Raffaele Acierno

Resveratrol inhibits the epidermal growth factor-induced epithelial mesenchymal transition in MCF-7 cells

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of a mesenchymal phenotype by tumour cells. Herein we show that exposure of MCF-7 cells to epidermal growth factor (EGF) resulted in morphological alterations characteristic of epithelial-to-mesenchymal transition (EMT). EGF treatment resulted in increased motility along with an up-regulation of transcription factors Slug, Zeb1, Zeb2, and mesenchymal markers Vimentin and N-cadherin. Treatment of MCF-7 cells with a combined stimulation of EGF and resveratrol, a naturally occurring stilbene with antitumor properties, failed to alter cell morphology, motility and overexpression of EMT markers induced by EGF. Using specific chemical inhibitors, we demonstrated that EGF-induced EMT is mediated by extracellular signal-regulated kinase 1/2 (ERK 1/2) signalling pathway and that resveratrol is able to repress EGF-induced ERK activation. In summary, these data provide new evidence of the inhibitory effect of resveratrol on EGF-induced EMT cell transformation.

Control of cardiovascular function in the icefish Chionodraco hamatus: involvement of serotonin and nitric oxide

The involvement of nitric oxide (NO) in the branchial circulation and cardiac performance of the Antarctic hemoglobinless icefish Chionodraco hamatus was investigated using isolated and perfused head and working heart preparations. In the branchial vasculature under basal (i.e. unstimulated conditions), the nitric oxide synthase (NOS) inhibitor L-NIO (L-N(5)-(1-iminoethyl) ornithine, 10(-5) and 10(-4) M), caused a marked vasoconstriction (20%), indicating a basal nitrergic vasodilator tone, while the dose-response curve of the NO donor SIN-1 (3-morpholinosydnonimine) showed a dose-dependent vasodilator effect. Acetylcholine induced a dose-dependent branchial vasoconstriction mediated by muscarinic receptors, since the effects were abolished by pre-treatment with atropine (10(-4) M). Serotonin (5-HT) induced a dose-dependent branchial methysergide-sensitive vasoconstriction which was abolished by pre-treatment with L-NIO, indicating a NO-dependent mechanism. On the isolated heart, the NOS inhibitor L-NMMA (N(G)-monomethyl-L-arginine) 10(-4) M produced a small, but significant decrease of heart rate and, as a consequence, a decrease of power output, while the NO donor sodium nitroprusside (SNP) 10(-4) M elicited increases of stroke volume, stroke work and power output, suggesting an exogenous NO-dependent positive inotropism. Exposure of the bulbus arteriosus to L-NMMA was without any appreciable effect. In contrast, SNP produced a notable relaxation of the bulbus wall with a marked increase of its stiffness, as indicated by the increase of systolic and diastolic dP/dt max (23 and 104%, respectively).

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2‐DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty‐six spots corresponding to 174 different proteins have been identified on 2‐DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.

SUMMARY H+/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H+/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (Km,app) of 0.806±0.161 mmol l-1. Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H+/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)+ RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H+/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0°C for the former and 25°C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

RFID-based tracing systems for drugs: Technological aspects and potential exposure risks

Radio Frequency Identification (RFID) is a very promising wireless technology able to trace and track individual objects. The pharmaceutical supply chain is a challenging scenario, where an item-level traceability is crucial to guarantee transparency and safety in the drug flow. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies. In order to face these challenges, multidisciplinary skills are required. A recent research project has attempted to coordinate heterogeneous activities focused on drug traceability. One of these is related to the evaluation of potential effects of exposure to electromagnetic fields on drugs. This paper aims to briefly describe both the main features of the defined framework for the item-level tracing of drugs on the whole supply chain and the most interesting results obtained by the evaluation of the potential effects of RFID systems on drugs. In particular, the potential alterations of the molecular structure of a commercial human insulin preparation have been analyzed by using investigative techniques such as Reverse Phase-High Pressure Liquid Chromatography and in vitro cell proliferation assays. The experimental results are strongly encouraging the use of RFID-based technologies for item-level tracing systems in the pharmaceutical supply chain.

Adaptation of intestinal cell membrane enzymes to low temperatures in the Antarctic teleost Pagothenia bernacchii

The enzymatic activity (expressed as milliunits per milligram total proteins) of three intestinal brush-border membrane enzymes, leucine aminopeptidase, alkaline phosphatase and maltase, measured over a range of temperatures between 1.5 and 37 °C, has been found to be much higher in the Antarctic fish Pagothenia bernacchii than in the temperate fish Anguilla anguilla. To explain this experimental observation the apparent Michaelis-Menten constant, the maximal velocity, the activation energy values and the thermal stability of these three enzymes were measured. The apparent Michaelis-Menten constant values of leucine amino peptidase and alkaline phosphatase were different in the intestine mucosal homogenate of the two fish at each measured temperature (from a minimum of 2.5 to a maximum of 37 °C). However, the values found at 2.5 °C for the Antarctic species and 15 °C for the eel where comparable. Furthermore, its value was unchanged in eel intestine apical membranes, both in the presence and without enzyme lipid microenvironment. While the maximal enzymatic activities of the leucine aminopeptidase and maltase did not decrease without their enzyme lipid microenvironment, produced by treatment with Triton X-100, the impairment of alkaline phosphatase maximal activity cannot be significantly differentiated from a non-specific inhibitory effect of the detergent. The activation energy values of leucine amino peptidase, alkaline phosphatase and maltase were lower in the Antarctic fish (11.7, 5.6 and 11.8 kcal·mol-1, respectively) than in the eel (13.6, 7.6 and 13.1 kcal·mol-1, respectively). The thermal stability of alkaline phosphatase and maltase is different in Pagothenia bernacchii and Anguilla anguilla intestinal homogenate.

Lipid and fatty acid composition of intestinal mucosa of two Antarctic teleosts

Abstract The fatty acid composition of intestinal mucosa of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii, was investigated in comparison with two temperate water species: Anguilla anguilla and Dicentrarchus labrax. Higher amounts of unsaturated and monounsaturated fatty acids were present in the Antarctic species, while the polyunsaturated fatty acid levels were not significantly different in all fish species. A higher content of phosphatidylethanolamine (a phospholipid rich in unsaturated fatty acids and with a relatively low melting point) and a lower content of sphingomyelin (a phospholipid rich in saturated fatty acids and with relatively high melting point) were observed in T. bernacchii in comparison with A. anguilla.

PBMCs protein expression profile in relapsing IFN-treated multiple sclerosis: A pilot study on relation to clinical findings and brain atrophy

This cross-sectional study investigated with two-dimensional gel electrophoresis coupled to MALDI-TOF and MRI the relationship between PBMCs protein expression profile and whole-brain atrophy in 16 unselected RR-MS IFN-treated patients compared with 6 RR IFN-untreated and 12 matched healthy control subjects. Grey/white matter fraction, T1/T2 lesion load and clinical variables were considered too. Twenty six proteins showed significant differential expression among RR IFN-treated patients and control samples. Four of these (IN35, GANAB, PP1B, SEPT2) resulted correlated with clinical and MRI findings in RR IFN-treated MS patients. Future clinical applications remain to be validated by other techniques and confirmed by a larger study.

Buffer capacity in the blood of the hemoglobinless Antarctic fish Chionodraco hamatus

Blood acid-base homeostasis of the hemoglobinless Antarctic teleost Chionodraco hamatus was analyzed by measuring the titratable buffer capacity (β) and carbonic anhydrase (CA) activity in blood smaples. Results were compared with those obtained in the red-blooded temperate fish Anguilla anguilla. Data show that the titratable blood buffer capacity of icefish, in the range of red-blooded teleost species, was significantly higher than that of A. anguilla. Furthermore, plasma inorganic phosphate and reactive sulfhydryl content was significantly higher in the Antarctic species. No enzymatic activity of CA was detected in blood samples of Antarctic fish, suggesting the absence of a blood CA-isozyme.