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Biomedical subjects

F Rossi

Publications and source records attributed to F Rossi.

At least 253 records · Page 14Linked to original sources
PubMed1996

Modulation of DNA topoisomerase I activity by p53.

The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by most anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increase in catalytic activity occurs in parallel with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 h of drug treatment. Furthermore, topoisomerase I co-immunoprecipitates with nuclear p53, suggesting that the activation of topoisomerase I may be a result of a direct interaction between the two proteins. In vitro experiments with purified recombinant proteins show that p53 increases the catalytic activities of topoisomerase I as measured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and phosphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoisomerase I sediments at a higher molecular weight in the presence of p53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, protecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in vivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I.

Animals↗
PubMed1996

Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Adenosine Triphosphate↗
PubMed1996

Lineage commitment of transformed haematopoietic progenitors is determined by the level of PKC activity.

Our previous work showed that haematopoietic precursors transformed by the E26 avian leukemia virus undergo multilineage differentiation in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA). Treatment of the cells with high concentrations of PMA (100 nM) favours myelomonocytic differentiation, while lower concentrations (20 nM) induce predominantly eosinophil differentiation. Here we have investigated the role of protein kinase C (PKC) in this process and found that 100 nM, but not 20 nM, PMA dramatically down-regulates total cellular PKC activity, indicating that high PMA concentrations result in less efficient signalling than lower PMA concentrations. Consistent with these findings is the observation that very low PMA concentrations (1 nM), which presumably only moderately activate PKC, induce myeloid differentiation. This suggests the existence of two PKC thresholds which play a role in lineage commitment. To test the model, alpha- and epsilon-PKC isoforms were expressed in E26-transformed progenitors. These cells exhibited myelomonocytic differentiation even in the absence of PMA, while treatment with concentrations of PMA as high as 100 nM led to the differentiation of predominantly eosinophils and failed to downregulate the exogenous PKC. Our results suggest that different levels of PKC activity result in three different phenotypes: (i) no PKC activity maintains the progenitor phenotype; (ii) low PKC activity favours myelomonocytic differentiation; (iii) high PKC favours eosinophil differentiation.

Animals↗
PubMed1996

Tyrosine phosphorylation and subcellular redistribution of p125 ras guanosine triphosphatase-activating protein in human neutrophils stimulated with FMLP.

In this paper, we show that the p125 ras guanosine triphosphatase-activating protein (p125 GAP) is present in the cytosol of human neutrophils and is transiently tyrosine phosphorylated and translocated to the membranes upon cell activation with formyl-methionyl-leucyl-phenylalanine (FMLP). When concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), which both induced a long-lasting respiratory burst, were used as stimuli, tyrosine phosphorylation and translocation of p125 GAP did not occur.

Cell Membrane↗
PubMed1996

Expression of runtB is modulated during chondrocyte differentiation.

The runt locus in Drosophila encodes a nuclear protein involved in embryo segmentation, sex determination/X dosage compensation, and neurogenesis. runt homologues have been identified in higher vertebrates. The encoded proteins share a domain of 128 amino acids called the runt domain. It has been reported that this domain mediates DNA binding and heterodimerization. Here, we analyze runtB expression during chondrocyte differentiation "in vitro" and "in vivo." We have first isolated, from a chondrocyte library, a cDNA clone coding for a runtB chicken homologue and containing a complete open reading frame. The predicted protein product is 84% identical to the mouse PEBP2alphaB2 isoform. By RT-PCR analysis we have also cloned the chicken cDNA fragment coding for delta alphaB2, the exon sequence included in the B1 isoform mRNA. On Northern blot analysis of cultured chondrocytes, runtB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Moreover, runt polypeptides were demonstrated in chondrocytes both in vivo and in vitro. These results suggest that runt plays a role in chondrogenic differentiation.

Amino Acid Sequence↗
PubMed1996

Nef-CD4 physical interaction sensed with the yeast two-hybrid system.

HIV-1 Nef protein has been known to induce downmodulation of CD4 receptor. In order to test whether the two proteins physically interact, the yeast two-hybrid system was exploited. A Saccharomyces cerevisiae strain carrying a GAL4-responsive lacZ fusion gene was cotransformed with plasmids in which the Nef and the CD4 cytoplasmic domain (CD4cd) coding sequences were fused to either the DNA binding (DB) or the activation (A) moiety of the GAL4 transcriptional activator. Both the DB-Nef + A-CD4cd and the DB-CD4cd + A-Nef combinations activated the reporter gene, weakly but specifically, as inferred by comparison with a number of controls. Reporter activation was similary observed when DB-Nef was cotransfected with the fusion A-CD4cd(aa 1-23). On the contrary, the combination DB-Nef + A-CD4cd(aa 24-40) was inactive. Also, mutating the CD4cd Leu20-Leu21 motif (known to be essential for both physiological and Nef-induced CD4 endocytosis) to Ala20-Ala21 abolished the GAL4 activity of DB-Nef + A-CD4cd. None of six DB-Nef derivatives in which Nef was partially deleted activated specifically the reporter when coexpressed with A-CD4cd. These findings suggest that CD4cd and Nef directly interact and that a largely complete Nef is required for the interaction. CD4cd aa 1-23 are sufficient for binding; in particular, the Leu20-Leu21 motif is essential. One can infer from these data that: (i) Nef-induced CD4 downmodulation involves a direct CD4-Nef contact and (ii) CD4cd Leu20-Leu21 is required in Nef-induced downmodulation, not simply as an endocytosis signal, but also as an essential component of the Nef-binding moiety.

Amino Acid Sequence↗
PubMed1996

Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.

On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.

Adult↗
PubMed1996

Translocation of p190rho guanosine triphosphatase-activating protein from cytosol to the membrane in human neutrophils stimulated with different agonists.

In this paper, we investigated the subcellular distribution of p190rho guanosine triphosphatase-activating protein (p190 GAP) in human neutrophils stimulated with different agonists. The results show that in neutrophils treated with formyl-methionyl-leucyl-phenylalanine (FMLP) (1) p190 GAP was translocated from the cytosol to the membranes; (2) the translocation of p190 GAP took place only at doses of FMLP that induced the translocation of rac 1 and rac 2 and the activation of the NADPH oxidase; and (3) the kinetic of translocation of p190 GAP paralleled that of rac 1 and rac 2. However, when the agonist was concanavalin A (ConA) or phorbol 12-myristate 13-acetate (PMA), rac 1 and rac 2, but not the p190 GAP, were translocated.

Cell Membrane↗
PubMed1996

The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin.

The EOS47 antigen is a 100-kD cell surface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified the EOS47 protein to homogeneity and used peptide sequence information to clone EOS47-encoding cDNAs. The open reading frames from these cDNAs predict a 738 amino acid protein with homology to human melanotransferrin, a membrane-found, transferrin-like protein that is expressed at high levels by a subset of melanomas, tumor cell lines, fetal intestine, and liver, but not by most normal adult tissues. The predicted protein sequence of EOS47 displays a 61% sequence identity with melanotransferrin and conservation of all 28 cysteine residues, indicating a similar tertiary structure. The finding that EOS47 lacks several of the iron-coordinating amino acids present in all transferrins suggests that it may be impaired in its ability to bind iron. In nonhematopoietic tissues, EOS47 is expressed at high levels by epithelial brush borders of small intestine and kidney and at lower levels by cells lining the sinusoids of the liver. Within hematopoietic tissues, EOS47 is restricted to a subpopulation of cells (1% to 5%) in bone marrow and early spleen and fluorescence-activated cell sorting of EOS47+ cells leads to a dramatic ( > 30-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting that the antigen is expressed by newly formed eosinophils and that expression ceases shortly before these cells emigrate from the bone marrow into the peripheral blood. Our results show that melanotransferrin is a stage-specific marker of eosinophils and should be useful for their isolation and further characterization.

Amino Acid Sequence↗
PubMed1996

Solution and solid state structure of an aib-containing cyclodecapeptide inhibiting the cholate uptake in hepatocytes.

The conformational analysis of synthetic cyclodecapeptide c(Pro-Phe-phe-Aib-Leu)2 related to the cyclolinopeptide A, in the solid state and solution, has been carried out by x-ray diffraction and nmr spectroscopy. The structure of the monoclinic form obtained from methanol [a = 11.351 (5) A, b = 27.455 (2) A, c = 12.716 (8) A, beta = 99.65 (3) degrees; space group P2(1); Z = 2] shows the presence of six intramolecular NH...CO hydrogen bonds, with formation of four turns (three of type I and one of type III) and two C16 ring structures. All peptide units are trans. The solution structure, as found by nmr, indicates that, at room temperature, the peptide is conformationally homogeneous; the structure determined is perfectly symmetrical and topologically similar to that found in the solid state. The cyclodecapeptide exhibits similar biological activity to cyclolinopeptide A.

Animals↗
PubMed1996

Monosodium glutamate affects mealtime food selection in diabetic patients.

Lunch intake was followed in 31 matched pairs of hospitalized diabetic patients over four consecutive days. Pairs of patients were matched for type and duration of diabetes, gender, age and body mass index. Lunches were composed of appetizer, meat, vegetables, starch, cheese, bread and dessert; water, coffee, tea and lemon were available. One patient per pair was randomly ascribed to the experimental group and was served vegetable and starch dishes added with 0.6% monosodium glutamate (MSG). Lunch intake was measured by weighing amounts served and left-overs. Patients in the experimental group ingested more starch food than their matched controls, and less lemon juice and yogurt. However, the total energy load at lunch was not different between groups. This effect on meal time food selection replicates earlier observations made on elderly persons. It is suggested that manipulating palatability of various foods within a meal, and especially by using MSG, is an efficient way to affect food selection in the meal, without inducing hyperphagia.

Adult↗
PubMed1996

Tissue and developmental specificity of a polysialo-ganglioside species in the amphibian Xenopus.

Xenopus embryos contain a considerable amount of a polysialo-ganglioside not yet fully characterized; in this paper, we will refer to it as ganglioside XI. Preliminary experiments indicate asialo-GMI as the core structure of the ganglioside XI and palmitic and oleic acid as the fatty acids of the ceramide moiety. Further analyses by comparative 2D-TLC with adult fish and chick embryo brains indicate the pentasialilated ganglioside GP1c as the possible structure of XI. In the adult Xenopus, XI characterizes the ganglioside pattern of the central nervous system while is absent in all the other tested tissues. At least two other more polar (presumably richer in sialic acid) bands are often visible under XI, both in embryos and in brain and spinal cord tissues of adult Xenopus. The persistence of polysialo-gangliosides in the brain and spinal cord of adult amphibians could serve to guarantee a proper functioning of the central nervous system at low body temperature.

Animals↗
PubMed1996

Opposing effects on blood pressure following the activation of metabotropic and ionotropic glutamate receptors in raphe obscurus in the anaesthetized rat.

The microinjection of L-glutamate (1-6 nmol/rat) and N-methyl-D-aspartate (NMDA 1-10 nmol/rat), ionotropic glutamate receptor (iGluR) agonists, into the nucleus raphe obscurus caused a concentration -dependent increase of arterial blood pressure. In contrast, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD, 14-42 nmol/rat), a metabotropic glutamate receptor (mGluRs) agonist, caused a concentration-dependent decrease in blood pressure. Pretreatment with D,L-2-amino-phosphono valeric acid (2-APV, 5 nmol/rat) a selective NMDA iGluR antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK801, 0.9 nmol/rat), a noncompetitive NMDA iGluR antagonist, blocked both the glutamate and NMDA pressor responses, while pretreatment with (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 0.05 nmol/rat), a mGluR1 antagonist, increased the glutamate-induced pressor effects and blocked the fall in blood pressure induced by t-ACPD. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 0.4 nmol/rat) a non-NMDA iGluR antagonist, did not affected the glutamate-induced hypertension. These observations indicate opposing roles for ionotropic and metabotropic receptors in the glutamate-induced blood pressure changes elicited from the nucleus raphe obscurus. Moreover, we suggest that the glutamate-induced hypertension may be due to the activation of NMDA ionotropic receptor subtypes and the metabotropic receptors may influence this activation through a reduction of excitability at level of synapses.

6-Cyano-7-nitroquinoxaline-2,3-dione↗