Biomedical subjects
Publications and source records attributed to G Graziani.
Fractal analysis is introduced in the field of environmental health physics. In particular, it is applied to the complex and inhomogeneous deposition pattern of radioactivity after the Chernobyl accident. The patchiness of 137Cs hot spots is quantified by a fractal dimension as low as 1. The problem of finding hot spots that might be of health concern is discussed.
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Fibroblast growth factors (FGFs) are polypeptide mitogens that induce the proliferation of a wide variety of cell types. Of the seven family members, the best characterized are basic and acidic FGF. In addition to their mitogenic effects, they participate in angiogenesis, differentiation and maintenance of survival of neurons, cell migration and embryonal development. Of all family members, keratinocyte growth factor (KGF) is unique in that it is a specific mitogen for epithelial cells and does not interact with the FGF receptor of fibroblasts. To study the interactions between KGF and its receptor, we isolated KGF and FGF receptors from keratinocytes and fibroblasts, respectively. In the course of this study, we isolated five different variants of the FGF receptor from human fibroblasts and showed that all were derived from a single genetic locus. Four of these variants encode transmembrane receptors and can be divided into two subgroups that differ from one another with respect to the number (two or three) of immunoglobulin (Ig)-like domains. Within each subgroup, one receptor differed from the other by the presence of a two-codon insertion. Thus, all the variations among the four isoforms are localized to their ligand binding domains. The fifth isoform encodes a molecule truncated just 3' to the first Ig-like domain and thus could be secreted from the cell. The transcripts encoding the long and short isoforms were found to be expressed in many cell types, but their relative levels of expression varied greatly depending on the cell type. These findings indicate that alternative splicing generates diverse FGF receptor isoforms in human cells.
Among the expression vectors, the baculovirus system has been successfully developed and it has been shown to be suitable as a helper-independent viral expression vector for high level of production of recombinant proteins in cultured insect cells. The high efficiency of this system derives from the viral strong promoter of the polyhedrin gene. Recombinant viruses containing foreign DNA inserted into the polyhedrin gene, will no longer produce polyhedrin and will form plaques morphologically different from the plaques produced by the wild type virus. We have expressed, at high level, the dbl and proto-dbl proteins using the baculovirus system. dbl and proto-dbl gene products produced in insect cells resulted to have the same post-translational modifications and subcellular localization observed in dbl and proto-dbl transfectants.
To study the transforming activity of the dbl oncogene and its effect on normal development in vivo, we linked dbl cDNA to promoters with different cell type specificities and used the constructs to generate transgenic mice. The promoters included the mouse alpha A-crystallin promoter, the rat insulin II promoter, and a mouse metallothionein promoter. We also generated transgenic mice carrying a recombinant cosmid clone that contains the entire dbl gene. Mice with the crystallin promoter construct developed cataracts and expressed the dbl protein in their lenses. The architecture of the lenses suggested a block to the normal pattern of differentiation and elongation of the secondary fiber cells. Mice with the insulin II promoter expressed dbl protein in the pancreas but showed no evidence of diabetes and no apparent pancreatic beta-cell defects. Similarly, mice with the metallothionein promoter expressed dbl protein in heart and testes, but showed no pathologic abnormalities in these tissues even after treatment with heavy metals. However, one family of mice carrying the metallothionein promoter construct showed cataracts and a dramatic fibroblastic dysplasia of the lens. One family with the cosmid-dbl gene showed a nearly identical lenticular dysplasia, but with a slower developmental time course. Thus, although the dbl oncogene did not induce neoplasia in any of the mice studied, it is apparently capable of interfering with the ability of the lens epithelial cells to differentiate into lens fiber cells, and of inducing metaplasia of the epithelial cells into fibroblastic cells.
Proto-dbl is a human proto-oncogene, whose oncogenic activation was initially detected by DNA transfection. We report significant sequence similarity between the predicted proto-dbl product and the products of CDC24, a Saccharomyces cerevisiae cell division cycle gene required for correct budding and establishment of cell polarity, and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia (CML). Of 925 residues of the predicted proto-dbl protein, a stretch of 238 residues showed 29% and 22% identity over a region of similar length of the CDC24 and bcr proteins, respectively. When evolutionarily conservative substitutions were taken into account, the similarities were 68.8% and 71.6% for proto-dbl/CDC24 and proto-dbl/bcr gene products, respectively. Moreover, all three sequences were predicted to be markedly hydrophilic over this region. Very small deletions within the conserved region completely abolished transforming activity of dbl, while extensive deletion outside of this region had no effect. Even substitutions over a small stretch of close similarity with the other proteins substantially impaired transforming activity. Cells transformed by the dbl oncogene, like cdc24 mutants arrested at the nonpermissive temperature, form multinucleate cells. Thus, our findings indicate that the conserved region is an essential domain that may reflect important functional similarities among these otherwise highly divergent molecules.
In a previous study we demonstrated that cyclosporin-treated renal transplanted patients have a reduced 1,25(OH)2D3 synthesis in comparison with azathioprine-treated transplanted patients. To assess the impact of this defect on intestinal calcium transport we compared the plasma calcium variation and the urinary calcium excretion in 14 cyclosporin-treated and in 12 azathioprine-treated patients, in fasting conditions and 4 hours after an oral calcium load (1 g). In ten cyclosporin patients we also correlated cyclosporin plasma values with plasma 1,25(OH)2D3 values before and after a 25(OH)D3 oral load. After the oral calcium load, plasma and urinary calcium increased significantly in the azathioprine group, while remaining unchanged in the cyclosporin group. A negative correlation between plasma concentrations of cyclosporin and the increment in 1,25(OH)2D3 after 25(OH)D3 oral load was also observed. Thus, our data suggest that cyclosporin impairs 1-alpha hydroxylase activity and alters the response to an oral calcium load.
We investigated the renal and humoral effects of short-term administration of ibopamine, an orally active dopamine agonist, in patients with liver cirrhosis. The patients were divided into two groups on the basis of sodium excretion with a constant sodium intake of 40 mEq/d. We also compared the effects of ibopamine with those induced by intravenous infusion of dopamine hydrochloride (3 micrograms/kg per minute) in similar patients. Ibopamine caused significant increases in urine output, glomerular filtration rate, and sodium excretion throughout the 4 hours of the trial in patients with basal sodium excretion rate greater than 20 mmol/d. These renal effects were associated with a significant reduction in plasma aldosterone concentration. In contrast, only a transient increase in glomerular filtration rate and a diminution in plasma aldosterone concentration were observed after ibopamine in the patients with a basal sodium excretion rate less than 20 mmol/d. The infusion of dopamine had renal effects similar to those of ibopamine in both groups of patients. These results indicate that in cirrhotic patients with normal sodium excretion, ibopamine exerts a diuretic and natriuretic effect similar to that of dopamine infusion. However, these properties of dopaminergic agents are apparently lost in patients with avid sodium retention.
Since 1987 we have treated 17 patients with bone sarcomas of the extremities (14 osteosarcomas, 3 malignant fibrous histiocytomas), with 2 cycles of neoadjuvant chemotherapy, consisting in iv high dose Methotrexate, Cis-Platin in 3 days in continuous infusion, and iv Adriamycin every 29th day. We obtained "good necrosis" (greater than 90%) in 13 patients (76.5%). We briefly report the possible correlations between angiographic findings after the first cycle and response to chemotherapy. All patients but one (94%), underwent conservative surgery. To date no patients have had local recurrence; 12 patients (70.5%) are disease free at a median follow up of 21.5 months; in 5 patients lung metastases appeared. To define overall survival and disease-free survival a longer follow up is required.
In 11 chronic haemodialysis patients we investigated whether the increase in haematocrit during recombinant human erythropoietin (rHuEPO) treatment might alter the long-term efficiency of haemodialysis. After correction of anaemia with rHuEPO (mean Ht 35 +/- 2% vs 19 +/- 2% at baseline) (p 0.001), mean predialysis creatinine and urea did not change, while predialysis phosphate (1.77 +/- 0.38 vs 1.51 +/- 0.29 mmol/l) were significantly increased (p 0.01). In six of the 11 rHuEPO treated patients a post- versus pre-dialysis haemoconcentration (haematocrit 44% vs 35%) not attributable to different ultrafiltration regimes, was observed. In these 6 patients mean predialysis phosphate, creatinine and urea tended to be higher, but not significantly, in comparison to he remaining 5 patients who did not haemoconcentrate. Dialyser clearances and total extractions for urea, creatinine, phosphate and inulin were compared to those of 11 matched haemodialysis patients with anaemia. No differences were observed either for small and middle molecule clearances or their extractions between rHuEPO and anaemic patients. In conclusion, dialysis efficiency is not affected if haematocrit values are kept about 35%.
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The evaluation of dialysis adequacy still remains an unsolved problem. The mathematical model proposed by Gotch and Sargent is now currently used to quantify the dialysis need in urysiemic patients. In this study we evaluated the reliability of this kinetic model in 9 patients admitted to high efficiency hemodialysis. Only 5 out of the 9 patients showed a Protein Catabolic Rate normalized (PCRn) within 0.8-1.6 gr/kg/die a range considered optimal for hemodialysis patients. The other patients showed a PCRn higher than 1.6 gr/kg/die and were therefore excluded from this study. The volume of urea distribution (V) measurements deduced from urea kinetic model showed a great variability with a very high mean value (more than 78.3 +/- 6.3%). We therefore suggest that for the evaluation of dialysis adequacy in patients submitted to high efficiency hemodialysis an arbitrary value of urea distribution equal to 60% of the body weight should be used. However further clinical and biochemical evaluations are needed to confirm whether this model might be considered as a reliable parameter of dialysis adequacy.
The translational product of the dbl oncogene is a 66 kDa (p66) protein with no apparent sequence similarity to any of the known oncogene products, whereas the human dbl proto-oncogene encodes a translational product of 115 kDa (p115). We compared proto-dbl p115 and dbl p66 with respect to their subcellular localization, biogenesis and post-translational modifications. Like p66, p115 was found to be a cytoplasmic phosphoprotein present in both cytosol and crude membrane preparations. Membrane fractionation studies revealed that p115 as well as p66 were primarily associated with fractions enriched in plasma membranes, suggesting that this subcellular compartment is a likely site of action of dbl proteins. The membrane-associated forms of p115 and p66 were fairly resistant to solubilization by nonionic detergents, suggesting that dbl proteins associate with the cytoskeletal matrix. p115 was also found to be phosphorylated primarily on serine residues. However, p115 was phosphorylated to a lesser extent as compared to the phosphorylated form of p66. The half-life of proto-dbl p115 was significantly shorter (1 hour) than that of dbl p66 (5-6 h). The higher stability of p66 is likely due to the acquisition of unrelated human sequences and/or to the deletion of the N-terminal region of proto-dbl.
The dbl proto-oncogene can transform NIH3T3 cells when overexpressed, but its transforming activity is about 50 to 70 fold lower than that of the dbl oncogene. The dbl oncogene encodes a protein of 478 amino-acids while proto-dbl encodes a protein of 925 amino-acids. The genesis of dbl involved the loss of the first 497 amino-acids of proto-dbl and the acquisition of a new N-terminus from another human locus. The last 428 amino-acids of proto-dbl and dbl product are identical with the exception of a single conservative amino-acid change. Any of these alterations could be responsible for the greater transforming activity of dbl. In order to define the role of these alterations more precisely, we constructed two deletion mutants, one derived from proto-dbl and the second from dbl in which only their last 428 amino-acids were retained. Under the control elements of the same promoter, the transforming activity of each of these mutants was similar to that of the dbl oncogene, i.e. 60-80 fold greater than that of proto-dbl. This finding suggests that the loss of the first 497 amino-acid of proto-dbl, rather than the acquisition of a new N-terminus, is crucial to the enhanced transforming activity of the dbl oncogene. Both mutant proteins were equally distributed between the membrane and cytosolic fractions, a pattern similar to that of their corresponding parental proteins. These results suggest that the subcellular distribution of proto-dbl is determined by its C-terminal 428 amino-acids. Unlike their parental proteins, neither mutant was phosphorylated, indicating that phosphorylation is not required for dbl transforming activity. In addition to the lack of phosphorylation, each mutant protein had a half-life of 5-6 h while the half-life of proto-dbl was about 1 h. Thus, our data suggest that the N-terminal half of proto-dbl can down regulate its transforming activity and that sequences within this region are responsible for rapid turnover of the protein.
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It is well known that glucocorticoids depress the natural killer (NK) activity of human peripheral blood lymphocytes when used both in vivo and in vitro. Since interferons enhance natural cytotoxicity, potential interaction between beta-interferon and hydrocortisone hemisuccinate has been investigated using mononuclear cells of peripheral blood obtained from 17 healthy donors. At the end of in vitro treatment mononuclear cells were tested for NK activity against K562 cells in a 4 h 51Cr-release assay. The results suggest that beta-interferon at the optimal treatment schedule (i.e. before and after exposure to hydrocortisone) is capable of abrogating the hydrocortisone-mediated impairment of NK function. These findings provide valuable suggestions for optimal treatment schedules with beta-interferon (i.e. beta-interferon treatment before and after exposure of effector cells to hydrocortisone) for overriding the suppressive effects of glucocorticoid therapy on natural immunity.
Natural human interferon beta (beta-IFN) was tested during the early phase of in vitro infection with HTLV-I virus of human cord blood mononuclear cells (CBL), to evaluate whether its antiviral and immunomodulating effects might prevent spreading of infection in the host. beta-IFN was found to reduce HTLV-I transmission and integration in CBL cultures. Moreover, beta-IFN had no effect in preventing virus transmission and integration in K562 and a very limited effect in HL60 and Molt-4 human tumour lines, suggesting a cell-type specific mode of action. beta-IFN induced a 'priming' response on CBL, since overnight pretreatment of recipient cells or one single treatment at the onset of the coculture were almost equally effective in protecting against HTLV-I infection. During the early days post infection (p.i.), IFN-treated CBL showed a pattern of phenotypic markers that was closer to that of non-infected CBL. In contrast, untreated CBL exposed to HTLV-I showed a percent increase of Tac+, M3+ and Leu 11+ subpopulations. Cell-mediated immune responses of CBL were depressed after coculturing with HTLV-I producer MT-2 cells. beta-IFN was able to boost the cell-mediated cytotoxicity of fresh and infected CBL against both K562 and MT-2 target cells. Leukocyte blastogenesis in mixed lymphocyte/tumour cell cultures, evaluated in terms of 3H-thymidine incorporation during the first week p.i., was also enhanced by IFN when macrophages and lymphocytes were reconstituted at an optimal 1:20 ratio. It is conceivable that this overall enhancement of the immune response induced by beta-IFN could contribute to reduce HTLV-I infection in vitro.
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