Claudio Giovannini

Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

ABSTRACT Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.

Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type‐2 Astrocyte Cell Lineage

Abstract: We have used postnatal rat cerebellar astrocyte‐enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type‐2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ‐[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N‐Methyl‐d‐aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l‐Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l‐glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d‐[3H]aspartate was taken up by both type‐1 and type‐2 astrocytes, but only type‐2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type‐2 astrocytes were also expressed by the immature, bipotential progenitors of type‐2 astrocytes and oligodendrocytes.

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ‐Aminobutyric Acid Release

Using cerebellar, neuron‐enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D‐[3H]aspartate release, whereas N‐methyl‐D‐aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20–100 μM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 μM. Kainate and dihydrokainate (20–100 μM) inhibited the initial rate of D‐[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N‐methyl‐DL‐aspartate were ineffective. D‐[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D‐[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50–200 μM) and, to a lesser extent, 2,3‐cis‐piperidine dicarboxylic acid (100–200 μM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20–100 μM) also stimulated γ‐[3H]aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D‐[3H]aspartate neuronal uptake by interfering with the acidic amino acid high‐affinity transport system.

Protocatechuic Acid and Human Disease Prevention: Biological Activities and Molecular Mechanisms

Epidemiological evidence has shown that a high dietary intake of vegetables and fruit rich in polyphenols is associated with a reduction of cancer incidence and mortality from coronary heart disease. The healthy effects associated with polyphenol consumption have made the study of the mechanisms of action a matter of great importance. In particular, the hydroxybenzoic acid protocatechuic acid (PCA) has been eliciting a growing interest for several reasons. Firstly, PCA is one of the main metabolites of complex polyphenols such as anthocyanins and procyanidins that are normally found at high concentrations in vegetables and fruit, and are absorbed by animals and humans. Since the daily intake of anthocyanins has been estimated to be much higher than that of other polyphenols, the nutritional value of PCA is increasingly recognized. Secondly, a growing body of evidence supports the concept that PCA can exert a variety of biological effects by acting on different molecular targets. It has been shown that PCA possesses antioxidant, anti-inflammatory as well as antihyperglycemic and neuroprotective activities. Furthermore, PCA seems to have chemopreventive potential because it inhibits the in vitro chemical carcinogenesis and exerts pro-apoptotic and anti-proliferative effects in different tissues. This review is aimed at providing an up-dated and comprehensive report on PCA giving a special emphasis on its biological activities and the molecular mechanisms of action most likely responsible for a beneficial role in human disease prevention.

Mitochondria hyperpolarization is an early event in oxidized low‐density lipoprotein‐induced apoptosis in Caco‐2 intestinal cells

We investigated the mechanisms underlying the pro‐apoptotic activity exerted by oxidized low‐density lipoproteins (oxLDL) in Caco‐2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that: (i) oxLDL induced mitochondrial‐mediated apoptosis by provoking first an increase in mitochondrial membrane potential, followed, later, by the typical apoptosis‐associated depolarization (type II apoptosis); accordingly, (ii) caspase‐9 inhibition significantly hindered apoptosis while caspase‐8 inhibition did not; and finally (iii) dietary phenolic antioxidizing compounds exerted a significant protective antiapoptotic activity. These results point to mitochondrial hyperpolarization as ‘sensitizing feature’ in apoptotic proneness of Caco‐2 intestinal cells to oxLDL exposure.

Wheat gliadin induces apoptosis of intestinal cells via an autocrine mechanism involving Fas^Fas ligand pathway

Wheat gliadin and other cereal prolamins have been said to be involved in the pathogenic damage of the small intestine in celiac disease via the apoptosis of epithelial cells. In the present work we investigated the mechanisms underlying the pro‐apoptotic activity exerted by gliadin‐derived peptides in Caco‐2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that digested peptides from wheat gliadins (i) induce apoptosis by the CD95/Fas apoptotic pathway, (ii) induce increased Fas and FasL mRNA levels, (iii) determine increased FasL release in the medium, and (iv) that gliadin digest‐induced apoptosis can be blocked by Fas cascade blocking agents, i.e. targeted neutralizing antibodies. This favors the hypothesis that gliadin could activate an autocrine/paracrine Fas‐mediated cell death pathway. Finally, we found that (v) a small peptide (1157 Da) from durum wheat, previously proposed for clinical practice, exerted a powerful protective activity against gliadin digest cytotoxicity.

Effects of dietary virgin olive oil phenols on low density lipoprotein oxidation in hyperlipidemic patients.

The aim of this study was to assess the effects of the dietary intake of extra virgin olive oil on the oxidative susceptibility of low density lipoproteins (LDL) isolated from the plasma of hyperlipidemic patients. Ten patients with combined hyperlipidemia (mean plasma cholesterol 281 mg/dL, triglycerides 283 mg/dL) consumed a low-fat, low-cholesterol diet, with olive oil (20 g/d) as the only added fat, with no drug or vitamin supplementation for 6 wk. Then they were asked to replace the olive oil they usually consumed with extra virgin olive oil for 4 wk. LDL were isolated at the beginning, and after the 4 wk of dietary treatment. LDL susceptibility to CuSO4-mediated oxidation was evaluated by measuring the extent of lipid peroxidation. We also determined fatty acid composition and vitamin E in plasma and LDL and plasma phenolic content. Extra virgin olive oil intake did not affect fatty acid composition of LDL but significantly reduced the copper-induced formation of LDL hydroperoxides and lipoperoxidation end products as well as the depletion of LDL linoleic and arachidonic acid. A significant increase in the lag phase of conjugated diene formation was observed after dietary treatment. These differences are statistically correlated with the increase in plasma phenolic content observed at the end of the treatment with extra virgin olive oil; they are not correlated with LDL fatty acid composition or vitamin E content, which both remained unmodified after the added fat change. This report suggests that the daily intake of extra virgin olive oil in hyperlipidemic patients could reduce the susceptibility of LDL to oxidation, not only because of its high monounsaturated fatty acid content but probably also because of the antioxidative activity of its phenolic compounds.

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone‐induced differentiation have been evaluated for the first time in 3T3‐L1 preadipocytes. Unlike control cells, oxLDL‐treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor‐1 (Pref‐1) mRNA expression at late times. By silencing Pref‐1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref‐1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref‐1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.

Modulation of Non-N-Methyl-D-Aspartate Receptors in Cultured Cerebellar Granule Cells

Abstract: Kainic acid (KA), quisqualic acid (QUIS), and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) stimulated D‐[3H]aspartate release from cultured cerebellar granule cells in a concentration‐dependent way. The EC50 values were 50 μM for KA (Gallo et al., 1987) and 20 μM for both QUIS and AMPA, but the efficacy of QUIS appeared to be greater than that of AMPA. The release of D‐[3H]aspartate induced by KA, QUIS, and AMPA was blocked, in a dose‐dependent way, by the new glutamate receptor antagonist 6‐cyano‐2,3‐dihydroxy‐7‐nitroquinoxaline (CNQX); IC50 values were 0.7 μM in the case of AMPA (50 μM) and 1 μM in the case of KA (50 μM). AMPA (50–300 μM) inhibited the effect of 50 μM KA on D‐[3H]aspartate release. At 300 μM AMPA, the effect of KA plus AMPA was not antagonized by the KA receptor antagonist kynurenic acid (KYN). In contrast, when KA was used at an ineffective concentration (10 μM), the addition of AMPA at concentrations below the EC50 value (10–20 μM) resulted in a synergistic effect on D‐[3H]aspartate release. In this case, the evoked release of D‐[3H]aspartate was sensitive to KYN. KA stimulated the formation of cyclic GMP, whereas QUIS, AMPA, and glutamate were ineffective. The accumulation of cyclic GMP elicited by KA (100 μM) was prevented not only by the antagonists CNQX (IC50= 1.5 μM) and KYN (IC50= 200 μM), but also by the agonists AMPA (IC50= 50 μM), QUIS (IC50= 3.5 μM), and glutamate (IC50= 100 μM). We conclude that AMPA, like QUIS, may act as a partial agonist at KA receptors. Moreover, CNQX effectively antagonizes non‐N‐methyl‐D‐aspartate receptor‐mediated responses in cultured cerebellar granule cells.

Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes

The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator‐activated receptor γ‐independent CD36 overexpression, by up‐regulating nuclear factor erythroid 2 (NF‐E2)‐related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up‐regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin‐resistance, data herein presented highlight the significance of oxLDL‐induced CD36 overexpression within the cellular defence response.