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The use of N-(n-butyl)thiophosphoric triamide (NBPT), as a urease inhibitor, is one of the most successful strategies utilized to increase the efficiency of urea-based fertilization. To date, NBPT has been added to the soil incorporated in fertilizers containing either urea or the inhibitor at a fixed percentage on the urea weight. The possibility of using NBPT physically separated from urea-based fertilizers could make its use more flexible. In particular, a granulated product containing NBPT could be utilized in soils treated with different urea-based fertilizers including livestock urine, the amount depending on soil characteristics and/or the urea source (e.g., mineral fertilizer, organo-mineral fertilizer, or animal slurry). In this study, a multilayer soil column device was used to investigate the influence of an experimental granular product (RV) containing NBPT and a garlic extract, combining the ability to protect NBPT by oxidation and nitrification inhibition activity, on (a) spatial variability of soil urease and nitrification activities and (b) timing of urea hydrolysis and mineral-N form accumulation (NO(2)(-), NO(3)(-), NH(4)(+)) in soil treated with urea. The results clearly demonstrated that RV can, effectively, inhibit the soil urease activity along the soil column profile up to 8-10 cm soil layer depth and that the inhibition power of RV was dependent on time and soil depth. However, nitrification activity is not significantly influenced by RV addition. In addition, the soil N transformations were clearly affected by RV; in fact, RV retarded urea hydrolysis and reduced the accumulation of NH(4)(+)-N and NO(2)(-)-N ions along the soil profile. The RV product was demonstrated to be an innovative additive able to modify some key ureic N trasformation processes correlated with the efficiency of the urea-based fertilization, in a soil column higher than 10 cm.

Background/aims: The functional roles of endogenous Notch3 and Notch1 for protecting human hepatocellular carcinoma (HCC) lines against doxorubicin-induced death have been investigated. We previously reported aberrant Notch3 and Notch4 up-regulation in HCC and we have extended these observations to include Notch1., Methods: Notch1 expression was assessed by immunohistochemistry and immunoblotting. Notch3 and Notch1 expression were ablated in multiple HCC lines by stable retroviral transduction of short hairpin RNAs (shRNAs). Effects on doxorubicin sensitivity were evaluated with respect to cell growth, expression of specific cell cycle effectors and multiple apoptotic parameters., Results: Notch3 depletion increased p53 expression, doxorubicin uptake, DNA damage, the apoptosis inducing effects of doxorubicin and also impeded the cell cycle progression of HCC cells. Ablating p53 expression in Notch3 knockdown (KD) cells largely abolished their enhanced doxorubicin sensitivity; and Notch3 KD in p53(-/-) Hep3B cells failed to influence their response to doxorubicin. Although up-regulated in most HCC, Notch1 (unlike Notch3) did not contribute to the doxorubicin resistance of HCC lines., Conclusions: Our in vitro results represent the first evidence that Notch3 silencing in combination with chemotherapeutics could conceivably provide a novel strategy for HCC treatment that deserves further exploration.

Oxidized LDL (oxLDL) increase in patients affected by type-2 diabetes, obesity, and metabolic syndrome. Likewise, insulin resistance, an impaired responsiveness of target tissues to insulin, is associated with those pathological conditions. To investigate a possible causal relationship between oxLDL and the onset of insulin resistance, we evaluated the response to insulin of 3T3-L1 adipocytes treated with oxLDL. We observed that oxLDL inhibited glucose uptake (-40%) through reduced glucose transporter 4 (GLUT4) recruitment to the plasma membrane (-70%), without affecting GLUT4 gene expression. These findings were associated to the impairment of insulin signaling. Specifically, in oxLDL-treated cells insulin receptor (IR) substrate-1 (IRS-1) was highly degraded likely because of the enhanced Ser(307)phosphorylation. This process was largely mediated by the activation of the inhibitor of kappaB-kinase beta (IKKbeta) and the c-Jun NH(2)-terminal kinase (JNK). Moreover, the activation of IKKbeta positively regulated the nuclear content of nuclear factor kappaB (NF-kappaB), by inactivating the inhibitor of NF-kappaB (IkappaBalpha). The activated NF-kappaB further impaired per se GLUT4 functionality. Specific inhibitors of IKKbeta, JNK, and NF-kappaB restored insulin sensitivity in adipocytes treated with oxLDL. These data provide the first evidence that oxLDL, by activating serine/threonine kinases, impaired adipocyte response to insulin affecting pathways involved in the recruitment of GLUT4 to plasma membranes (PM). This suggests that oxLDL might participate in the development of insulin resistance.

Adrenal myelolipoma is an uncommon tumour of the adrenal gland, usually unilateral, frequently associated with hypertension and obesity, with a benign biological behaviour and without hormonal activity, first described in 1905. The neoplasm consists of adipose tissue and myelopoietic cells of the bone marrow. These tumours have a very slow but continued growth and their volume and weight vary significantly from small lesions of a few grams to huge masses weighing up to several kilograms. If symptoms occur, surgery should be performed without delay, especially for large myelolipomas that are at high risk of spontaneous rupture with haemorrhage and life-threatening shock. In this report a case of a 43-year-old male with a 22 x 18 x 9 cm giant myelolipoma, weighing 3500 g and originating from the right adrenal gland is described. The large mass dislocating and compressing the inferior vena cava, was removed surgically. The early postoperative course and the late outcome were favourable without recurrence after 30 months. The different aetiological hypotheses of this rare neoplasm and its clinical features, diagnosis and treatment are discussed.

Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti-cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub-G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation-induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes.

Background: Tricyclic antidepressants and serotonin reuptake inhibitors are considered to be equally effective, but differences may have been obscured by internally inconsistent measurement scales and inefficient statistical analyses., Aims: To test the hypothesis that escitalopram and nortriptyline differ in their effects on observed mood, cognitive and neurovegetative symptoms of depression., Method: In a multicentre part-randomised open-label design (the Genome Based Therapeutic Drugs for Depression (GENDEP) study) 811 adults with moderate to severe unipolar depression were allocated to flexible dosage escitalopram or nortriptyline for 12 weeks. The weekly Montgomery-Asberg Depression Rating Scale, Hamilton Rating Scale for Depression, and Beck Depression Inventory were scored both conventionally and in a more novel way according to dimensions of observed mood, cognitive symptoms and neurovegetative symptoms., Results: Mixed-effect linear regression showed no difference between escitalopram and nortriptyline on the three original scales, but symptom dimensions revealed drug-specific advantages. Observed mood and cognitive symptoms improved more with escitalopram than with nortriptyline. Neurovegetative symptoms improved more with nortriptyline than with escitalopram., Conclusions: The three symptom dimensions provided sensitive descriptors of differential antidepressant response and enabled identification of drug-specific effects.

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

Proliferating cell nuclear antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including DNA synthesis and repair, cell cycle regulation and apoptosis. An alteration in PCNA structure might contribute to DNA-damage accumulation in cancer cells. This study was aimed to evaluate the PCNA pattern of expression, in terms of aggregation status, isoforms and post-translational modifications, in human hepatocellular carcinoma (HCC) and cirrhosis as well as in HCC cell lines. Twelve HCCs and surrounding cirrhotic tissues were analysed, along with HepG2, Hep3B and SNU-398 cell lines. Normal liver specimens and cirrhosis without HCC were included as controls. Both DNA-bound and DNA-unbound PCNA fractions were analysed, and PCNA pattern of expression was displayed on two-dimensional gel electrophoresis followed by western blot. Results were confirmed by mass spectrometry. To compare HCCs vs surrounding tissues, immunolabelling and immunostaining were performed. In 6 of 12 HCCs and in cell lines, we found three major PCNA acidic forms, corresponding to monomers, probably dimers and trimers, and a basic isoform. In the six remaining HCCs, only a PCNA acidic form associated with multiple basic isoforms was detected. Importantly, the PCNA basic form was not found in cirrhotic tissues. To clarify the nature of the detected PCNA isoforms, ubiquitin-specific immunoblotting as well as phosphatase treatment were employed. A PCNA-ubiquitylated form in cell lines and PCNA-phosphorylated isoforms in 6 of 12 HCCs were detected. Finally, in the DNA-bound fraction we detected only an acidic PCNA monomeric form. We conclude that human hepatocellular carcinoma expresses specific PCNA isoforms compared to those found in cirrhosis, implicating a role for PCNA functional alterations in hepatocarcinogenesis.

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 gamma-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.

Small and large cell neuroendocrine carcinomas of the urinary bladder are rare and usually coexist with urothelial carcinoma in elderly patients. Here we report the clinical case of a young smoker who was referred to our institution for a primary pure neuroendocrine carcinoma of the bladder, and review the existing literature on small and large cell neuroendocrine carcinomas of the urinary bladder.

Polyphenols, occurring in fruit and vegetables, wine, tea, extra virgin olive oil, chocolate and other cocoa products, have been demonstrated to have clear antioxidant properties in vitro, and many of their biological actions have been attributed to their intrinsic reducing capabilities. However, it has become clear that, in complex biological systems, polyphenols exhibit several additional properties which are yet poorly understood. Apoptosis is a genetically controlled and evolutionarily conserved form of cell death of critical importance for the normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. The malfunction of the death machinery may play a primary role in various pathological processes, since too little or too much apoptosis can lead to proliferative or degenerative diseases, respectively. Cancer cells are characterized by a deregulated proliferation, and/or an inability to undergo programmed cell death. A large body of evidence indicates that polyphenols can exert chemopreventive effects towards different organ specific cancers, affecting the overall process of carcinogenesis by several mechanisms: inhibition of DNA synthesis, modulation of ROS production, regulation of cell cycle arrest, modulation of survival/proliferation pathways. In addition, polyphenols can directly influence different points of the apoptotic process, and/or the expression of regulatory proteins. Although the bulk of data has been obtained in in vitro systems, a number of clinical studies suggesting a preventive and therapeutic effectiveness of polyphenols in vivo is available. However, a deeper knowledge of the underlying mechanisms responsible for the modulation of apoptosis by polyphenols, and their real effectiveness, is necessary in order to propose them as potential chemopreventive and chemotherapeutic candidates for cancer treatment.

In this study, we investigated the alterations of the redox balance induced by the lipid fraction of oxLDL in Caco-2 intestinal cells, and the effects of tyrosol and protocatechuic acid, two dietary phenolic compounds. We found that oxidized lipids extracted from oxLDL (LipE) induced oxidative stress by determining, 6 h after treatment, ROS overproduction (about a 100% and a 43% increase of O*2 and H2O2 production, respectively, P<.05: LipE vs. control) and, 12 h after treatment, GSH depletion (about a 26% decrease, P<.05: LipE vs. control), and by impairing the activities of superoxide dismutase, catalase and glutathione peroxidase. In response to the induced oxidative stress, we observed significant overexpression of glutathione peroxidase (6 h after treatment: P<.05), glutathione reductase and gamma-glutamylcysteine synthetase (12 h after treatment: P<.05). Notably, when GSH depletion occurred, p66Shc protein expression increased by about 300% with respect to control (P<.001; LipE vs. control). These effects were fully counteracted by dietary phenolics which inhibited ROS overproduction and GSH consumption, rendered the reactive transcription of glutathione-associated enzymes unnecessary and blocked the intracellular signals leading to the overexpression and rearrangement of p66Shc signalling molecule. Altogether, these results suggest that the impairment of the antioxidant system hijacks intestinal cells towards an apoptotic-prone phenotype via the activation of p66Shc molecule. They also propose a reappraisal of dietary polyphenols as intestinal protecting agents, indicating the antiapoptotic effect as a further mechanism of action of these antioxidant compounds.

High serum levels of IL-6 correlate with poor outcome in breast cancer patients. However, no data are available on the relationship between IL-6 and mammary stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in the MCF-7 breast cancer cell line and in primary human mammospheres (MS), multicellular structures enriched in stem/progenitor cells of the mammary gland. MS from node invasive breast carcinoma tissues expressed IL-6 mRNA at higher levels than did MS from matched non-neoplastic mammary glands. In addition, IL-6 mRNA was detected only in basal-like breast carcinoma tissues, an aggressive breast carcinoma variant showing stem cell features. IL-6 treatment triggered Notch-3-dependent upregulation of the Notch ligand Jagged-1 and promotion of MS and MCF-7-derived spheroid growth. Moreover, IL-6 induced Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promoted a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, autocrine IL-6 signaling relied upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, these data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

Background: Notch signalling is altered in several solid tumours and it plays a role in growth inhibition and apoptosis of hepatocellular carcinoma (HCC)-derived cell lines, bile duct development and hepatocyte regeneration., Aims: This study aims to analyse the expression of Notch3, Notch4 and HES1 and HES6 as Notch-target genes in HCC, matched non-neoplastic tissue and HEPG2 cells., Results: Notch3 and Notch4 are not expressed in normal liver and in chronic hepatitis surrounding HCC. Cirrhotic tissue stains negative for Notch3, while Notch4 is expressed by hepatocytes at the edge of regenerative nodules and in cell planes adjacent to fibrous septa. HCC tissue displays Notch3 and Notch4 abnormal accumulation, respectively, in 78% and 68% of the cases. The endothelium of hepatic veins with neoplastic permeation is frequently Notch4 positive. An upregulation of Notch3 mRNA was found in 95% of HCCs vs cirrhosis (P=0.0001), while Notch4 mRNA was downregulated in 80% of HCCs. HES6 mRNA expression was higher in HCC tissue when compared with cirrhosis (P=0.007), paralleling Notch3 mRNA expression. The HEPG2 cell line displays high Notch3 and low Notch4 protein and mRNA levels., Conclusions: These descriptive findings suggest an aberrant expression of Notch3 and Notch4 in HCC and allow the hypothesis of an activation of Notch signalling by Notch3.

Background and Aim: Extra virgin olive oil has been associated with a reduced incidence of risk factors for coronary heart disease also owing to the presence of antioxidant biophenols. This study compared the protective effects of tyrosol and hydroxytyrosol, two biophenols greatly different in antioxidant power, on J774 A.1-mediated oxidation of LDL., Methods and Results: Cell-mediated oxidation of LDL was evaluated by TBARS formation, and relative electrophoretic mobility increase. Redox imbalance was studied by: (i) cytofluorimetric determination of intracellular ROS and GSH, and (ii) evaluation of GSH-related enzyme activities and gene expressions by colorimetric and RT-PCR analyses, respectively. The cellular uptake of the biophenols was evaluated by HPLC. Both biophenols inhibited cell-mediated oxidation of LDL but to a different extent (100% hydroxytyrosol vs 40% tyrosol), and counteracted the impairment of antioxidant cellular defence, i.e., GSH and related enzymes. Tyrosol was effective in inhibiting about 30% of ROS production only at later time-points (12h for superoxide, 24h for hydrogen peroxides). Interestingly, both biophenols were effective when added to the cells for 2h and removed before LDL treatment. This was probably related to cell-biophenol interactions: hydroxytyrosol was rapidly found inside the cells (1.12+/-0.05ng/mg cell protein) and disappeared within 18h, while tyrosol accumulated intracellularly with time (0.68+/-0.09 vs 1.72+/-0.13ng/mg cell protein at minute 5 and hour 18, respectively)., Conclusions: In spite of its weak antioxidant activity, tyrosol was effective in preserving cellular antioxidant defences, probably by intracellular accumulation. These findings give further evidence in favour of olive oil consumption to counteract cardiovascular diseases.

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in approximately 70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer.

Zenker's diverticula commonly occur in the elderly, and quality of life is often impaired by typical symptoms such as dysphagia, regurgitation, halitosis, cough and aspiration pneumonia, malnutrition and weight loss. The "gold standard" treatment for pharyngo-oesophageal diverticula is the resection of the sac via left lateral cervicotomy and cricopharingeal myotomy. In the last decade, with the fast development of minimally invasive techniques, an endoscopic stapled approach has been proposed. This procedure rapidly encountered the favour of gastroenterologists because patients with Zenker's diverticulum often present serious co-morbidities and seem to benefit from the minimally invasive technique; but the crucial point in the treatment of Zenker's diverticula, in addition to the sac resection, is the myotomy of the cricopharyngeal muscle fibres and this could not be safely and completely achieved in endoscopic stapling owing to the risk of vascular lesions and incomplete sectioning of the sac. Moreover, many studies have reported similar results between open and endoscopic procedures in terms of postoperative morbidity and mortality, showing better functional outcomes in surgical patients even if elderly and presenting co-morbidities. In this report, the case of a 95 year-old patient, one of the oldest operated on for this disease and reported in the literature, is described. He was affected by a massive 8 cm Zenker's diverticulum and an oesophageal motility disorder (dyskinesia), with significant co-morbidity. Surgical diverticulectomy combined with cricopharyngeal myotomy was performed with excellent early and late results.

The relationship between apoptosis induced by gamma radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by (1)H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of gamma-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of gamma-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.

The comprehension of the basic biology of stem cells is expected to provide a useful insight into the pathogenesis of cancer. In particular, there is evidence that hypoxia promotes stem cell renewal in vitro as well as in vivo. It therefore seems reasonable that stem cell survival and hypoxia response are strictly connected at molecular level. We here report that the 66-kDa isoform of the SHC gene (p66Shc) is induced in a breast cancer cell line by the exposure to hypoxic environment and that it controls the expression of the stem cell regulatory gene Notch-3. Then, we show that p66Shc/Notch-3 interplay modulates self-renewal (by inducing the Notch-ligand Jagged-1) and hypoxia survival (by inducing the hypoxia-survival gene carbonic anhydrase IX) in mammary gland stem/progenitor cells, expanded in vitro as multicellular spheroids (mammospheres). We conclude that mechanisms that regulate stem cell renewal and hypoxia survival are integrated at the level of the p66Shc/Notch3 interplay. Because Notch-3, Jagged-1, and carbonic anhydrase IX are dysregulated in breast cancer, and because p66Shc is an aging-regulating gene, we envision that these data may help in understanding the relationship among aging, cancer, and stem cells.

1H MRS signals of glutathione and of free glutamate were examined in samples from cultured tumour cells, namely MCF-7 from mammary carcinoma and TG98 from malignant glioma, with the aim of relating signal intensities to aspects of GSH metabolism. Spectra of cells harvested at different cell densities suggest that GSH and glu signal intensities are related to cell density and proliferation and their ratio is dependent on the activity of the gamma-glutamyl cysteine synthetase. The hypothesis is confirmed by experiments performed on cells treated with buthionine sulfoximine that inhibits the enzyme activity.

Polyphenols have been demonstrated to have clear antioxidant activities in vitro. However, in complex biological systems, they exhibit additional properties which are yet poorly understood. Apoptosis is a genetically controlled and evolutionarily conserved form of cell death of critical importance for the normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. The malfunction of the death machinery may play a primary role in various pathologic processes, leading to proliferative or degenerative diseases. Polyphenols can directly interact with specific steps and/or proteins regulating the apoptotic process in different ways depending on their concentration, the cell system, the type or stage of the pathological process. A growing body of in vitro evidence has provided interesting insights in the comprehension of the cellular and molecular mechanisms responsible for the modulation of apoptosis. However additional and harder studies are needed to better elucidate the mechanisms of action and the real in vivo effectiveness of polyphenols in order to propose them as potential candidates for chemoprevention and treatment of cancer and cardiovascular diseases.

Fruit and beverages such as tea and red wine represent the main sources of polyphenols. Despite their wide distribution, the healthy effects of dietary polyphenols have come to the attention of nutritionists only in the last years. The main factor responsible for the delayed research on polyphenols is the variety and the complexity of their chemical structure. Emerging findings suggest a large number of potential mechanisms of action of polyphenols in preventing disease, which may be independent of their conventional antioxidant activities. To establish evidence for the effects of polyphenol consumption on human health and to better identify which polyphenols provide the greatest effectiveness in disease prevention, it is first of all essential to determine the nature and the distribution of these compounds in our diet, and secondly to better know their bioavailability.

We describe the case of a young woman with a recurrent right Bernard-Horner's syndrome associated with the onset of intermittent supraclavicular mass. Surgical excision of the mass revealed the endometrial pattern of the tumor and the close relationship between Bernard-Horner's syndrome and thoracic endometriosis. No other case of this syndrome has been reported in the literature.

Over the past few years, incidental thyroid microcarcinoma has become a frequent disease and its incidence in some reports is considerable. Discovering new cases depends on the extended indications for total thyroidectomy for benign disease, on progress in the field of diagnostic instruments (ultrasound, scintigram, fine needle biopsy for cytology, CT scan, MRI), and on the pathology examination of very thin slices of specimens. In spite of the high incidence reported in some autopsy series, suggesting that this tumour may have a good prognosis, some Authors report an overall incidence of up to 11% of local recurrence, metastasis or mortality. For all these reasons the surgical treatment of incidental thyroid microcarcinoma is still controversial. The aim of this study was to estimate the incidence and examine the clinical-pathological findings of incidental thyroid microcarcinoma in a series of 100 consecutive thyroidectomies and to evaluate whether complete removal of the gland should be adopted in all cases. In the present series the incidence of incidental thyroid microcarcinoma was 21.6% (19/88). Total thyroidectomy was considered the treatment of choice for diffuse benign disease and appeared to be necessary for both the diagnosis and treatment of incidental thyroid microcarcinoma.

We hereby present the case of a 55-year-old woman with an iatrogenic tracheal laceration that occurred during double lumen intubation for left pneumonectomy. Conservative treatment was performed and the patient was discharged 12 days after surgery. No major sequelae after 3 months of follow-up were observed.

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.

Background: The aim of this study was to examine the expressions of the bcl-2, bax, fas and c-myc apoptosis-related genes in benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) to determine whether significant differences exist within each disease and between the two groups of patients. The correlation between gene expression and tumour diameter, stage, Gleason score and serum PSA was also investigated., Patients and Methods: Tissue specimens from 51 cases of BPH and 27 cases of CaP were examined for bcl-2, bax, fas and c-myc expression by reverse transcriptase-PCR (RT-PCR)., Results: In BPH, bcl-2 and bax gave the weakest signals (p < 0.001). In CaP, bcl-2 was the least expressed gene (p < 0.001). In both patient groups, fas and c-myc were the most highly expressed genes (p < 0.05). Both bcl-2 and bax were expressed at higher levels in CaP than in BPH (p < 0.02). The bcl-2/bax ratio was lower in CaP than in BPH (p < 0.001). Bcl-2 was more highly expressed in high Gleason grade (> 7) tumours (p < 0.05). In the BPH group, bax showed a positive relationship with fas (p < 0.01), while the bcl-2 level inversely correlated with that of c-myc (p < 0.05)., Conclusion: Our data showed that all the apoptosis-related genes were expressed in both BPH and CaP. The stronger expression of bax and the lower bcl-2/bax ratio observed in CaP may suggest a pro-apoptotic stimulus, while the higher bcl-2 levels appear to counterbalance the tendency to cell death.

Background: By mediating local cell-cell interactions, the Notch signaling pathway seems to control a variety of processes from cell fate decisions during development, to stem cell renewal and to differentiation in many adult tissues. Hence, perturbed Notch signaling may be involved both in the development and the spread of cancer. The expression and the functional role of some major components of the Notch signaling pathway in human hepatocellular carcinoma (HCC) are poorly characterized., Materials and Methods: Notch3, HES1, Jagged1 and Delta1 were analyzed both at the RNA and protein levels in the HepG2 liver cell line derived from human HCC., Results: The results of this study demonstrated, for the first time, that both Jagged1 and Delta1 ligands and the downstream effector gene HES1 are expressed in the HepG2 actively proliferating cell line. Moreover, a high expression of Notch3 intracellular domain, indicative of constitutively activated Notch signaling, was the only detectable Notch3 subunit in HepG2., Conclusion: These findings suggest that Notch3 may be involved in mechanisms controling the differentiation and the spread of HCC and that Notch3 activation may be dependent on both Jagged1 and Delta 1 ligands.

Inhibition of apoptosis in tumour cells may depend on intracellular reduced glutathione (GSH) level. In this work, GSH levels were studied by (1)H MRS in MCF-7 and HeLa cells, characterised by a different radiosensitivity. Annexin-V test showed that the fraction of apoptotic HeLa cells after irradiation is much higher than in control, although MCF-7 cells did not show a significant apoptosis. MRS signals from GSH (G) show lower intensity in HeLa with respect to MCF-7 cells; the opposite is true for free glutamic acid [glu (g)]. After irradiation, the G/g ratio decreases in MCF-7, although remaining approximately constant in HeLa cells. Buthionine sulfoximine (BSO) treated MCF-7 cells show an increase in the percentage of apoptotic cells; in parallel, G/g ratio behaves as in HeLa. This study indicates that GSH level may act as predictive marker of apoptosis by irradiation.

Among diet antioxidants, polyphenols, naturally occurring in vegetables, fruits and plant-derived beverages such as tea, red wine, and extra virgin olive oil, are the most abundant ones. In vitro cell culture experiments have shown that polyphenols possess antioxidant properties, and it is thought that these activities can contribute to the prevention of several oxidative stress-associated diseases. It has however become clear that the mechanisms of action of polyphenols go beyond their intrinsic reducing capabilities, being able to exert other additional effects that are as yet poorly understood. This article gives an overview of the most recent data on the subject and describe the additional functions that polyphenols can have in biological systems, focusing on their effects on glutathione and its related enzymes. Evidence is provided of a tight connection between exogenous and endogenous antioxidants that appear to act in a coordinated fashion. Experimental data indicate that polyphenols may offer an indirect protection by activating endogenous defense systems. It is reasonable to hypothesize that this is achieved, at least in part, through antioxidant responsive elements (ARE) present in the promoter regions of many of the genes inducible by oxidative and chemical stress. The latest studies strongly suggest that dietary polyphenols can stimulate antioxidant enzyme transcription through ARE.

Much attention has been devoted in the past to monitor changes of mobile lipid (ML) (1)H MRS signals in spectra of tumour cells. The purpose of this work is to exploit ML signals to provide information on cell metabolism after irradiation, comparing tumour cells characterised by different radiosensitivity and relating MRS findings to changes in cell proliferation and delays in cell cycle phases. Irradiated HeLa cells present less intense ML signals with respect to controls. The opposite is true for MCF-7 cells. A G(2) arrest is observed for both cell lines after irradiation. In HeLa cells, G(1) decreases and S phase is maintained; a sub G(1) peak is also visible. In MCF-7 cells, G(1) is decreased and S phase is strongly reduced, while no sub G(1) is present. The observed changes in ML are tentatively associated to cell cycle regulation of phospholipid synthesis. Mathematical modelling of ML variations is in progress.

Growth arrest and DNA damage 45-alpha (GADD45-alpha) is a nuclear protein involved in maintenance of genomic stability, DNA repair, and suppression of cell growth through interaction with nuclear elements, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and PCNA. In this study, GADD45-alpha expression was assessed in 28 cases of hepatocellular carcinoma (HCC) and matched cirrhosis tissues, and correlated with the presence of DNA-bound PCNA and CDKN1A as markers of DNA repair, as well as with clinicopathologic variables including histopathologic grade, tumor size, nodularity, viral status, alpha-fetoprotein serum levels, and p53 and Ki67 immunostaining. GADD45-alpha and CDKN1A messenger RNA (mRNA) were analyzed by reverse transcriptase-polymerase chain reaction. GADD45-alpha protein expression was evaluated by Western blot (WB) and enzyme-linked immunosorbent assays (ELISAs). PCNA and CDKN1A DNA-bound fractions were determined by WB. GADD45-alpha mRNA was down-regulated in 20 of 26 HCCs with respect to matched cirrhosis, but no correlation was found with the corresponding protein levels assessed by both WB and ELISA. GADD45-alpha and CDKN1A protein levels were related to each other both in cirrhotic and in neoplastic tissues, and a concordant up- or down-regulation was observed in HCCs with respect to cirrhosis. DNA-bound PCNA and CDKN1A were present in 5 HCCs and were associated with higher GADD45-alpha protein levels assessed by ELISA. No significant association was found in HCCs between GADD45-alpha protein expression and histopathologic grading, nodule size, focality, and proliferation, whereas a positive correlation was found with alpha-fetoprotein serum levels. In conclusion, GADD45-alpha mRNA was down-regulated with respect to matched cirrhosis in most HCCs; however, no correlation was found between mRNA and protein levels. GADD45-alpha protein levels were higher in HCCs with DNA-bound CDKN1A and PCNA, suggesting a possible role in DNA repair.

Polyphenols are wide variety of compounds that occur in fruits and vegetables, wine, tea, extra virgin olive oil, chocolate and other cocoa products. Several polyphenols have been demonstrated to have clear antioxidant properties in vitro, and many of their biological actions have been attributed to their intrinsic reducing capabilities. However, this concept appears now to be a simplistic way to conceive their activity. Evidence is indeed accumulating that polyphenols might exert several other specific biological effects that are as yet poorly understood. In this article we review the most recent data on the subject and describe the additional functions that polyphenols can have in biological systems, focusing on their effects on glutathione and its related enzymes. Experimental data indicate that polyhenols may offer an indirect protection by activating endogenous defense systems. Several lines of evidence suggest a tight connection between exogenous and endogenous antioxidants that appear to act in a coordinated fashion. It is reasonable to hypothesize that this is achieved, at least in part, through antioxidant responsive elements (AREs) present in the promoter regions of many of the genes inducible by oxidative and chemical stress. The latest studies strongly suggest that dietary polyphenols can stimulate antioxidant transcription and detoxification defense systems through ARE.

Over the last few years, incidental thyroid microcarcinoma (TMC) has become a frequent disease and its incidence in some reports is considerable. The discovery of new cases depends on the progress of the diagnostics (US scan, fine needle biopsy and cytology, CT, MRI), on the extended indications to thyroidectomy for benign disease and on the attention in pathologic examination of the specimen. The clinical evolution of this disease is not well known: in spite of a high incidence reported in some autoptic series, suggesting that this tumour could have a good prognosis, some authors report an overall incidence of up to 11% of local recurrence, metastasis and mortality. For these reasons the treatment of TMC is still controversial today. Aim of this study was to estimate the incidence and the clinico-pathological findings of TMC over a one year period of total thyroidectomies for diffuse benign thyroid diseases, and to evaluate, on the basis of the frequency of incidental microcarcinoma, if the surgical procedure of complete removal of the gland should be adopted in any case. In this series no patient had pre-operative diagnosis or tentative diagnosis of carcinoma and the incidence of TMC at the final histologic examination was 27.4%. Total thyroidectomy confirmed to be the treatment of choice for diffuse benign diseases and appeared necessary to obtain both, diagnosis and treatment of incidental TMC.

Goals: The aim of this work is to assess how the clinical features of celiac disease have changed in Italy after the widespread introduction of serologic tests in 1993., Study: Twenty Italian Clinical Centers collected information from 1982 until 2002 on 1968 patients older than 18 years diagnosed with celiac disease., Results: The results show that the incidence of atypical and silent cases of celiac disease has increased after the wider availability of serological test, which has allowed earlier diagnosis and treatment., Conclusions: This paper provides a view on the evolution of the clinical features of celiac disease in Italy over 2 decades. The relevance of the analysis is supported by the fact that Italy is one of the countries with the highest incidence of celiac disease. These findings underline the importance of a timely diagnosis of celiac disease.

Background: Although cellular oxidative stress is a major cause of DNA damage, it is still not clear to what degree it affects genetic instability and malignant progression in established prostate carcinoma (PCa)., Materials and Methods: We examined the expression of CDKN1A and Gadd45 proteins acting on cell cycle checkpoints and DNA repair in PCa relative to the presence of oxidative DNA damage, as measured by the detection of the DNA adduct 8-hydroxy-2-deoxyguanosine (8-OHdG.). Sixteen PCa and 28 benign prostate hyperplasias (BPH) were analyzed. RT-PCR was used to evaluate WAF1 and Gadd45 transcripts. Western blot and ELISA were used to analyze proteins and 8-OHdG adducts. Proliferation was studied by Ki67 image cytometry; telomerase activity was detected by TRAP- ELISA., Results: Multivariate factor analysis provided evidence that, in PCa, DNA checkpoint proteins were associated with 8-OHdG and did not prevent neoplastic cells proliferation. Conversely, in BPH, oxidative DNA damage was inversely correlated with DNA checkpoint proteins and proliferation, suggesting the presence of energy-depleted senescent cells., Conclusion: Although in non-malignant tissue extensive oxidative DNA damage drives cells to a metabolic blockage, in PCa neoplastic cells it activates repair mechanisms favoring the escape from senescence and the expansion of DNA-damaged clones.

It has been reported that oxidized LDL (oxLDL) are involved in the pathogenesis of atherosclerosis, and that macrophages as well as other cells of the arterial wall can oxidize LDL in vitro, depending on the balance between intracellular prooxidant generation and antioxidant defense efficiency. Because of their possible beneficial role in the prevention of atherosclerosis and other oxidative stress-associated diseases, phenolic compounds naturally occurring in vegetables, fruits, and beverages are receiving increased attention. In the present work, we investigated the mechanisms underlying the protective effect exerted by extra virgin olive oil biophenols, namely, protocatechuic acid and oleuropein, on LDL oxidation mediated by murine J774 A.1 macrophage-like cells. The biophenols were added to the cells with LDL and left in the medium during the entire experimental period, or for a period of 2 h and then removed from the medium before the addition of LDL. The effect of biophenols alone was also tested. In both experimental procedures, these antioxidants had the following effects: 1). completely prevented the J774 A.1-mediated oxidation of LDL; 2). counteracted the time-dependent variations in intracellular redox balance, inhibiting the production of O(2)(.-) and H(2)O(2) and the decrease in glutathione (GSH) content; 3). restored glutathione reductase (GR) and peroxidase (GPx) activities; and 4). restored the mRNA expression of gamma-glutamylcisteine synthetase (gammaGCS), GR, and GPx to control values. More importantly, we observed significant overtranscription and increased activities of two antioxidative enzymes, GPx and GR, compared with controls when the biophenols were present in the medium for 2 h and then removed before LDL exposure, or when the cells were exposed to the antioxidants alone for up to 24 h. Our findings suggest that the activation of mRNA transcription of GSH-related enzymes represents an important mechanism in phenolic antioxidative action.

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.

Background: Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA-synthesis and repair. During DNA-synthesis and repair the only active PCNA fraction is tightly bound to DNA. Similarly, during DNA-repair, a fraction of p21 colocalizes with PCNA in a detergent-insoluble form., Aim: The aim of the study was to analyze to what extent the presence of DNA-bound PCNA and p21 correlates with cell proliferation and DNA-repair in hepatocellular carcinoma (HCC)., Methods: Twenty-six HCCs and surrounding cirrhosis were studied. The DNA-bound and detergent-soluble fractions of PCNA and p21 were analyzed by immunoblotting. P53 and Ki67-Labeling Index (Ki67-LI) were evaluated by immunocytochemistry., Results: Soluble fractions of PCNA and p21 were found in all samples. One out of 26 cirrhotic samples displayed a DNA-bound fraction of PCNA while no case expressed DNA-bound p21. Fourteen HCCs showed a DNA-bound PCNA fraction. A highly significant correlation was found between Ki67-LI and DNA-bound PCNA but not with detergent-soluble PCNA. DNA-bound p21 and PCNA, indicating ongoing DNA repair activity, were present in 6 of these 14 HCCs and correlated with a high histological grade and high Ki67-LI., Conclusions: Our results suggest that in HCC PCNA participates both in DNA synthesis and repair and that highly proliferating HCCs may display a sustained DNA-repair.

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.

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., (Copyright 2003 Federation of European Biochemical Societies)

Background/aims: The aim of this study was to evaluate functional results after Billroth I, Billroth II and Roux-en-Y reconstruction in subtotal gastrectomy., Methodology: Forty-five patients were randomized between 1990 and 1995 and stratified in 3 different groups: 15 BI, 15 BII and 15 Roux. They were investigated by esophagogastroduodenoscopy with multiple biopsies and upper gastrointestinal scintiscanning, to evaluate gastroesophageal reflux and dynamics of gastric emptying. They also answered a questionnaire: Gastrointestinal Quality of Life. Index., Results: A reflux esophagitis was found in 5 BI, in 7 BII and in 2 Roux (p < 0.001). No gastric lesions were found in 6 BI, in 5 BII and in 12 Roux, (BI vs. Y, p < 0.05; BII vs. Y, p < 0.001). Chronic superficial gastritis was present in 9 BI, in 4 BII and in 3 Roux (BI vs. Y, p < 0.05). Dynamic scintiscan demonstrated the presence of gastroesophageal reflux in 5 BI and gastric emptying was fast (37' < T 1/2 < 86'), but incomplete (60' residual activity: 49-62%). Gastroesophageal reflux was evident in 7 BII with slow (28' < T 1/2 < 143') and incomplete (60' residual activity: 48-72%) gastric emptying. Gastroesophageal reflux was detected in 2 Roux and radioactive bolus progression in the Roux limb was fast (24' < T 1/2 < 53') and complete (60' residual activity: 42-52%) (BI vs. Y; BII vs. Y, p < 0.001). There was no statistical significance between Gastrointestinal Quality of Life Index score in the 3 groups., Conclusions: The Authors affirm that Roux-en-Y is the technique of choice in subtotal gastrectomy, if compared with BI and BII.

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.

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.

Objective: To evaluate whether it is possible to detect telomerase activity in cells exfoliated from prostate biopsies immediately before fixation., Methods: A total of 115 transrectal biopsies of prostate tissue from 49 patients were touch-imprinted on an RNase-free microscope slide and then fixed. Touch imprints were immediately frozen and used to extract telomerase. Telomerase activity was determined by a telomeric repeat amplification protocol (TRAP) using a PCR-ELISA method. Inflammation and epithelial cells in each biopsy were quantitated by image cytometry., Results: A total of 90/115 extracts had a proteic content suitable for analysis. Telomerase activity was detected in 18/26 (70%) carcinomas, 2/9 (22%) low-grade prostatic intraepithelial neoplasia (PIN) lesions, and 1/3 (33%) high-grade PIN lesions. In 4 of 7 patients with telomerase-positive tumors, telomerase activity was also found in a distant site devoid of morphologically detectable cancer cells. Telomerase activity was detected in touch imprints from fragments with less than 1 mm(2) of epithelial tissue, and was not associated with the extent of inflammation., Conclusions: From the technical stand point, the touch-imprint method may provide a useful adjunct for telomerase detection in prostate biopsies. With this procedure the bioptic fragment is left intact for histological examination. Diagnostically, the presence of telomerase activity in sites distant from the original tumor might suggest the presence of tumor cells that are morphologically undetectable.

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.

Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H(2)N-gln-gln-pro-gln-asp-ala-val-gln-pro-phe-COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the '1157.5' Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the '1157.5' Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide '1157.5' Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the '1157.5' Da peptide is capable of preventing such an effect.

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell-cell and cell-substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with alpha-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as alpha-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.