Artificial nose, NIR and UV-visible spectroscopy for the characterisation of the PDO Chianti Classico olive oil.

An authentication study of the Italian PDO (Protected Designation of Origin) olive oil Chianti Classico, based on artificial nose, near-infrared and UV-visible spectroscopy, with a set of samples representative of the whole Chianti Classico production area and a considerable number of samples from other Italian PDO regions was performed. The signals provided by the three analytical techniques were used both individually and jointly, after fusion of the respective variables, in order to build a model for the Chianti Classico PDO olive oil. Different signal pre-treatments were performed in order to investigate their importance and their effects in enhancing and extracting information from experimental data, correcting backgrounds or removing baseline variations. Stepwise-Linear Discriminant Analysis (STEP-LDA) was used as a feature selection technique and, afterward, Linear Discriminant Analysis (LDA) and the class-modelling technique Quadratic Discriminant Analysis-UNEQual dispersed classes (QDA-UNEQ) were applied to sub-sets of selected variables, in order to obtain efficient models capable of characterising the extra virgin olive oils produced in the Chianti Classico PDO area.

Role of angiogenesis inhibitors in colorectal cancer: sensitive and insensitive tumors.

Angiogenesis is a key factor in the carcinogenesis process. In oncological practice, angiogenesis inhibition, mainly through the blockade of the VEGF family and its receptors, has been robustly demonstrated to produce clinical benefits and, in specific disease subsets such as colorectal cancer, to extend the overall survival of treated patients. VEGF is a multifunctional growth factor that mediates its functions through cognate receptors on endothelial cells and it has been discovered for its capability to induce macromolecule hyperpermeability in veins and venules. Several approaches have been taken to target angiogenesis in cancer: drugs that target one or more soluble ligands of the VEGF family, drugs that selectively inhibit one or more receptors of the VEGF receptor family, and drugs that inhibit VEGF receptor(s) among other, non VEGF-related targets. At present, two compounds have shown significant clinical activity, bevacizumab, Avastin® and aflibercept, Zaltrap®, and only one of these (bevacizumab) has so far been registered for use in clinical practice. In the present review, we explore and summarize the main features of the angiogenetic process, concerning in particular a common and potentially lethal disease as colorectal cancer. We overview the molecular pathways that characterize angiogenesis, focusing on VEGF family, the current applications and limitations of its blockade in oncology, and the hypothetical future perspectives of anti-angiogenic therapy.

Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

Inhibition of a protein-protein interaction between INI1 and c-Myc by small peptidomimetic molecules inspired by Helix-1 of c-Myc: identification of a new target of potential antineoplastic interest.

c-Myc is a transcription modulator proto-oncogene. When overexpressed, it becomes an important contributor to the multi-hit process of malignant transformation. In two earlier papers in this journal (see refs. 19 , 20) we reported that retro-inverso peptidomimetic molecules inspired by the Helix-1 of c-Myc motif could be sequence-specific antiproliferative agents active in the low micromolar range. We also found that our peptides were not opening the four-alpha-helix Myc:Max bundle. Their antiproliferative activity in cancer cell lines needs the presence of side chains projecting outside of the bundle in the corresponding native H1 motif. This observation suggested interference with an external partner. In this study we investigated the INI1:Myc interaction. INI1 is a subunit of the SWI/SNF complex (component of the enhanceosome surrounding Myc:Max heterodimer). The INI1:Myc interaction was confirmed via pull down, ELISA, and fluorescence anisotropy assays. According to the length of INI1 fragments used, we calculated Kds ranging between 1.3x10(-6) and 4.8x10(-7) M. The three different techniques applied showed that the INI1:Myc interaction was also the target of our retro-inverso peptidomimetic molecules, which seem to bind specifically at INI1. A Myc binding, 21aa INI1 fragment (minimum interacting sequence), could inspire the synthesis of a new class of more selective c-Myc inhibitors.

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems.

In 1998 we reported that an L-peptide derived from H1 of c-Myc (Int-H1-S6A,F8A), linked to an internalization sequence from the third a-helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro-inverso (RI-) peptides can assume a 3D conformation very close to their corresponding L-forms and can be recognized by the same monoclonal antibody. We synthesized a RI-peptide form of our original L-peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI-form of the original Antennapedia internalization sequence was perfectly capable of carrying a D-peptide into human cells. We have studied three different potentially active peptides. L-peptides: Int-H1wt, Int-H1-S6A,F8A. D-peptides: RI-Int -H1-S6A,F8A. We have also studied three presumed control peptides: Int and RI-Int (no H1 motif), H1-S6A,F8A (no internalization sequence). Both 'active' and 'control' peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 mM) of the control peptide RI-Int, non-Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c-Myc (and molecules of the family), we chose an iso-amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F-->A). From a family of 73 H1 motifs belonging to (H1-Loop-H2) hu man sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N-Myc, L-Myc, c-Myc, H1-S6A,F8A of c-Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int-H1isoamph and the corresponding RI-Int-H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF-7 cells) a colon cancer line (HCT-116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso-amphipathic-modified H1 sequence were always very clearly (3-10 times) less active than the corresponding peptides carrying a conserved "H1 of Myc" motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF-7 cells treated with L-peptides; MCF-7 cells treated with RI-peptides; HCT-116 cells treated with L-peptides; PBLs treated with L-peptides; PBLs treated with RI-peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF-7 cells. On a molar basis, RI-peptides were about 5-10 times more potent and 30-35 times more stable in complete culture medium, than their corresponding L-forms. RI-Int can probably internalize longer peptido-mimetic molecules (for instance molecules mimetic of (H1-Loop-H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors-two steps closer to a potential drug.

Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence.

c-Myc is a nuclear protein with important roles in cell transformation, cell proliferation, and gene transcription. It has been previously shown that a 14-amino acid (aa) modified peptide (H1-S6A,F8A) derived from the helix 1 (H1) carboxylic region of c-Myc can interfere in vitro with specific c-Myc DNA binding. Here, we have linked the above Myc-derived 14-aa peptide to a 16-aa sequence from the third helix of Antennapedia (Int). It has been repeatedly reported that this 16-aa Antennapedia peptide is able to cross mammalian cell membranes and to work as a vector for short peptides. Using fluorescent (dansylated or rhodaminated) peptides, we have shown that the fusion peptide with the Antennapedia fragment (Int-H1-S6A,F8A) but not the c-Myc derived fragment alone (H1-S6A,F8A) was capable of internalization inside MCF-7 human breast cancer cells. Int-H1-S6A,F8A and H1-S6A,F8A were the only two peptides capable of inhibiting coimmunoprecipitation of the c-Myc/Max heterodimer in vitro. We have treated (continuously for 10-11 days) MCF-7 cells with four different peptides: Int, H1-S6A,F8A, Int-H1-S6A,F8A, and Int-H1wt [a peptide differing from Int-H1-S6A,F8A by 2 aa (S6 and F8) in the H1 region]. In intact MCF-7 cells, Int-H1-S6A,F8A was the only active peptide capable of inducing the following biological effects: (a) inhibition of cloning efficiency on plates; (b) inhibition of cell growth and induction of apoptosis in subconfluent/confluent cells; and (c) inhibition of transcription of two c-Myc-regulated genes (ODC and p53). Int-H1-S6A,F8A was active in the 1-10 microM range. Int-H1-S6A,F8A may represent a lead molecule for peptidomimetic compounds that have a similar three-dimensional structure but are more resistant to peptidases and, therefore, suitable for in vivo treatment of experimentally induced tumors.

Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice.

A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the overexpression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493-497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.

Identification of a new member (ZNF183) of the Ring finger gene family in Xq24-25.

Four genes were mapped to the Xq24-25 region by searching the EST and the non-redundant database with short tracts of genomic sequences. These were random STSs present in the STS database or sequences derived from CpG islands (EagI-based STSs). One of the four matches corresponded to the full length transcript from the intronless glutamate dehydrogenase gene. The second was the human homolog of the bovine NADH ubiquinone oxidoreductase MWFE subunit gene (GDB symbol: NDUFA1). The other two, ZNF183 and ITBA4, were novel genes whose function cannot directly be inferred from their sequence analysis. However, a known motif, the C3HC4 Ring finger domain, shared by various tumor suppressors, DNA repair genes and cytokine receptor-associated molecules, is present at the C terminus of the ubiquitously expressed ZNF183 gene. ITBA4 is expressed at various levels in different tissues and is alternatively processed in brain. Similarity search did not detect any significant match in databases. These results, together with others previously reported by our laboratory, suggest that comparison of genomic and transcribed sequences which are continuously accumulating in databases, can provide 'virtual' mapping of a substantial number of ESTs to the specific genomic region which the STSs have been derived from.

A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds.

Transgenic mice for genotoxicity testing have been developed, although no such models have been produced for the evaluation of toxic, nongenotoxic chemical compounds. We have developed a transgenic mouse model for the analysis of toxic inorganic compounds. We engineered a mouse lineage with the human growth hormone (hGH) gene under the control of the human hsp70 promoter, in which a plasma-detectable hGH response can be elicited by exposure to heat shock. In primary cell cultures from these mice, hGH release was observed following treatment with several toxic inorganics. Transgenic mice injected intraperitoneally with sodium arsenite, cadmium chloride, copper sulphate, or methylmercurium chloride showed significant hGH levels in plasma.

Partial regression, yet incomplete eradication of mammary tumors in transgenic mice by retrovirally mediated HSVtk transfer 'in vivo'.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

Identification of two forms (31-33 and 48 kD) of the urinary soluble p55 tumor necrosis factor receptor that are differentially N- and O-glycosylated.

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

Identification of an epitope of tumor necrosis factor (TNF)-receptor type 1 (p55) recognized by a TNF-alpha-antagonist monoclonal antibody.

The relationships between epitope topography and agonistic/antagonistic effects of anti-TNF receptor type 1 (TNF-R1) antibodies on TNF-alpha cytotoxic activity have been studied. To this purpose various monoclonal antibodies (mAbs) against the soluble form of TNF-R1 (sTNF-R1) have been generated and characterized. Epitope topography studies identified at least four distinct epitopes located outside (4E10) or within (or close to) the TNF-alpha binding site of urinary sTNF-R1 (7H3, 4C1, 9B11). mAbs 7H3 and 4C1 were able to neutralize the inhibition of human TNF-alpha cytotoxicity on L-M cells by sTNF-R1, while 4E10 was unable. Moreover, 7H3 and 4C1 were able to antagonize the TNF-alpha cytotoxicity on human U937 cells, while they were uneffective on mouse L-M cells, suggesting that these antibodies recognize, in a species-specific mode, also the membrane form of the human receptor. No agonistic effects were observed when these antibodies were used in the absence of TNF-alpha. Epitope topography studies carried out using overlapping decapeptides covering most of the sTNF-R1 sequence showed that residues 143-148 of the fourth cysteine-rich domain of the receptor (FFLREN) contain antigenic determinants recognized by the antagonist antibody 7H3. These results suggest that at least part of residues 143-148 of sTNF-R1 are surface exposed on the soluble as well as on the membrane forms of TNF-R1 and are accessible to TNF-alpha antagonists.