Biomedical subjects

R Graziani

Publications and source records attributed to R Graziani.

At least 19 recordsLinked to original sources

PubMed2002

Synthesis, characterization and in vitro cytotoxicity of new organotin(IV) derivatives of N-methylglycine.

The synthesis and characterization of new coordination compounds of some diorganotins(IV) with N-methylglycine (sarcosine) are reported; all these derivatives mainly tend to assume a chelate structure. As single crystals were not obtained, a large number of experimental techniques were used to accomplish a definitive characterization and determination of their structure. Results obtained by (1)H/(119)Sn NMR, FT-IR and (119)mSn-Mössbauer spectroscopy and thermogravimetric analysis allow us to deduce the pentacoordination for 1:1 (Sn/sarcosine) derivatives [R(2)SnCl(2)(Sar)](+)Cl(-) (R=Me, n-Bu) in a trigonal-bipyramidal structure, and the hexacoordination for 1:2 complexes [R'(2)Sn(Sar)(2)](2+)2Cl(-) (R'=Me, n-Bu, Ph) in an octahedral structure; however, the probability of partially or totally non-chelate structures for some adducts increases with the steric hindrance of the R/R' groups and the number of the sarcosine molecules bound to the tin atom, so that they give rise to fluxional equilibria in solution. Finally, the synthesized compounds have been tested for in vitro cytotoxic activity against human adenocarcinoma HeLa cells showing, in some cases, strong activity even at low concentration.

Antineoplastic Agents↗

PubMed2002

Spiral computed tomography assessment of resectability of pancreatic ductal adenocarcinoma: analysis of results.

BACKGROUND: Despite more recent technical advances, single detector spiral computed tomography is still the most widely used imaging technique for the detection and staging of pancreatic adenocarcinoma. Many reports have recently focused on single detector spiral computed tomography imaging findings indicative of unresectability AIM: To evaluate the ability of single detector spiral computed tomography in the pre-operative staging of ductal adenocarcinoma of the pancreas in selecting surgically resectable versus unresectable cases, considering different parameters (vascular and local infiltration, liver metastases, lymphadenopathy, and peritoneal carcinomatosis). PATIENTS AND METHODS: A total of 100 patients diagnosed with ductal adenocarcinoma of the pancreas, pathologically verified, and examined with single detector spiral computed tomography were considered. Of these, 63 underwent surgery; 37 were excluded (imaging evidence of unresectability in biopsy-proven adenocarcinomas). Single detector spiral computed tomography studies, performed at the time of surgery, were retrospectively reviewed by two radiologists. The final results were reached by consensus, without being aware of the operation. All prospective clinical readings were also considered, and tabulated. RESULTS: In the patients undergoing surgery, 44 proved to be unresectable and 19 resectable. Considering the above-mentioned parameters, the prospective assessment of resectability demonstrated a rather low sensitivity (68%), with high specificity (95%). Accordingly, a high positive predictive value (97%) was achieved, while the negative predictive value was 56%. After the retrospective evaluation, the value of sensitivity increased (84%) to the expense of the specificity (84%). Moreover, a positive predictive value of 92.5%, and a negative predictive value of 69.5% were obtained. CONCLUSIONS: The demonstration of many signs of unresectability of pancreatic ductal adenocarcinoma reported in the literature yields a better sensitivity in the diagnosis, but unfortunately, is associated with an inevitable reduction in specificity.

Adenocarcinoma↗

PubMed2001

Platinum(II) and palladium(II) complexes with dithiocarbamates and amines: synthesis, characterization and cell assay.

The [M(ESDT)Cl]n (M = Pd or Pt; ESDT = EtO2CCH2(CH3)NCS2, methylamino-acetic acid ethyl ester-dithiocarboxylate) species have been reacted with various amines (py, pyridine; PrNH2, n-propylamine; c-BuNH2, cyclobutylamine; en, ethylenediamine) in dichloromethane or chloroform with the aim to obtain mixed ligand complexes. The neutral complexes [M(ESDT)(L)Cl] (L = py, PrNH2 or c-BuNH2) and the ionic species ([M(ESDT)(L)2]Cl and [M(ESDT)(En)]Cl) have been isolated, and characterized by IR and proton NMR spectroscopies. The crystal structure of [Pd(ESDT)(PrNH2)Cl] has been determined by X-ray crystallography. The behaviour of the complexes in various solvents was described on the basis of the proton NMR spectra. The complexes and the dithiocarbamato intermediates have been tested for in vitro cytostatic activity against human leukemic HL-60 and HeLa cells.

Amines↗

PubMed2001

CT features of malignant mucinous cystic tumors of the pancreas.

This study is aimed at evaluating which pre-operative findings at CT are prevailingly associated with histologically malignant appearance of mucinous cystic tumor (MCT) of the pancreas. The CT examinations of 52 female patients affected with pathologically proved MCT were retrospectively evaluated by two radiologists, blinded to the histopathological assessment of the biologic behavior (either benign or malignant). A multivariate logistic regression analysis was performed in order to identify the most important features associated with the malignant nature of MCT. Calcifications in the wall and/or in the septa, thick wall, and septations resulted in the most important features associated with malignancy. The simultaneous presence of all these radiological signs constitutes an almost "certain" marker of malignancy, being the risk equal to 0.95, whereas the simultaneous presence of at least two of them entails a risk of malignancy ranging from 0.56 to 0.74, according to the type of morphological features. On the other hand, the absence of the mentioned radiological features entails a negligible risk of malignancy (0.02). Mucinous cystic tumors characterized by multilocular macrocystic architecture, with thick wall and calcifications in the wall and/or the septa, present the highest risk of malignancy, and advocate prompt surgical intervention. When those signs are absent, the probability of malignancy is lower. In this case a non-operative management is possible.

Adult↗

PubMed1998

Surgical treatment of pancreatic metastases from renal cell carcinomas.

Pancreatic metastases from a renal cell carcinoma are rare and may occur long after manifestation of the primary disease. Resection of the metastases should be regarded as the best treatment. In our center, owing to the slow evolution of these secondaries, we perform resections capable of limiting the destruction of the pancreatic parenchyma as far as possible. The use of 'atypical' resections of the pancreas is characterized by a higher incidence of postoperative complications, particularly fistulas. Despite this, we believe that adjusted resection is to be advocated because of the possibility of additional remote secondaries, the shorter duration of surgery, the preservation of the glandular parenchyma and intact adjacent organs, such as duodenum, stomach, and spleen, and the fact that there have been no reports on local recurrences.

Aged↗

PubMed1997

Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF alpha-receptor.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.

Antigens, CD↗

PubMed1997

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Animals↗

PubMed1997

Blunt pancreatic trauma. Role of CT.

PURPOSE: To define the evolution patterns of blunt pancreatic trauma, and to point out the CT features most significant for the diagnosis. MATERIAL AND METHODS: Ten cases of pancreatic trauma, observed over a period of about 10 years, were analyzed in retrospect. The cases were divided into 3 groups according to the time that had elapsed between trauma and first CT: early phase (within 72 h: n=3/10); late phase (after 10 days: n=3/10); and following pancreatic drainage (n=4/10). RESULTS: In the early phase, one case showed a blood collection surrounding the pancreatic head and duodenum, and displacing the mesenteric vessels to the left. In the 2 other cases it was possible to demonstrate a tear in the pancreas at the neck, perpendicular to the main pancreatic axis. In the late phase in all 3 cases, one cystic lesion was present at the site of the tear, either surrounding the gland or embedded - more or less deeply - within the parenchyma. One of the lesions subsided spontaneously; the 2 others required surgery. In the postoperative phase, an external fistula was demonstrated in 2 cases following percutaneous drainage of pancreatic cysts; the fistula was fed by a cystic lesion in the pancreatic neck. In the 2 other cases a pseudocyst developed. CONCLUSION: Early demonstration of a parenchymal tear was difficult. At a later stage the diagnosis was easier owing to the demonstration of cystic lesions within the parenchyma at the site of the tear. The surgical drainage of this lesion does not usually lead to healing since an external fistula or a pseudocyst may develop.

Adult↗

PubMed1997

Cystic dystrophy of the duodenal wall: radiologic findings.

PURPOSE: To determine the radiologic characteristics of cystic dystrophy of the duodenal wall. MATERIALS AND METHODS: Ten patients with cystic dystrophy of the duodenal wall and chronic pancreatitis underwent ultrasonography (US) (n = 10), computed tomography (CT) (n = 10), endoscopic US (n = 5), and endoscopic retrograde cholangiopancreatography (ERCP) (n = 9). Cystic dystrophy of the duodenal wall was classified as either cystic or solid. The imaging findings were retrospectively analyzed and compared with findings at pancreatoduodenectomy (n = 10). RESULTS: The more frequent cystic type (n = 7) of cystic dystrophy of the duodenal wall was characterized by the presence of easily recognizable cystic lesions (diameter, more than 1 cm), located within the thickened wall of the second portion of the duodenum. The solid type (n = 3) of cystic dystrophy of the duodenal wall demonstrated fibrous thickening of the duodenal wall within which small cysts (diameter, less than 1 cm) were present. The intraduodenal cysts were usually elongated or bilobate with a thick wall. The thickening of the duodenal wall appeared as a solid layer between the duodenal lumen and the pancreas, hypoechoic at US, isoattenuating at unenhanced CT, and hypoattenuating in the early phase (after initiation of infusion of contrast material) and isoattenuating in the late phase (after completion of infusion) at contrast material-enhanced CT. Findings at retrospective analysis of CT and endoscopic US images were characteristic. CONCLUSION: Imaging modalities, notably CT and endoscopic US, helped establish the diagnosis of cystic dystrophy of the duodenal wall.

Adult↗

PubMed1996

Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130.

The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.

Animals↗

PubMed1996

Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.

Amino Acid Sequence↗

PubMed1996

Intraductal mucin-producing tumors of the pancreas: imaging findings.

PURPOSE: To evaluate the radiologic characteristics of intraductal mucin-producing tumors of the pancreas. MATERIALS AND METHODS: Sixteen patients with intraductal tumors underwent ultrasound (US); (n = 15), computed tomography (CT); (n = 16), endoscopic retrograde cholangiopancreatography (ERCP); (n = 12), and intraoperative pancreatography (n = 2). Findings were compared with those from surgery (n = 14) or biopsy (n = 2). RESULTS: Lesions were classified as either main duct type or branch duct type tumors. Main duct tumors were characterized at US and CT by either diffuse or segmental dilatation of the Wirsung duct. Pancreatography showed ductal dilatation and filling defects caused by mucin deposits. At US and CT, branch duct tumors, which were mainly located at the uncinate process, were seen as fluid-filled masses with central septa and the pancreatic duct was dilated. ERCP showed partial or complete opacification of the lesion. In four patients, endoscopy showed protrusion of the papilla into the duodenal lumen and mucin leaking from its dilated orifice. CONCLUSION: Imaging modalities, especially US and ERCP, enable early diagnosis of mucin-producing pancreatic tumors.

Adenocarcinoma, Mucinous↗

PubMed1995

Definition of a composite binding site for gp130 in human interleukin-6.

The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.

Antigens, CD↗

PubMed1995

In vitro binding of ciliary neurotrophic factor to its receptors: evidence for the formation of an IL-6-type hexameric complex.

Ciliary neurotrophic factor (CNTF) is a cytokine sharing structural and functional similarities with interleukin-6 (IL-6) and other helical cytokines that utilize the common signalling chain gp130. While IL-6 induces gp130 dimerization, CNTF, after the initial interaction with the specific, non-signalling receptor subunit, CNTFR, induces the formation of gp130/LIF-receptor heterodimers. Through immunoprecipitation experiments with tagged soluble receptor molecules, we recently demonstrated that IL-6 drives the formation of a hexameric receptor complex with a defined topology and composed of two IL-6, two IL-6R alpha and two gp130 molecules. Here, we apply the same strategy to study the assembly in vitro of the CNTF receptor complex. We present evidence that both the cytokine and the specific binding chain undergo dimerization in the presence of gp130. Furthermore, although gp130 and LIFR are able to bind independently to the CNTF/CNTFR sub-complex, they never form homodimers but only heterodimers. We propose that CNTF assembles a hexameric receptor complex composed of two CNTF, two CNTFR, one gp130 and one LIFR molecule, and present a model of the reciprocal interaction of these molecules based on similarities with the IL-6 hexameric complex.

Amino Acid Sequence↗

PubMed1995

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.

Amino Acid Sequence↗

PubMed1994

Oncostatin M binds directly to gp130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors.

Oncostatin M (OM) and interleukin 6 (IL-6) are functionally related cytokines, which trigger similar biological responses because they share gp130 as a common signal transducing transmembrane receptor. While IL-6 recruits gp130 only upon binding to its specific receptor subunit (IL-6R alpha), reconstitution and cross-linking experiments on cell membranes suggest that OM can directly interact with gp130 and that this interaction is necessary but not sufficient to stimulate cells. However, the issue of the direct binding between gp130 and OM, in the absence of any additional membrane component, remained essentially unclarified. In this paper we show that, uniquely among the family of cytokines that transduce through gp130, OM directly binds in vitro with a 10(-8) M affinity sgp130, a soluble form of gp130. Moreover, titration of sgp130 with OM inhibits the formation of a ternary complex comprising IL-6, sIL-6R alpha, and sgp130. These in vitro properties of OM are consistent with the additional finding that on human hepatoma Hep3B cells, which express gp130 but not functional OM receptors, OM does not mimic IL-6 activity, but rather behaves, at high doses, as an IL-6 antagonist.

Animals↗

PubMed1994

Nonradioactive receptor binding assay for ciliary neurotrophic factor.

A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.

Amino Acid Sequence↗

PubMed1994

Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli.

Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells. After the addition of a six histidine tag at the N-terminus, the coding region of mature Onc M was cloned in the pT7.7 expression vector. Histidine tagged Onc M was overexpressed in bacterial cells and purified to homogeneity in one step on a metal chelating column. We found that recombinant 6xHis-OncM remains fully active in a growth inhibition assay. Structural characterization of the purified protein was performed by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry. Thermal and pH stability dependence of Onc M was assessed by circular dichroism spectroscopy; the helical content is about 50%, in agreement with the four helix bundle fold postulated for cytokines that bind haematopoietic receptors of type I.

Amino Acid Sequence↗