-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.

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) supression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, but not of cytoplamic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.

Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.

Effects of elicitors and Ca2+ deprivation on the levels of sterols and 1α,25-dihydroxy vitamin D3 in cell cultures of Solanum malacoxylon

1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3], the hormonal form of vitamin D3 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 1α,25(OH)2D3 in plant cells, we monitored the changes of cellular levels of 1α,25(OH)2D3 in cell suspension cultures of Solanum malacoxylon Sendt. under different experimental conditions. We also measured the levels of cholesterol, the assumed precursor of vitamin D3, and other end-product sterols such as β-sitosterol, stigmasterol and campesterol. It was found that when cells are incubated in a calcium-deprived medium, the level of 1α,25(OH)2D3 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 1α,25(OH)2D3 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 1α,25(OH)2D3 is discussed in light of these results.