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Biomedical subjects

G Vecchio

Publications and source records attributed to G Vecchio.

At least 73 records · Page 4Linked to original sources
PubMed1995

Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells.

The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

Animals↗
PubMed1995

The saccharide chain of lupin seed conglutin gamma is not responsible for the protection of the native protein from degradation by trypsin, but facilitates the refolding of the acid-treated protein to the resistant conformation.

Native glycosylated and enzymically deglycosylated conglutin gamma (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40-residue peptide was cleaved from the N-terminus of conglutin gamma light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7-h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse-phase HPLC analyses of the glycosylated and deglycosylated conglutin gamma forms. Therefore, it was concluded that the saccharide chain of conglutin gamma increased the rate of formation of a trypsin-resistant conformation upon refolding of the acid-treated protein, without playing any direct role in the protection of the native protein from proteolysis.

Amino Acid Sequence↗
PubMed1995

Transformation of thyroid epithelium is associated with loss of c-kit receptor.

The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.

Cell Line↗
PubMed1995

Effect of gemfibrozil treatment in hypercholesterolemia on low density lipoprotein (LDL) subclass distribution and LDL-cell interaction.

Gemfibrozil, a widely used fibric acid derivative, corrects hypercholesterolemia in a non-negligible fraction of patients. To investigate the mechanism of the cholesterol-lowering activity of fibric acids, a study was performed in 12 type IIa hyperlipidemic patients treated with gemfibrozil for 12 weeks. Changes in low density lipoprotein (LDL) structure and composition, agonist capacity of LDL against the LDL-receptor in human skin fibroblasts, LDL-receptor activity in mononuclear cells, lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, were evaluated. Plasma total and LDL cholesterol levels decreased by 17% and 20% after 12 weeks of treatment, the reduction being directly correlated with the baseline levels (r = 0.75 and 0.78, respectively). The mean LDL diameter increased significantly, from 25.5 to 26.1 nm, while the relative content of small LDL particles (< 25.1 nm) increased from 23.4% to 32.8% of total LDL. Neither the apolipoprotein (apo) B secondary structure nor the affinity of LDL for the LDL-receptor of fibroblasts were affected. The LDL-receptor activity in patients' mononuclear cells increased 3-fold, the rise being unrelated to the plasma cholesterol reduction. LCAT activity did not change, while CETP activity was reduced by 25% (P = 0.13) after treatment. These findings indicate that gemfibrozil causes significant changes in LDL structure that do not, however, affect the LDL interaction with peripheral cells.

Adult↗
PubMed1995

Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B.

Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma are dominantly inherited cancer syndromes. All three syndromes are associated with mutations in RET, which encodes a receptor-like tyrosine kinase. The altered RET alleles were shown to be transforming genes in NIH 3T3 cells as a consequence of constitutive activation of the RET kinase. The MEN2A mutation resulted in RET dimerization at steady state, whereas the MEN2B mutation altered RET catalytic properties both quantitatively and qualitatively. Oncogenic conversion of RET in these neoplastic syndromes establishes germline transmission of dominant transforming genes in human cancer.

3T3 Cells↗
PubMed1995

Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic transformation.

Tumors are believed to arise as a result of an accumulation of mutations in critical genes involved in the control of cell proliferation. Thyroid neoplasms represent a good model for studying the role of these mutations in epithelial cell multistep carcinogenesis because they comprise a broad spectrum of lesions with different degrees of malignancy. Recent reports have described the involvement of specific genetic alterations in different types of thyroid neoplasms. Papillary carcinomas are characterized by the activation of the receptor tyrosine kinases RET and TRK-A proto-oncogenes. Ras point mutations are frequently observed in tumors with follicular histology and a high prevalence of p53 point mutations have been found in anaplastic carcinomas. A definition of molecular defects characterizing thyroid tumors will be helpful in establishing sensitive and specific detection strategies and, in addition, to define genetic and environmental factors important for their pathogenesis.

Carcinoma↗
PubMed1995

Expression of the RET oncogene induces differentiation of SK-N-BE neuroblastoma cells.

Expression of the RET proto-oncogene, a cell surface receptor for an as yet unknown ligand, is associated with tumors, tissues, and cell lines of neural crest origin. Accumulating evidence suggests that RET activity is involved in the process of neuronal differentiation. Moreover, induction of phenotypic differentiation of neuroblastoma cell lines is associated with the rapid accumulation of RET transcripts. To verify the role of RET in neuronal differentiation, we introduced into the human neuroblastoma cell line SK-N-BE four versions of the RET oncogene, activated by different mechanisms: RET/PTC1 and RET/PTC3, which are activated by rearrangement with heterologous genes; and two activated RET mutants, which carry the single amino acid substitution found associated to the inheritance of the multiple endocrine neoplasia type 2A (retMEN2A allele) and type2B (retMEN2B allele), respectively. We demonstrate that, after transfection with the RET oncogenes, SK-N-BE cells display a reduced growth rate and acquire a neurite-bearing phenotype accompanied by enhanced expression of the axonal growth-associated protein, GAP-43, and the high molecular weight neurofilament, NF200. These results indicate that, when activated, RET is able to cause growth inhibition and to promote neuronal differentiation of neuroblastoma cells.

Blotting, Northern↗
PubMed1994

Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I.

The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173-->Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly alpha-helical structure predicted to be approximately 65% in the lipid-free and approximately 78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of alpha-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher alpha-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the alpha-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer.

Amino Acid Sequence↗
PubMed1994

Seminal ribonuclease inhibits tumor growth and reduces the metastatic potential of Lewis lung carcinoma.

The role of some RNases as antitumoral agents has been recently emphasized. We have previously demonstrated a striking inhibitory effect of bovine seminal RNase on the in vitro growth of tumor cells of metastatic origin. This has prompted us to test the effects of this protein in vivo on the induction of metastatic foci in mice lungs after i.m. injection of a highly metastatic Lewis lung carcinoma cell line. The results presented here, while confirming and expanding upon those previously reported on the antitumor effects of bovine seminal RNase in vivo on primary thyroid epithelial tumors, indicate for the first time that bovine seminal RNase can also be regarded as a potent antimetastatic agent on in vivo spontaneous metastases.

Animals↗
PubMed1994

Focusing of alkaline proteases (subtilisins) in pH 10-12 immobilized gradients.

Isoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9-11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10-12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 +/- 0.15; that of Durazym as 10.95 +/- 0.20 and that of the pI marker cytochrome c as 10.6 +/- 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10 degrees C, instead of the tabulated values at 25 degrees C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a value of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10 degrees C) of 11.15.

Amino Acid Sequence↗
PubMed1994

Inhibition of photohemolysis by copper(II) complexes with SOD-like activity.

Red blood cell lysis photosensitized by Ketoprofen, 2-(3-benzoxyphenyl)propionic acid (KPF), was investigated in the presence of some copper(II) complexes with linear and cyclic dipeptides as well as functionalized beta-cyclodextrins with SOD-like activity with the aim of ascertaining their protective activity towards the photoinduced cell damage. The comparison between the decrease of photolytic activity caused by these complexes and their superoxide dismutase activity showed an appreciable correlation. The correct determination of species existing in experimental conditions was obtained through a simulation approach based on the knowledge of the stability constants of the complexes.

Amino Acid Sequence↗
PubMed1994

Pravastatin effectively lowers LDL cholesterol in familial combined hyperlipidemia without changing LDL subclass pattern.

Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder among young survivors of myocardial infarction. Elevations of plasma total and low-density lipoprotein (LDL) cholesterol and the prevalence of small, dense LDL particles are both involved in the high coronary risk of FCHL patients. We investigated the ability of pravastatin to favorably correct plasma lipid and lipoprotein levels and LDL structure in FCHL patients. Twelve patients with FCHL, documented by studies of first-degree relatives, received pravastatin (40 mg/d) for 12 weeks. Pravastatin significantly lowered plasma total and LDL cholesterol levels by 21% and 32%, respectively. Triglyceride levels did not change, and apolipoprotein B (apoB) concentrations decreased by 9% (P = NS). High-density lipoprotein (HDL) cholesterol increased by 6% because of a significant 73% rise of HDL2 cholesterol. LDL were smaller (diameter, 24.5 +/- 0.5 nm), less buoyant, and apoB-rich (cholesteryl ester-apoB ratio, 1.64 +/- 0.46) in the selected patients compared with patients with familial hypercholesterolemia or healthy control subjects. LDL became even smaller (23.8 +/- 0.6 nm) and richer in apoB (cholesteryl ester-apoB ratio, 1.27 +/- 0.52) after pravastatin treatment. Although pravastatin favorably altered plasma lipid and lipoprotein levels in FCHL patients, the abnormal LDL particle distribution and composition were not affected. Because of the apparent resistance of the small, dense LDL to drug-induced modifications, a maximal lipid-lowering effect is needed to reduce coronary risk in FCHL patients.

Adult↗
PubMed1994

Cloning and characterization of H4 (D10S170), a gene involved in RET rearrangements in vivo.

H4(D10S170) is a gene which we isolated because of its frequent rearrangement with the RET proto-oncogene in vivo. Its fusion to RET generates the RET/PTC1 oncogene, which has been detected in about 20% of human thyroid papillary carcinomas. We have cloned and sequenced the cDNA corresponding to the H4(D10S170) gene from a human normal thyroid cDNA library. The nucleotide sequence of the H4(D10S170) 3 kb transcript shows no significant homology to known genes and contains an open reading frame (ORF) of 585 amino acids. H4(D10S170) predicted protein has no transmembrane domain and shows extensive regions in the alpha helical conformation, which are 30% homologous to the alpha-helical domains of several proteins including tropomyosin, vimentin, keratin and the tail region of myosin heavy chain. A putative SH3 binding site is present at the carboxy terminus, which suggests that H4(D10S170) might be a cytoskeletal protein.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma.

The RET proto-oncogene encodes a transmembrane receptor of the tyrosine kinase family and has frequently been found activated in human thyroid carcinomas of the papillary subtype. In most cases the activation consisted of the fusion of its tyrosine-kinase domain with the 5'-terminal region of a gene designated H4 or D10S170. We have named the resulting H4/RET chimeric oncogene RET/PTC. Another activated form of the RET oncogene has subsequently been found in a thyroid carcinoma and is now referred to as RET/PTC2. Here we report the identification and cloning of a novel rearranged version of the RET oncogene in a human thyroid papillary carcinoma. In this case the tyrosine-kinase domain of RET was fused to a sequence 790 bp long belonging to a new gene that we have named RFG (RET Fused Gene). This novel chimeric oncogene has been designated RET/PTC3. In order to have more insights into the function of RFG we have completely cloned and sequenced its cDNA. RFG predicted amino-acid sequence does not have any significant homology to any already known genes and is ubiquitously expressed in human and mouse tissues. Finally we provide evidence indicating that the rearrangement leading to the generation of RET/PTC3 occurred in vivo in the original tumor DNA.

Amino Acid Sequence↗
PubMed1993

New platinum(II) complexes of beta-cyclodextrin diamine derivatives and their antitumor activity.

The new platinum(II) complexes of beta-cyclodextrin functionalized at the primary position with ethylenediamine or with propylenediamine were synthesized and characterized by mass spectrometry, electronic spectrophotometry, and multinuclear NMR spectroscopy. Platinum(II) complexation makes the cavity more asymmetric. These complexes were tested in vitro for their cytotoxic activity. The relevance of the low activity observed regarding the interaction between the cell and the cyclodextrin cavity is discussed.

Antineoplastic Agents↗
PubMed1993

Determination of superoxide dismutase-like activity of copper(II) complexes. Relevance of the speciation for the correct interpretation of in vitro O2- scavenger activity.

The superoxide dismutase activity of several copper(II) complexes of linear and cyclic dipeptides has been measured. The results of a classic indirect method (xanthine-xanthine oxidase) have been compared with those obtained by generation of the superoxide radical through 2-(3-benzoylphenyl)propionic acid (ketoprofen) photolysis. A simulation approach, based on the knowledge of the stability constants of the different complex species existing in experimental conditions, has allowed us to obtain the correct speciation and to use these data to calculate the pertinent catalytic constants.

Catalysis↗
PubMed1993

Oncogenes of DNA and RNA tumor viruses and the origin of cellular oncogenes.

Oncogenes were first described as the genes of DNA tumor viruses responsible for in vivo and in vitro neoplastic transformation induced by such viruses. The first oncogenes described were the oncogenes of polyoma, SV40 and papilloma viruses. The polyoma virus oncogene has been designated the 'middle' T (tumor) antigen, whereas the SV40 oncogene is designated the 'large' T antigen. The large T of the polyoma virus can also be considered an oncogene, because it is capable of 'immortalizing', but not transforming primary cell cultures. Other oncogenes of DNA tumor viruses are the E1A of adenoviruses and the E7 gene of papilloma viruses. It has been proposed that some of the oncogenes of DNA tumor viruses act by binding the products of tumor suppressor genes, such as the retinoblastoma gene product Rb. In general, the products of the oncogenes of DNA tumor viruses need the cooperation of other oncogenes to establish a completely transformed phenotype. Oncogenes have been discovered in all replication defective RNA tumor viruses, whereas they are not present in the genomes of replication competent RNA tumor viruses, which can also give rise to a neoplastic phenotype, in vivo, but by a different mechanism. The discovery of oncogenes in RNA tumor viruses has led to the concept that these oncogenes are actually present in the genomes of all eucaryotic organisms and have been transduced and, in some cases, modified by a retrovirus. The characterization of the oncogenes of acute transforming retroviruses has led to the present classification of oncogenes, which include genes coding for proteins having different functional properties, but all characterized by having some role in the pathway which regulates cell proliferation. The experimental approach of DNA transfection has led to the discoveries of some oncogenes already known from the genomes of retroviruses and also of some new oncogenes which had never been transduced by retroviruses. The knowledge derived from retrovirus research and from the use of the DNA transfection technique has opened a whole new field of science thereby contributing greatly to our understanding of the mechanisms underlying neoplastic transformation and also to the understanding of normal cell physiology.

Animals↗