Implications of antioxidant enzymes in human gastric neoplasms.

The present study is the first to evaluate the expression and activity of MnSOD, Cu/ZnSOD and catalase in human gastric samples, since ROS play a significant role in the pathogenesis of different forms of malignancy inducing mutations and various diseases such as gastric cancer. Biopsies and surgical samples from 53 patients (male/female 22/31, mean age 56.5+/-15.8 years) consisted of 15 healthy, 12 autoimmune atrophic gastritis, 10 Helicobacter pylori (HP) infection, 8 HP-negative chronic gastritis (CG) and 8 adenocarcinoma cases. Enzyme activity and expression were evaluated by spectrophotometry and immunoblotting after specific extraction in phosphate buffer. We found that MnSOD activity was increased in adenocarcinoma, CG and HP tissues (p<0.05-0.001), while Cu/ZnSOD was significantly lower in adenocarcinoma and HP tissues (p<0.001) when compared to the healthy control. MnSOD and Cu/ZnSOD were expressed to a significantly higher degree in adenocarcinoma and HP tissues (p<0.05 and <0.001 respectively) and to a significantly lower degree in CG tissues with respect to the healthy patients (p<0.05 and <0.001). A significant decrease in CAT activity in adenocarcinoma and HP tissues was observed (p<0.01 and <0.05). Gastric human neoplasms showed significant changes in antioxidant enzymes, that represent the first line in antioxidant protection against radical attack. The difficulties in correlating the antioxidant enzyme with the neoplasms was related to the complexity of the biochemical pathways that regulate the cellular redox balance. Our results are important in enhancing the understanding of the role that these enzymes play in the promotion/suppression of the carcinogenesis cascade in human gastric mucosa.

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[a]P.

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.

Chloramphenicol influence on antioxidant enzymes with preliminary approach on microsomal CYP1A immunopositive-protein in Chamelea gallina.

Chloramphenicol (CA) is a largely used antibiotic and it is an inhibitor of protein synthesis that also induces ROS production. In this work there were investigated activities and expressions in the Adriatic bivalve Chamelea gallina of some antioxidant and detoxification proteins like superoxide dismutase (Mn-SOD, Cu/Zn-SOD), catalase (CAT) and Cytochrome P450 (CYP1A). Clams exposed to 5mgl(-1) of chloramphenicol were sampled 2, 4 and 8 days after treatment (CA2, CA4 and CA8). SODs, CAT, and CYP1A activity and/or expression were detected in pooled digestive glands by Western blotting and by spectrophotometrical analysis. Enzymes activities increase during the entire antibiotic exposure. With respect to the control Cu/Zn-SOD expression increases, while Mn-SOD expression decreases significantly after 4 days. Two CYP1A immunopositive-proteins (57.7 and 59.8kDa) were detected. The lower band significantly decreases in CA8, the upper one also in CA4 condition. High levels of Mn-SOD, CAT activity and Cu/Zn-SOD expression, indicate intense ROS production while Mn-SOD expression inhibition might be ascribable to mitochondrial alterations due to CA and indirectly to ROS. CYP1A1 action determines H2O2 production that would contribute to a CYP1A1 gene promoter down regulation, a response to oxidative stress with the antioxidant enzymes activation as a final result. This study highlights the close association, in C. gallina, in presence of chloramphenicol, between SOD/CAT and CYP system, and it appear particularly interesting to the lack of similar researches on mollusc species.

Evaluation of lung maturity by amniotic fluid analysis in equine neonate.

The aim of this study was to gather useful new data for evaluation of lung maturity in the neonatal foal. Because equine neonatal intensive therapy is very expensive, a precocious diagnosis could help to express a prognosis and to offer a respiratory support early after birth, increasing the survival rate and reducing complications. Amniotic fluid was collected at parturition on n=18 mares. Lamellar bodies were isolated in the amniotic fluid and measured with transmission electron microscopy (TEM). Furthermore two tests on amniotic fluid that are commonly used in humane medicine were utilized: lecithin/sphingomyelin ratio (L/S) and lamellar body count (LBC). L/S ratio was determined using thin layer chromatography (TLC) and, for the first time in equine amniotic fluid, with high performance liquid chromatography (HPLC). LBC was performed with an automated blood cell counter. The mean of the L/S ratio obtained in mature foals was 2.5 with TLC and 2.7 with HPLC. The mean LBC in the same group was 48x10(3)/microL. The Spearman's Rank correlation test found a significant correlation between TLC and Apgar score (R=0.66, p<0.01), between TLC and cord pH (R=0.65, p<0.05), between HPLC and Apgar score (R=0.63, p<0.01) and between cord pH and Apgar score (R=0.82, p<0.01). The Student's t-test did not found a significant difference between L/S ratio performed with TLC and with HPLC. These methods may be useful for evaluation of lung maturity in the equine species, but further studies on a large number of mature and premature foals are necessary to establish equine pulmonary maturity standards.

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis.

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical-pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.

Chemical and biochemical parameters of cultured diatoms and bacteria from the Adriatic Sea as possible biomarkers of mucilage production.

Bacteria and diatom strains from the Adriatic Sea were investigated, under standard and altered environmental conditions, for carbohydrate production and for the presence of specific biomarkers. Algae from P-depleted cultures showed an increase in extracellular carbohydrate production, a significantly lower chlorophyll a content and unchanged total lipid levels. However, the fatty acid composition of algal cultures was severely affected by low P levels, in that, total saturated and monounsaturated fatty acids increased and total polyunsaturated fatty acids decreased. Marine heterotrophic bacteria resulted enriched by 4 to 6 orders of magnitude in mucilage samples respect to surrounding seawater, unlike other groups of bacteria such as the non-halophylic heterotrophs. The major fatty acids detected in bacteria were 16:0 and 18:1n-7; the uneven fatty acids 17:0i, 17:0 and 17:1 also constituted an important component of various strains and, as a result, the total monounsaturated fraction represented the main component of total fatty acids. All the mucilage samples analysed shared the same general fatty acid composition features with a high amount of saturated components, especially 16:0; typical marine polyunsaturated fatty acids, such as 20:5n-3 and 22:6n-3, were found at very low levels. With regard to the sterol composition, the analysed algal species and bacteria showed that different compounds prevailed in the different species, and under P-deprivation sterol distribution resulted differently affected in the various algal species. In mucilage samples an overall prevalence of cholesterol was observed and, among 4alpha-methylsterols, constantly present, dinosterol prevailed in all samples. Vibrational IR spectroscopic analyses confirmed the main results obtained with the GC analysis: a higher unsaturation degree in nutrient replete diatom cultures than in P-depleted ones, a lower amount of P-containing compounds in the latter, bacterial lipid profiles with a high amount of free carboxylic acids and/or ketones and a low unsaturation degree and, finally, mucilage samples with a very low unsaturation degree. All these results allowed some speculations on the involvement of the various microbial and phytoplankton components in mucilage genesis.

Cd-metallothionein and metal-enzymes interactions in the goldfish Carassius auratus.

Goldfish injected with cadmium chloride synthesized metallothionein. Ten days after the first injection, cadmium reached a maximum in the metallothionein peak (2 micrograms/ml) obtained after gel filtration of liver cytosol. Pyruvate kinase activity was inhibited from the beginning of the experiment; after the fourth day, the enzyme activity again started to increase but did not reach the control level. Alkaline phosphatase and fructose biphosphatase did not show any apparent inhibition. From the results here reported, a detoxifying role of metallothionein could be suggested.

Phospholipid composition of photosynthetic membranes of Rhodopseudomonas capsulata.

The phospholipids and the fatty acids present in membranes of cells of Rhodopseudomonas capsulata, grown photosynthetically in anaerobiosis, were analyzed by thin layer chromatography and gas chromatography-mass spectrometry. The three phospholipids detected, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol, contained about 80% of a single monounsaturated C18 fatty acid, cis-vaccenic acid. These membranes offer therefore a naturally occurring model system endowed with an extremely simplified phospholipid complement. The data indicate moreover that the biosynthetic pathway of unsaturated fatty acids present in these facultative aerobic bacteria proceeds only via the 3-hydroxydecanoyl acyl carrier protein dehydratase (E.C. 4.2.1.60).