Biomedical subjects

G Vecchio

Publications and source records attributed to G Vecchio.

At least 55 records · Page 3Linked to original sources

PubMed1997

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution.

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance.

Alanine↗

PubMed1997

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-beta-cyclodextrin.

A modified beta-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-beta-cyclodextrin, CDampy) was synthesized by C6-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d.. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show and difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes ("cis effect"), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI.

Alanine↗

PubMed1997

Histamine-modified beta-cyclodextrins for the enantiomeric separation of dansyl-amino acids in capillary electrophoresis.

Two novel monosubstituted beta-cyclodextrins (CD) bearing the histamine moiety linked to the upper CD rim, either through the amino group or the imidazole nitrogen 1N, CD-hm and CD-mh, were successfully used as chiral selectors in capillary electrophoresis for the enantiomeric discrimination of dansyl (Dns)-amino acids. Good results were obtained by using low concentrations of the selectors (1-3 mM). The effect of pH on the chiral discrimination was studied in order to modulate the number and the position of the positive charges. By increasing the pH from 5 to 7.5, chiral discrimination decreased along with the deprotonation of the imidazolyl moiety. Inversion of the migration order was observed with the two CDs, depending on the relative position of the charged moieties on the upper rim. Ion pair interaction coupled to inclusion complexation seems to account for the discrimination process. The effects of the temperature, CD concentration and capillary length on chiral resolution were also examined.

Carbohydrate Sequence↗

PubMed1996

Ligand stimulation of a Ret chimeric receptor carrying the activating mutation responsible for the multiple endocrine neoplasia type 2B.

Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.

3T3 Cells↗

PubMed1996

Human apolipoprotein A-II inhibits the formation of pre-beta high density lipoproteins.

The role of human apolipoprotein A-II (apoA-II) in the remodeling of human high density lipoproteins (HDL) was investigated during incubation of native and reduced-carboxamidomethylated (RCM) HDL3 with a lipoprotein-depleted plasma fraction (LPDP) in the presence of triglyceride-rich particles (TGRP) isolated from Intralipid. Reduction-carboxamidomethylation of HDL3 entirely converts the disulfide-linked apoA-II dimers into monomers, without affecting the structure, composition and particle size distribution of HDL3. Following incubation with LPDP and TGRP, unmodified HDL3 are mainly converted into large, HDL2 particles (diameter: 9.90 +/- 0.07 nm), enriched in triglycerides and depleted of cholesteryl esters. RCM-HDL3 are converted into both large HDL2 (9.86 +/- 0.07 nm) and small (7.53 +/- 0.06 nm) HDL3. The small products are protein-rich and cholesterol-poor, and consist of two different particles: a component with pre-beta mobility, containing only apoA-I, and a component with alpha mobility, containing both apoA-I and apoA-II. Kinetic studies suggest that a two-step process is involved in the formation of small, pre beta-HDL3, by which changes in lipid composition cause alterations in lipoprotein structure/stability, favoring the dissociation of apolipoproteins and reduction of particle size. These findings indicate that apolipoprotein structure is a major determinant of HDL remodeling, apoA-II potentially counteracting the anti-atherogenic properties of apoA-I by inhibiting the formation of small, pre-beta-migrating HDL.

Adult↗

PubMed1996

Development of mammary and cutaneous gland tumors in transgenic mice carrying the RET/PTC1 oncogene.

RET/PTC1 is a chimeric oncogene created by the fusion of the tyrosine kinase domain of RET to the 5'-terminal region of another gene named H4. So far, this oncogene has been found activated only in human papillary thyroid carcinomas. In order to investigate its transforming properties in vivo, we have produced transgenic mice carrying RET/PTC1 under the control of the H4 promoter. The transgene was expressed in several tissues, consistently with the ubiquitous expression of the wild type H4 gene. Mammary adenocarcinomas and, less frequently, hyperplasia of sebaceous glands and rare benign skin tumors, named pilomatrixomas, developed in these mice. The tumors were shown to express the transgene both at the RNA and protein level. These results demonstrate that the transforming ability of the RET/PTC1 oncogene is not restricted to the thyroid epithelium in vivo. Despite its ubiquitous expression, however, RET/PTC1 was able to induce only a limited number of tumor types; specifically mammary epithelium was affected by transgene expression, thus suggesting that RET/PTC1 is able to couple with transforming pathways specific for these glandular cells.

Age Factors↗

PubMed1996

A potential pathogenetic mechanism for multiple endocrine neoplasia type 2 syndromes involves ret-induced impairment of terminal differentiation of neuroepithelial cells.

Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growth-inhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.

Adrenal Gland Neoplasms↗

PubMed1996

Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice.

Gene rearrangements activating the RET proto-oncogene are frequently associated with human thyroid carcinomas belonging to the papillary subtype. These arrangements cause the fusion of the tyrosine-kinase domain of RET to the 5'-terminal region of different genes creating the RET/PTC chimeric oncogenes. Here we report the generation of transgenic mice lines expressing the RET/PTC1 oncogene under the control of the thyroid-specific rat thyroglobulin promoter. RET/PTC1-transgenic mice developed thyroid tumors displaying the histological aspect of papillary carcinomas. These tumors were slowly progressive and did not cause premature death of the animals. Two additional mice developed areas of thyroid hyperplasia. Immunohistochemical and reverse-transcriptase polymerase chain reaction analyses confirmed the thyroid-specific expression of the transgene. Given the frequency of activating rearrangements of RET in human papillary thyroid carcinomas we conclude that this animal system could be a good model for studying the neoplastic progression of thyroid carcinomas.

Animals↗

PubMed1996

Modifications occur at different structural levels during the heat denaturation of beta-lactoglobulin.

Heat-induced modifications in the tertiary and quaternary structure of beta-lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near-ultraviolet-CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65-70 degrees C. Addition of iodoacetamide during the heating/cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein. Reaction of the native beta-lactoglobulin dimer with iodoacetamide or dithiobis(2-nitrobenzoic acid) was only observed upon heating at temperatures higher than 40 degrees C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate-limiting step in the reaction of beta-lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (Ea = 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive species in heat-treated beta-lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like free monomer that has an exposed sulfhydryl group on its surface.

Circular Dichroism↗

PubMed1996

Synthesis and structural characterization of 6I,6II-diamino-6I,6II-dideoxy-cyclomaltoheptaose, a difunctionalized beta-cyclodextrin.

6I,6II-Diamino-6I,6II-dideoxy-cyclomaltoheptaose was prepared using the regioselective procedure described by Tabushi. The difunctionalized beta-cyclodextrin crystallizes as hexadecahydrate in the orthorhombic space group P2(1)2(1)2(1), with a = 11.395(3), b = 32.989(9), c = 17.560(5) A, V = 6601 A3, Z = 4. The structure was solved by molecular replacement techniques using the program PATSEE and was refined to a conventional final R = 0.058 for the 5031 observed reflections with I > or = 3 sigma(I). The beta-CD macrocycle presents only slight differences with respect to uncomplexed hydrated or methylated beta-CD. The macrocycle structure maintains an approximate seven-fold symmetry. The round shape of the cyclodextrin ring is stabilized by intramolecular O-H ... O H-bonds between the secondary hydroxyl groups of neighbouring glucose residues. Along the a axis, the beta-CD molecules are arranged in columns; the macrocycles form a herring-bone pattern, so that the cavity of each beta-CD molecule is closed at each end by neighbouring molecules. The macrocycles are directly linked to each other by H-bonds involving either primary and secondary hydroxyl or amino groups of symmetry-related molecules. The resulting layers are connected to each other by a dense intermolecular hydrogen-bond network, in which solvent molecules participate.

Carbohydrate Sequence↗

PubMed1996

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41.

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577-599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization.

Amino Acid Sequence↗

PubMed1996

Fourier-transform infrared conformational study of bovine insulin in surfactant solutions.

To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier-transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23-30) octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600-1700 cm-1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a beta-sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (> or = 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar.

Animals↗

PubMed1996

Presence of physiologically stimulated RET in adult rat brain: induction of RET expression during nerve regeneration.

The product of the RET proto-oncogene is a protein belonging to the receptor-like tyrosine kinase superfamily. RET is expressed in several neural crest-derived cell lineages and has been implicated in the correct development of the peripheral nervous system. To gain further insight into RET function, we investigated the presence of active RET in adult rat tissues. We show, by immunoblotting, that the products of the RET proto-oncogene (p155ret) are present in specific regions of adult rat brain, including the cerebellum, striatum, brainstem, hypothalamus, hippocampus, and olfactory bulb. Moreover, in the cerebellum, p155ret is phosphorylated in tyrosine residues, thus indicating that this brain structure contains p155ret in an activated state. Finally, the presence of RET in motoneurons prompted us to analyze the effects of hypoglossal nerve section on its expression. We observed a dramatic increase in p155ret in the motoneuron nuclei, thus suggesting that RET tyrosine kinase plays a role in the neuronal response to axotomy and/or during nerve regeneration.

Animals↗

PubMed1996

Transgenic mice carrying the human KRAS oncogene under the control of a thyroglobulin promoter: KRAS expression in thyroids analyzed by in situ hybridization.

We have previously generated transgenic mice bearing a molecular construct obtained by fusing the rat thyroglobulin promoter with the human Kirsten ras oncogene (KRAS). These mice showed thyroid abnormalities, although at very low incidence and after a long latency period. A six-month thyrotropin (TSH) stimulation of thyroid glands, followed by a two-month suspension, induced a significant increase in the number of lesions in transgenic mice as compared with a nontransgenic control group. Our goal was to follow the progression and the reversion of the tumorigenesis process in relationship with the levels of expression of the KRAS in this experimental model. In situ hybridization was used to detect expression of KRAS mRNA in sections of thyroids of the various groups of mice. A positive hybridization was observed in follicular cells of TSH-stimulated transgenic mice, whereas no expression could be appreciated in control nontransgenic mice. A positive signal was also observed in thyroid glands excised from transgenic mice after the 2-month suspension of treatment; however, the number of expressing cells was decreased compared with transgenic mice killed immediately after 6 months of a goitrogen regimen. Finally, every cell in the single thyroid carcinoma observed after the two-month suspension was positive for the transgene mRNA. This study further strengthens the role of the expression of mutated KRAS in the early stages of thyroid follicular cell transformation and indicates that when the expression of the mutated KRAS becomes independent of exogenous TSH stimulation, this event coincides with a further progression towards tumorigenesis.

Adenoma↗

PubMed1996

[Colorectal obstruction caused by cancer].

The authors report their experience, matured in 10 years, in the management of cancer of the rectum and large bowel complicated by obstruction; 425 patients out of 493 total cases of colorectal cancer presented an intestinal obstruction and underwent emergency surgery. In 386 cases it was possible to perform resection (91%). The surgical treatment of the patients with right colon obstruction usually consisted of a right hemicolectomy. The surgical treatment of left colon obstruction is still controversial; in the experience of the authors it was accomplished by Hartmann operation mainly until 1985; in recent years the authors have introduced a procedure of intraoperative anterograde irrigation of the colon and they have performed resection-anastomosis in a single stage successfully but only in selected patients. In 4 out of twelve cases of cancer of the splenic flexure a subtotal colectomy was performed with one stage ileo-sigmoid anastomosis crowned with success. The authors examine the operative and postoperative mortality of the patients with colorectal cancer and point out that 5 years survival is worse in patients with colorectal obstruction compared to elective operations (24% versus 41%).

Adult↗

PubMed1996

[Leiomyosarcoma of the small intestine and the colorectum. A case contribution].

Smooth muscle tumors of the alimentary tract are uncommon. A retrospective study was made of 7 patients treated for leiomyosarcoma of the small and large bowel to identify prognostic factors that influence results. The symptoms associated with these tumors varied according to the anatomic sites of the lesions and the position of the growth in relation to the intestinal lumen but the most common presenting clinical signs are bleeding or obstruction. The differential diagnosis between benign and malignant smooth muscle tumors is sometimes quite difficult. Clinical behavior of the myosarcomas of the gastrointestinal tract can be predicted to a large extent by the site of the tumor, the presence or absence of invasion of adjacent vital organs, and the histopathologic grade of malignancy. Although the mitotic activity of a gastrointestinal stromal tumor remains the most critical prognostic factor, tumors have been seen to recur locally and to metastasize even with rare or absent mitotic figures. Further studies are needed to pinpoint the factors that may be correlated to the prognosis.

Adult↗

PubMed1995

Mutated human Kirsten ras, driven by a thyroglobulin promoter, induces a growth advantage and partially dedifferentiates rat thyroid epithelial cells in vitro.

We have earlier shown that expression of the human activated Ki-ras, directed by the rat thyroglobulin (TG) promoter in the thyroid gland of transgenic mice, is able to induce thyroid benign tumors, albeit at low incidence. A likely explantation of our results is that the low levels of exogenous Ki-ras transcripts are not sufficient to induce multifocal tumors in the thyroid gland. We have performed experiments to analyze the effects of a similar construct in vitro upon thyroid-cell proliferation and differentiation. Transfection of FRTL-5 rat thyroid cells with the human Ki-rasval12 fused to the rat TG promoter is rapidly followed by reduced expression of the differentiation markers thyroglobulin, thyroperoxydase and thyrotropin receptor, but not by fully malignant cell transformation. The data reported support the hypothesis that Ki-ras mRNA levels are critical to the process of complete neoplastic transformation of thyroid epithelial differentiated cells in vitro.

Amino Acid Sequence↗

PubMed1995

Dbl expression driven by the neuron specific enolase promoter induces tumor formation in transgenic mice with a p53(+/-) genetic background.

The dbl oncogene, generated by the truncation of the amino-terminal portion of the proto-oncogene sequence, encodes a guanine-nucleotide-releasing factor. The transforming activity of this oncogene has never been demonstrated in vivo or in vitro except in the NIH 3T3 mouse fibroblast cell line. The expression of the proto-dbl transcript is confined to tissues and tumors of neuroectodermal derivation. Therefore, to study the transforming activity of the dbl oncogene in vivo, we have generated transgenic mice that express this oncogene in neuroepithelial tissues. Mice carrying the dbl oncogene did not develop a tumor. Successively, to establish whether dbl interacts with the tumor suppressor gene p53 in tumorigenesis, we have used a p53 deficient mouse strain. The results reported here indicate that dbl is capable of causing tumor formation in vivo when its expression is driven in an appropriate cellular and genetic environment.

Animals↗