Biomedical subjects
S Ferrari
Publications and source records attributed to S Ferrari.
Proliferation, differentiation arrest, and survival in leukemic blast cells.
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Antisense strategies to characterize the role of genes and oncogenes involved in myeloid differentiation.
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Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.
Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.
Osteosarcoma of the extremity metastatic at presentation: results achieved in 26 patients treated with combined therapy (primary chemotherapy followed by simultaneous resection of the primary and metastatic lesions).
From September 1986 to December 1989, 26 selected patients with high-grade osteosarcoma of the extremities metastatic at presentation were treated with primary chemotherapy (high doses of methotrexate, -cisplatinum and adriamycin) followed by surgery. Twenty-one cases underwent resections of the primary and metastatic tumor at the same time; owing to the disappearance of lung metastases after preoperative chemotherapy in 3 cases, only the primary tumor was operated on. Due to progression of the disease in 2 patients, no surgery was performed. Histologic examination of the resected specimen was performed to evaluate the percentage of necrosis produced by chemotherapy on the primary and metastatic tumor. After surgery, the patients received further chemotherapy with the same drugs used preoperatively plus ifosfamide and VP-16. The histologic response of the primary tumor was good (> 90% tumor necrosis) in 25% of the cases; in the resected metastatic nodules, 23% had good responses. A discrepancy between the histologic response of the primary and secondary tumor was observed in only 15% of the cases. These results seem to confirm the validity of the strategy (widely used today in the neoadjuvant treatment of non-metastatic osteosarcoma) of changing the postoperative treatment when the histologic response of the primary tumor is poor. At an average follow-up of 3.5 years, only 6 patients remained disease-free; 19 patients relapsed and 1 patient died for adriamycin cardiotoxicity. Of the 19 relapsed patients, 16 died and 3 are still alive but with uncontrolled disease. These results are much worse than those obtained in 144 cases of non-metastatic osteosarcoma of the extremities treated in the same period with the same preoperative chemotherapy (77% with good response in the primary tumor and 78% with continuous disease-free survival). The data suggest that a very effective neoadjuvant chemotherapy for nonmetastatic osteosarcoma of the extremities gives disappointing results in osteosarcoma of the extremities which is metastatic at presentation.
T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis.
T-cell receptor (TCR) beta-chain proteins appear early (approximately 15th week of gestation) during human thymic ontogenesis. These beta-chain proteins, which appear before terminal deoxynucleotidyl transferase (TdT), could be an expression of a fully rearranged (V-D-J), incompletely rearranged (D-J), or germline TCR beta-chain gene. The aims of this study, performed from the 15th week onward, were the following: (1) to investigate whether or not TCR beta gene rearranges at an early stage during human thymic ontogenesis; (2) to investigate whether complete presumptive functional (1.3 kb) TCR beta gene transcript is present at these early stages of development, or if incomplete (1 kb) or germ-line (1.1 kb) transcripts are expressed; (3) to examine the phenotype of TCR beta-chain+ cells with two-color fluorescence using monoclonal antibody (MoAb) beta F1 and MoAbs that recognize CD1, CD2, CD3, CD4, CD8, CD5, and CD7 antigens (rabbit anti-calf TdT antiserum was used to detect TdT); and (4) to demonstrate whether or not beta gene N-diversity regions are detectable as early as the 15th week and whether or not N-nucleotide insertions correlate to TdT expression. Fifteen- to 22-week fetal thymuses and pediatric thymuses were investigated. We demonstrated that TCR beta-chain gene rearranged as early as the 15th week in human thymus and that a complete functional TCR beta gene transcript was expressed at these early stages of human development. No other analyses to date have investigated TCR beta gene expression in early human thymus using molecular biology techniques. No significant differences were detectable between phenotypic analysis of fetal and pediatric samples, except for TdT expression, which appeared after the 20th week. Essentially all mCD3+ (OKT3+) cells were beta-chain+ at the different weeks investigated. A significant percentage of CD1+ cells were beta-chain+, and the percentage increased along with the age of development. After the 20th week, we identified three main populations: TdT+, cCD3+, beta F-(early thymic precursors); TdT+, CD1+, beta F1+ (intermediate maturity cortical thymocytes); and TdT-, mCD3+, beta F1++ (mature medullary thymocytes). Given these values, we may consider beta-chain expression an ordered process. beta gene N-nucleotide insertions were correlated to TdT expression, since N-regions increased considerably after the 20th week. A further increase of N-nucleotide insertions was detected from the 22nd week to the 32nd week.
Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation.
The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells.
The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins.
The mammalian nuclear protein HMG1 contains two segments that show a high sequence similarity to each other. Each of the segments, produced separately from the rest of the protein in Escherichia coli, binds to DNA with high specificity: four-way junction DNA of various sequences is bound efficiently, but linear duplex DNA is not. Both isolated segments exists as dimers in solution, as shown by gel filtration and chemical crosslinking experiments. HMG1-like proteins are present in yeast and in protozoa: they consist of a single repetition of a motif extremely similar to the DNA binding segments of HMG1, suggesting that they too might form dimers with structural specificity in DNA binding. Sequences with recognizable similarity to either of the two DNA binding segments of HMG1, called HMG boxes, also occur in a few eukaryotic regulatory proteins. However, these proteins are reported to bind to specific sequences, suggesting that the HMG box of proteins distantly related to HMG1 might differ significantly from the HMG box of HMG1-like proteins.
SRY, like HMG1, recognizes sharp angles in DNA.
HMG boxes are DNA binding domains present in chromatin proteins, general transcription factors for nucleolar and mitochondrial RNA polymerases, and gene- and tissue-specific transcriptional regulators. The HMG boxes of HMG1, an abundant component of chromatin, interact specifically with four-way junctions, DNA structures that are cross-shaped and contain angles of approximately 60 and 120 degrees between their arms. We show here also that the HMG box of SRY, the protein that determines the expression of male-specific genes in humans, recognizes four-way junction DNAs irrespective of their sequence. In addition, when SRY binds to linear duplex DNA containing its specific target AACAAAG, it produces a sharp bend. Therefore, the interaction between HMG boxes and DNA appears to be predominantly structure-specific. The production of the recognition of a kink in DNA can serve several distinct functions, such as the repair of DNA lesions, the folding of DNA segments with bound transcriptional factors into productive complexes or the wrapping of DNA in chromatin.
Myasthenia gravis associated with Charcot-Marie-Tooth neuropathy: report of a case.
We report the case of a 24 year old woman who developed myasthenia gravis in the course of a mild form of Charcot-Marie-Tooth neuropathy. We describe the clinical manifestations together with the neurophysiological, pathological, serological findings and response to therapy and discuss the unusual association in the light of the relevant literature.
Effects induced by olive oil-rich diet on erythrocytes membrane lipids and sodium-potassium transports in postmenopausal hypertensive women.
Since we have observed that monounsaturated fatty acids (MUFA) enriched diet modifies red cell membrane lipids and cation transport systems in normotensive subjects, we similarly evaluated a group of hypertensive patients undergoing an analogous dietary modification. In a group of 18 moderately hypertensive women, the diet was supplemented for two months with olive oil (about 45 g/day), which replaced an equal amount of seasoning fats. Before and after this period, red cell fatty acid composition was evaluated by gas-chromatography in order to verify diet compliance: a significant increase in oleic acid was observed, while the content of saturated and polyunsaturated fatty acids remained unchanged. After olive oil, maximal rates of Na-K pump (5580 +/- 329 vs 6995 +/- 390, p less than 0.001) and Na-K cotransport (Na-COT 544 +/- 52 vs 877 +/- 46, p less than 0.001: K-COT 790 +/- 76 vs 1176 +/- 66, p less than 0.001), cell Na content (9.58 +/- 0.4 vs 10.61 +/- 0.6, p less than 0.03) and passive permeability for Na (936 +/- 74 vs 1836 +/- 102, p less than 0.001) rose significantly. Although the reduction in maximal rate of the Li-Na CT after olive oil was not significant, it was the only cation transport parameter being correlated with the variations of membrane lipids, namely negatively with UFA (r = -0.528, p less than 0.05) and positively with SFA (r = 0.482, p less than 0.005). The change in maximal rate of Li-Na CT was also correlated with the variation of systolic and diastolic BP (r = 0.50, p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)
Induction of Calbindin-D28K by 1,25-dihydroxyvitamin D3 in cultured chicken intestinal cells.
Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene.
Effect of intra-arterial versus intravenous cisplatin in addition to systemic adriamycin and high-dose methotrexate on histologic tumor response of osteosarcoma of the extremities.
In neoadjuvant treatment of osteosarcoma of the extremities intra-arterial (IA) instead of intravenous (IV) administration of cisplatinum (CDDP) has been advocated to improve local response and consequently increase the percentage of limb salvages and the cure rate. Since the major local advantages from the IA use of CDDP have been recently questioned, a comparative prospective study was performed at the Rizzoli Institute between July 1990 and September 1991. Preoperative chemotherapy consisted of two cycles of high-dose methotrexate (HDMTX), followed 6 days later by CDDP and adriamycin (ADM), HDMTX and ADM were delivered intravenously while CDDP was delivered according to previous randomization, either intra-arterially or intravenously. Of the 79 patients who entered the study 40 received CDDP IA and 39 CDDP IV. The percentage of clinical and radiological response of the tumor as well as the percentage of limb salvage procedures performed was the same in the two groups. However the rate of good histological response (tumor necrosis greater than 90%) was significantly higher in the group of patients who received IA CDDP than in the patients who received the drug intravenously (77.5% vs 46%: p less than 0.01). No differences in terms of local or systemic side effects were found in the two groups. A longer follow-up is necessary to establish if this improved good response rate achieved with IA CDDP will lead to an improved cure rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Primary chemotherapy and delayed surgery for osteosarcoma of the extremities with synchronous metastases.
From September 1986 to September 1989 11 patients affected by osteosarcoma of the extremities with synchronous metastases were treated with two cycles of high-dose methotrexate i.v., cisplatin i.a. and adriamycin i.v. followed by simultaneous resection of the primary and metastatic tumor. A complete histological examination of the resected specimens was always performed to evaluate the percentage of necrosis produced by chemotherapy on both the primary and metastatic tumor. After surgery the patients received 3 more cycles of the same drugs as used preoperatively plus ifosfamide. The histological response of the primary tumor was 'good' (90% or more tumor necrosis) in 4 patients and 'poor' (less than 90% of tumor necrosis) in 7, while in the 34 metastatic nodules the resulting necrosis was good in 8 and poor in 26. A good correlation between the histological response in the primary and metastatic tumor was observed, particularly for poor responders (91% of poor responses also in metastatic nodules). At an average follow-up of 42 months, only 3 patients are alive, 2 disease-free, and one with uncontrolled disease. These data suggest that the prognosis of osteosarcoma of the extremities with synchronous metastases remains poor, even with a very aggressive treatment. Our results also seem to confirm the validity of the present strategy in the treatment of non-metastatic osteosarcoma: introducing new drugs post-operatively in poor responding patients can allow a better treatment of microscopic disease and can improve the prognosis for these patients.
High calcitonin levels in unconscious polytrauma patients.
We measured levels of calcitonin and other markers of calcium and phosphorus metabolism in both unconscious and conscious patients after multiple trauma. We found dramatic increases in calcitonin levels in unconscious patients, and smaller increases in conscious patients. In two cases, very high levels, more than 100 x normal, appeared to be related to more rapid healing of bone. Calcitonin levels were highest immediately after admission and decreased over the ensuing two weeks. The possible relationship between unconsciousness and the increased rate of healing of fractures is discussed.
Evolutionary distinct mechanisms regulate apolipoprotein A-I gene expression: differences between avian and mammalian apoA-I gene transcription control regions.
In mammals, the apolipoprotein (apo) A-I gene is expressed predominantly in liver and intestine, while in avian species it is expressed in all tissues. Although liver and intestine are the major sites of chicken apoA-I mRNA synthesis, there are appreciable amounts of apoA-I mRNA in kidney, ovary/testes, brain, lung, skeletal, and heart muscle. In this study, the nucleotide sequences of the chicken apoA-I gene and its 5' flanking region, as well as the sequences involved in the expression of this gene, have been determined. The gene spans 1.5 kilobases and contains 4 exons and 3 introns, closely resembling the mammalian apoA-I gene. To determine the sequences involved in the expression of the chicken apoA-I gene, plasmid constructs containing serial deletions of the 5' flanking region of the chicken apoA-I gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected in human hepatoma (HepG2), colon carcinoma (Caco2), epithelial (Hela), mouse embryonal fibroblast (NIH3T3) cells, and quail myoblasts (QMLA29). The shortest deletion construct, containing 60 bp of the 5' upstream region, was sufficient for maximal transcriptional activity in all cell lines tested. This region contains a short sequence (nucleotides -60 to -54) that is highly conserved in birds and mammals, and an Sp1 binding site. Although the sequence between nucleotides -232 and -101 of the 5' region of the chicken apoA-I gene is partially homologous to the hepatic cell-specific enhancer of the mammalian apoA-I gene (located between nucleotides -222 and -110 upstream of the human apoA-I gene transcription start site), this chicken sequence is transcriptionally inactive in HepG2 cells. These results suggest that differences in the cis-acting regulatory elements of the apoA-I gene play a fundamental role in determining the differences in the tissue-specific expression of this gene in avian and mammalian species.
Red blood cell susceptibility to lipid peroxidation, membrane lipid composition, and antioxidant enzymes in continuous ambulatory peritoneal dialysis patients.
OBJECTIVE: To investigate the overall susceptibility of red blood cells (RBC) to lipid peroxidation from patients on continuous ambulatory peritoneal dialysis (CAPD). METHODS: The following parameters were measured: RBC malondialdehyde (MDA) production after oxidative stress with H2O2, RBC antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and RBC membrane lipid composition. The levels of plasma vitamin E and serum selenium were also assayed. PATIENTS: Eleven patients on continuous ambulatory peritoneal dialysis. Twenty-one healthy blood donors of similar age were used as normal controls. RESULTS: The MDA formation after H2O2 stimulation was normal in CAPD patients (0.79 +/- 0.1 mumol/gHb versus 0.78 +/- 0.1 in the control group). RBC from CAPD patients also showed a normal SOD activity, a more than adequate vitamin E status, and a peculiar pattern of membrane lipids, with reduced polyunsaturated fatty acids (p less than 0.001) and increased monounsaturated fatty acids (p less than 0.001). Both RBC GSH-Px activity, a selenium-dependent enzyme, and serum selenium levels were significantly lower in CAPD patients, and a significant positive correlation (r = 0.68; p less than 0.02) between the two parameters was found. CONCLUSIONS: This study found a normal sensitivity to oxidant stress in RBC from a group of CAPD patients, despite an impaired GSH-Px activity. The peculiar lipid pattern of RBC membrane, characterized by reduced PUFA and increased MUFA content, may contribute, in addition to adequate SOD activity and vitamin E status, to normal RBC lipid peroxidation.
[Effects of hydration on plasma concentrations of methotrexate in patients with osteosarcoma treated with high doses of methotrexate].
High dose of methotrexate (HDMTX) with leucovorin rescue requires over-hydratation to avoid nephrotoxicity; nevertheless the relationship between hydratation and plasma MTX levels is unknown. We compared the effects of two different types of hydratation (2 lt/m2 vs 1.5 lt/m2) on plasma MTX levels in two groups of patients with osteosarcoma of the extremities and treated with HDMTX (8 g/m2 IV). Samples were obtained at the end of infusion of MTX and 14 and 38 hours after the start of MTX infusion. At the end of infusion of MTX the medium plasma MTX levels proved significantly higher in the group with low hydratation than in the group with high hydratation (585.5 microns/l vs 427.7 microns/l P less than 0.001). The values obtained at 14 and 38 hours, did not show significant differences. No significant differences were seen between the two groups in term of late elimination of MTX and correlated toxicity. These data show that a low hydratation regime allows higher plasma MTX levels at the end of infusion of HDMTX and does not increase the incidence of late elimination of MTX compared to a high hydratation regime.