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S Giordano

Publications and source records attributed to S Giordano.

At least 73 records · Page 4Linked to original sources
PubMed1994

A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family.

Signaling by tyrosine kinase receptors is mediated by selective interactions between individual Src homology 2 (SH2) domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. Here, we report the existence in the hepatocyte growth factor/scatter factor (HGF/SF) receptor of a multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV. Phosphorylation of this site mediates intermediate- to high-affinity interactions with multiple SH2-containing signal transducers, including phosphatidylinositol 3-kinase, phospholipase C gamma, pp60c-src, and the GRB-2-Sos complex. Mutation of the two tyrosines results in loss of biological function, as shown by abrogation of the transforming activity in the oncogenic counterpart of the receptor. The same bidentate motif is conserved in the evolutionarily related receptors Sea and Ron, suggesting that in all members of the HGF/SF receptor family, signal transduction is channeled through a multifunctional binding site.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Generation of a truncated hepatocyte growth factor receptor in the endoplasmic reticulum.

The hepatocyte growth factor (HGF) receptor (p190MET) is a tyrosine kinase composed of two disulfide-linked chains, alpha of 50 kDa and beta of 145 kDa. We have previously described an isoform (p140MET) containing a beta chain of 85 kDa, lacking the cytoplasmic kinase domain. The two receptor variants originate by post-translational processing of a common single-chain precursor of 170 kDa (Pr170). In the endoplasmic reticulum a fraction of Pr170 is cleaved at the cytosolic side generating an intermediate product of 120 kDa (Pr120). This molecule 1) is already detectable after 15 min of pulse labeling, 2) contains high mannose-branched oligosaccharides, and 3) accumulates upon treatments inhibiting the export from the endoplasmic reticulum. A second cleavage, occurring after 30 min of chase in the trans-Golgi network, converts the single-chain precursors Pr170 and Pr120 into the mature heterodimers p190MET and p140MET. This process is inhibited by brefeldin A treatment. Conditions leading to Pr170 accumulation in the endoplasmic reticulum, such as receptor overexpression, induce kinase activation and overproduction of Pr120. Conversely, cells expressing a kinase-defective HGF receptor lack the truncated isoform. The proteolytic cleavage of the cytoplasmic domain may thus represent a safety mechanism aimed at preventing ligand-independent intracellular activation of the HGF receptor kinase.

3T3 Cells↗
PubMed1994

Molecular characterization of fish neurolin: a growth-associated cell surface protein and member of the immunoglobulin superfamily in the fish retinotectal system with similarities to chick protein DM-GRASP/SC-1/BEN.

We have used the polymerase chain reaction to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis demonstrates that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion molecule DM-GRASP, SC-1, BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish. Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two are shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion molecule, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1994

Cloning of multiple forms of goldfish vimentin: differential expression in CNS.

In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal lambda gt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin alpha and vimentin beta. RNase protection assays indicate that vimentin alpha mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin beta mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin alpha is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.

Amino Acid Sequence↗
PubMed1994

Effects of defibrotide on physical performance and hemorheologic picture in patients with peripheral arteriopathy.

In a random double-blind study versus placebo, 60 ambulatory patients with peripheral occlusive disease of the lower limbs and claudicatio intermittens (Leriche's stage 2), were treated for 60 days with defibrotide (400 mg b.i.d., oral, n = 30) or placebo (n = 30). Patients in the defibrotide group received additional treatment with the same drug at the reduced rate of 400 mg once daily for another 120 days for maintenance (total treatment duration 180 days). All patients were assessed at intake and 60 days for relative and absolute walking distance (RWD and AWD) in a standard treadmill test and for the Winsor Index (WI) at rest and after exercise; patients of the defibrotide treatment group were retested in the same way at 90-180 days. In a subgroup of patients (defibrotide = 11, placebo = 12), blood samples were obtained for the assessment of whole blood and plasma viscosity at intake and after 60 days of treatment. These samples could not be collected properly in the remaining cases, for technical reasons. At day 60, we compared the effects of the two treatments on physical performance: mean (SE) values of RWD were for defibrotide 148 (9.7) and 179 (12.4) m in basal and post-treatment conditions, respectively, and 209 (16.2) and 212 (17.1) m for placebo. Similar changes were observed for AWD: for defibrotide 206 (13.4) and 241 (15.2) m and for placebo 270 (22.9) and 272 (23.1) m. The mean changes were significantly larger with defibrotide: for RWD + 33 (7.1) vs. + 0.3 (3.8) m (p < 0.01) and for AWD + 34 (9.2) and -2 (6.6) m (p < 0.01). The overall gain of walking distance after maintenance therapy with the reduced defibrotide dosage amounted to approximately + 50% over basal (after 180 days). Blood and plasma viscosity improved in patients on defibrotide but the change fell short of statistical significance versus placebo. All findings confirm the potential usefulness of defibrotide in the treatment of peripheral arterial disease, at the same time encouraging further studies of the involved mechanisms of action.

Aged↗
PubMed1994

Structural and functional domains critical for constitutive activation of the HGF-receptor (Met).

The MET gene, encoding the tyrosine kinase receptor for Hepatocyte Growth Factor, is a potentially harmful oncogene overexpressed in a significant fraction of human cancers. To study the molecular mechanisms responsible for oncogenic activation, the biochemical and biological properties of a number of MET constructs were analysed. The native heterodimeric receptor (alpha beta), the beta chain alone, as well as a kinase defective mutant did not transform rodent fibroblasts upon transfection. The cytoplasmic domain, truncated immediately below the transmembrane region, acquired constitutive tyrosine kinase activity in vivo, produced foci of transformation, and was tumorigenic in nude mice. Removal of the first 39 amino acids of the juxtamembrane domain resulted in loss of constitutive activation in vivo and transforming potential, without impairment of the in vitro kinase activity. Replacement of the juxtamembrane domain with 5' TPR sequences restored constitutive kinase activation and transforming properties. Site-directed mutagenesis of either of the two tyrosine residues involved in the positive regulation of the catalytic activity upon phosphorylation (Y1234 or Y1235 in the kinase domain of the HGF receptor), strongly impaired TRP-MET transforming potential. These data show that: (1) the truncated cytoplasmic HGF receptor has constitutive kinase activity and is oncogenic; (2) the first 39 amino acids of the juxtamembrane domain and (3) the regulatory tyrosines in the catalytic domain are required to unleash its transforming potential.

3T3 Cells↗
PubMed1993

Basic fibroblast growth factor expression is required for clonogenic growth of human glioma cells.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein, expressing potent mitogenic and angiogenic properties. Elevated levels of bFGF have been identified in human gliomas and glioma cell lines, suggesting that bFGF expression is involved in the aberrant growth patterns associated with these tumors. In the present study, the influence of bFGF on additional parameters of glioma cell malignancy was evaluated utilizing three distinct methods to suppress bFGF expression or activity including antisense oligonucleotide primers, a neutralizing monoclonal antibody or an inhibitor of the agonist action of bFGF: (1) The addition of 30 microM bFGF-specific antisense oligonucleotide primer to the human glioma cell line SNB-19 resulted in a 55% inhibition in colony formation in soft agar. This effect was dose-dependent and specific, as sense strand primer was ineffective in suppressing growth. In addition to exhibiting fewer colonies, antisense treatment significantly altered colony morphology. (2) SNB-19 cell growth in culture was suppressed in the presence of a neutralizing bFGF-specific monoclonal antibody. (3) Inositolhexakisphosphate, a newly identified antagonist of FGF binding and activity, suppressed SNB-19 cell growth in soft agar culture. These results demonstrate that bFGF may regulate glioma growth and progression independent of its role in tumor angiogenesis and that bFGF release or secretion may be required for these actions.

Astrocytes↗
PubMed1993

Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene.

The MET protooncogene encodes p190MET, a tyrosine kinase which is the receptor for a molecule known as scatter factor or hepatocyte growth factor (SF/HGF). This molecule has different biological activities, including stimulation of cell motility, promotion of matrix invasion and, in some cells, mitogenesis. We have cloned the full-length MET cDNA and transfected it into NIH 3T3 fibroblasts. Stable transfectants expressed the p190MET receptor together with two previously described truncated forms of 140 and 130 kDa lacking the tyrosine kinase domain. All three forms bound radiolabeled SF/HGF. The factor stimulated tyrosine kinase activity of the transfected p190MET and induced changes in cell shape, migration in Boyden chambers, and invasion of collagen matrices in vitro. The motile and invasive phenotype was transient and strictly dependent on the presence of SF/HGF. The factor did not stimulate either cell growth or thymidine incorporation in transfected cells, while it promoted colony formation in soft agar in the presence of 5% fetal calf serum. These data show that, in the presence of its ligand, the MET receptor expressed in fibroblasts induces cells to pursue a motogenic-invasive rather than a proliferative program.

3T3 Cells↗
PubMed1992

Limb anomalies following chorionic villus sampling: a registry based case-control study.

Using data from the Italian Multicentric Birth Defect Registry a case-control study was performed to verify if chorionic villus sampling (CVS) was associated with transverse limb defects (TLD), with or without features of oro-mandibular-limb hypogenesis complex (OMLHC), in the exposed offspring. The results show that the risk of TLD and OMLHC is increased following CVS, and is particularly high for CVS performed early in pregnancy, i.e., under 70 days of gestational age. These results, together with a review of other epidemiologic studies, biological data and clinical reports, strongly suggest a causative role of CVS as a risk factor for TLD and indicate that at this stage CVS before 70 days of gestational age should be discouraged as an option for prenatal diagnosis and that all patients wishing to undergo CVS should be informed about the possible risk of the procedure.

Abnormalities, Multiple↗
PubMed1992

Multiple molecular weight forms of basic fibroblast growth factor are developmentally regulated in the central nervous system.

Basic fibroblast growth factor (bFGF) is a heparin-binding protein implicated in the differentiation, proliferation, and maintenance of cells in the central nervous system (CNS). It is not clear how bFGF achieves this multiplicity of effects. Multiple molecular weight forms of bFGF have recently been identified, however, and each form may have distinct activities during CNS development. We have examined the pattern of bFGF expression during CNS development using protein immunoblot and RNA blot analyses. RNA blot analysis detected a major bFGF transcript of 3.7 kb in embryonic and adult rat brain; however, this message decreased in abundance during development. Three bFGF protein forms were identified on immunoblots of adult rat brain extract with approximate molecular weights of 18, 21, and 22 kDa. Embryonic rat brain extracts also contained the 18- and 21-kDa bFGF protein forms, but lacked the 22-kDa form. Expression of the 22-kDa form was first detected in the neonate and then steadily increased to adult levels by 1 month of age. Immunoblots of adult human brain extracts also showed the presence of three bFGF protein forms with approximate molecular weights of 18, 22, and 24 kDa. In human second trimester fetal brain extracts, only the 18-kDa bFGF protein was detected. Comparison of bFGF proteins in developing rat spinal cord, cerebellum, and cortex demonstrated that distinct patterns of bFGF protein forms exist in different regions of the CNS. Therefore, the expression of individual bFGF protein forms is regulated in the CNS with regard to both developmental stage and location. These data support the idea that different forms of bFGF may be associated with specific developmental events during the maturation and organization of the nervous system.

Amino Acid Sequence↗
PubMed1992

Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene.

MKN 45 is a poorly differentiated gastric carcinoma cell line from which the subclone GTL 16 was obtained. Both lines carry an amplification unit derived from chromosome 7 sequences and containing an activated c-met oncogene. Karyotypic analysis showed that GTL 16 derived from a subclone of MKN 45 after endoreduplication. Several clonal abnormalities are evident in both lines; some are frequently observed in gastrointestinal tumors (loss of 17p and monosomy 18). Other consistent anomalies include 6q-, t(8;10) and t(5;8), and inv(16). A marker chromosome (M1), which was previously shown to contain the c-met amplification unit, is constantly duplicated in all GTL 16 metaphases; in contrast, most unidentified markers are retained in only a single copy in GTL 16 cells. These data are in agreement with the hypothesis that the c-met oncogene activation in these gastric cancer cell lines might be related to a gene dosage effect.

Adenocarcinoma↗
PubMed1992

Pharmacokinetics of two oral liquid formulations of oxatomide in children.

A study on the pharmacokinetics of two suspensions of oxatomide at the concentrations of 25 and 2.5 mg/ml was carried out on 12 children (7M, 5F) with a mean age of 9 years and weight of 29 kg. The plasmatic concentration peak, the time to reach the peak and the area under the curve displayed no significant differences between the two formulations. The maximum concentrations and the peak time of oxatomide in children are similar to those observed in adults.

Administration, Oral↗
PubMed1991

C-terminal truncated forms of Met, the hepatocyte growth factor receptor.

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. p190MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p50 alpha) and 145 kDa (p145 beta). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (p140MET) is membrane bound and is composed of an alpha chain (p50 alpha) and an 85-kDa C-terminal truncated beta chain (p85 beta). The second protein (p130MET) is released in the culture supernatant and consists of an alpha chain (p50 alpha) and a 75-kDa C-terminal truncated beta chain (p75 beta). Both truncated forms lack the tyrosine kinase domain. p140MET and p130MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. p140MET is preferentially localized at the cell surface, where it is present in roughly half the amount of p190MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.

3T3 Cells↗
PubMed1991

Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor).

The MET proto-oncogene encodes a 190-kDa disulfide-linked heterodimeric receptor (p190 alpha beta) whose tyrosine kinase activity is triggered by the hepatocyte growth factor. The mature receptor is made of two subunits: an alpha chain of 50 kDa and a beta chain of 145 kDa, arising from proteolytic cleavage of a single-chain precursor of 170 kDa (pr170). In a colon carcinoma cell line (LoVo), the precursor is not cleaved and the Met protein is exposed at the cell surface as a single-chain polypeptide of 190 kDa (p190NC). The expression of the uncleaved Met protein is due to defective posttranslational processing, since in this cell line (i) the proteolytic cleavage site Lys-303-Arg-Lys-Lys-Arg-Ser-308 is present in the precursor, (ii) p190NC is sensitive to mild trypsin digestion of the cell surface, generating alpha and beta chains of the correct size, and (iii) the 205-kDa insulin receptor precursor is not cleaved as well. p190NC is a functional tyrosine kinase in vitro and is activated in vivo, as shown by constitutive autophosphorylation on tyrosine. The MET gene is neither amplified nor rearranged in LoVo cells. Overlapping cDNA clones selected from a library derived from LoVo mRNA were sequenced. No mutations were present in the MET-coding region. These data indicate that the tyrosine kinase encoded by the MET proto-oncogene can be activated as a consequence of a posttranslational defect.

Amino Acid Sequence↗
PubMed1991

c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase.

The putative tyrosine kinase receptor encoded by the oncogene c-met is activated (tyrosine-phosphorylated in vivo) in the human gastric carcinoma cell line GTL-16. The corresponding gene is amplified and over-expressed. In this study we show that c-met is part of an amplification unit measuring more than 3000 kb. The multiple copies of the amplicon are located on a novel chromosome different from chromosome 7. We have previously shown that the c-met protein present in GTL-16 cells is indistinguishable from that found in other cells. Kinase activation could be due to over-expression of the normal c-met protein or to the presence of activating mutation(s). To verify the primary structure of the c-met protein in GTL-16 cells we sequenced a series of overlapping cDNAs obtained from GTL-16 cell RNA by reverse transcription and polymerase chain reaction. Two differences were found in the c-met coding region with respect to the published human c-met cDNA: (1) the lack of 54 nucleotides corresponding to a stretch of 18 amino acids located in the extracellular domain of the receptor, and (2) the substitution of the codon specifying alanine 1209 (located in the tyrosine kinase domain) with one coding for glycine. However, we also obtained cDNAs identical to that just described from a number of control cell lines. These results suggest: (1) that the present c-met cDNA presumably reflects the sequence of the most abundant transcript in several cell types, and (2) that over-expression of the normal c-met protein, alone or in combination with an autocrine loop, is most probably responsible for the activation of the c-met kinase in GTL-16 cells.

Base Sequence↗
PubMed1991

Expression of the Met/HGF receptor in normal and neoplastic human tissues.

The MET oncogene encodes a transmembrane tyrosine kinase receptor. Recently, hepatocyte growth factor (HGF), a potent growth factor for hepatocytes involved in liver regeneration, has been proposed as a ligand. In this paper, the physiological role of the human Met/HGF receptor is investigated by studying its specific distribution in normal and neoplastic tissues. Northern blot analysis has shown that the MET gene is selectively expressed in several epithelial tissues. High levels of MET mRNA have been found in liver, gastrointestinal tract, thyroid and kidney. Western blot analysis has shown that the levels of the Met protein generally correspond to those of the mRNA. However, in the thyroid, where there is a high level of MET mRNA, the protein was barely detectable, suggesting translational or post-translational regulation. The protein was also detected in the brain. Normal or increased levels of MET mRNA and Met protein were consistently found in fresh samples of carcinomas as well as in epithelial tumor cell lines. In thyroid carcinomas of a specific histiotype the amount of Met protein, almost undetectable in the normal counterpart, was found to be increased more than 100-fold. The tissue distribution of the Met/HGF receptor indicates that this molecule is involved in growth control of epithelial cells other than hepatocytes and suggests that its increased expression may confer a growth advantage to neoplastic cells.

Adrenal Glands↗