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Biomedical subjects

M V Corrias

Publications and source records attributed to M V Corrias.

28 records · Page 2Linked to original sources
PubMed1987

Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation.

The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes.

Animals↗
PubMed1981

Poliovirus morphogenesis. I. Identification of 80S dissociable particles and evidence for the artifactual production of procapsids.

The current model of poliovirus morphogenesis postulates a fundamental role for procapsid, 80S shells that, upon interaction with viral RNA and subsequent proteolytic cleavage, give rise to complete virus particles. Although 80S sedimenting particles can, indeed, be isolated from cytoplasmic extracts of infected cells, their physical properties differ from those reported for procapsids. Far from being stable structures, they can be dissociated by pH 8.5 and 0.1% sodium dodecyl sulfate into slower-sedimenting subunits. The reasons for this discrepancy were investigated, and two main modalities leading to the appearance of procapsids in vitro were identified. The first involves a temperature-mediated conversion of dissociable 80S particles into stable 80S procapsids, and the second involves the self-assembly of endogenous 14S subunits, also primed by an increase in the temperature of cytoplasmic extracts.

Capsid↗
PubMedDate not supplied

An oligomer complementary to the 5' end region of MDR1 gene decreases resistance to doxorubicin of human adenocarcinoma-resistant cells.

Acquired resistance to doxorubicin and other anti-cancer drugs is generally dependent on gene amplification of a specific nucleotide sequence, the MDR1 gene. Verapamil, cyclosporin and other drugs have been used to circumvent the resistance in experimental models in vitro and/or in vivo. We have attempted to reverse the MDR phenotype by treating human adenocarcinoma resistant cells with 20 mers of synthetic unmodified oligodeoxynucleotide MDR1 antisenses. Five ODNs towards different mRNA regions and three different schedules of ODN antisense administration were tested. We found that FCS concentration greatly influenced the stability of ODN, whereas heat-inactivated FCS had no effect. The kinetics of ODN cellular uptake suggest the presence of a saturable receptor. Among the five antisense ODNs used, the most efficient was the oligomer (ODN-1) complementary to 20 bases upstream of the AUG initiation codon. No effect was observed with antisense complementary to the nucleotide binding sites. Administration of ODN-1 every 12 hr for 72 hr partially reversed the MDR phenotype. Approximately 60% of the cells lost their resistance to doxorubicin and did not form colonies in the presence of the drug. The MDR1 mRNA was transiently down-regulated so that the level of gp170 was slightly reduced. The incomplete switch off of MDR1 gene expression may be ascribed to the large abundance and great stability of MDR1 messenger RNA. Moreover, the inactivity of the two ODNs complementary to the NBS protein domains suggests that translation inhibition is ineffective. It is likely that ODN-4 and ODN-5 complement a large number of mRNAs competing for duplex formation, because these sequences are highly conserved among many proteins.

Adenocarcinoma↗
PubMedDate not supplied

Expression of multiple drug resistance gene, MDR1, and N-myc oncogene in an Italian population of human neuroblastoma patients.

Thirty-four patients of an Italian population affected by neuroblastoma (NB) were evaluated at diagnosis for multidrug resistance gene (MDR1) and N-myc oncogene amplification. No patients showed MDR1 amplification, while extra copies of the N-myc gene were found in 9 out of 34 patients (26%). N-myc amplification was correlated (p = 0.008) with a shorter progression-free survival. RNA was purified from fresh tumor biopsies and analysed in 29 NB samples. MDR1 gene expression was found to be increased in 5 out of 29 tumor samples at onset (17%) and in 1 out of 3 at relapse, but none of them expressed both MDR1 and N-myc genes simultaneously. No correlation was found between MDR1 or N-myc genes expression and tumor progression. MDR1 mRNA transcription may occur spontaneously after onset, suggesting that certain NB tumors could be resistant to antineoplastic drugs at onset. All 5 patients showing MDR1 mRNA transcription achieved complete or partial clinical remission after polychemotherapy. This was presumably due to inclusion in the therapeutic protocol of a high dose of Cisplatin, a drug not susceptible to the effects of the MDR1 gene product. Our findings show that cells which actively transcribe for the MDR1 gene are present in several untreated NB patients. No gene amplification was detected and probably the MDR1 gene expression is regulated at the transcriptional level.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

[Rational bases for new approaches to the therapy of pediatric solid tumors: immunotherapy and gene therapy].

Neuroblastoma is one of the commonest solid tumors in children. Conventional therapeutic approaches, such as surgery, chemotherapy and radiotherapy, fail to control tumor progression in stage III and IV patients. The search for novel therapeutic strategies should necessarily take into account immunotherapy and gene therapy. Here the theoretical bases for the development of such approaches are discussed. Studies carried out with neuroblastoma (NB) cell lines have shown that neoplastic cells express a wide array of potential tumor associated antigens (TAA) but are devoid of HLA molecules which are necessary for TAA presentation to the host immune system. Transfection of NB cells with the interferon gamma gene appears a promising approach, since this cytokine up-regulates the expression of class I HLA molecules in NB cells. Other cytokines of potential interest for gene transfer studies are interleukin 2 (IL2) and interleukin 12 (IL12).

Antigens, Neoplasm↗
PubMedDate not supplied

Characterization and tumorigenicity of human neuroblastoma cells transfected with the IL-2 gene.

Immunization of cancer patients with cytokine-engineered tumor cells is being currently tested in several trials. To test the feasibility of this approach in neuroblastoma (NB) patients we investigated the functional consequences of interleukin-2 (IL-2) gene transfer into NB cell lines. Two human NB cell lines were transfected with the plasmid expression vector RSV.5neo containing the human IL-2 cDNA, and their tumorigenicity was evaluated in a nude mice xenograft model after characterization of the growth patterns and phenotypic features in vitro. The combination of IL-2 gene transfection and the xenograft model in nude mice was chosen on the basis of the low or absent expression of HLA class I antigen in human NB tumors. Our aim was to evaluate the effectiveness of an immunization protocol that could elicit a nonspecific antitumor response. The IL-2 stable transfectants were morphologically identical to parental or vector-transfected cells but completely lost tumorigenicity and inhibited, through a bystander effect, the growth of parental cells injected simultaneously at the same site. Histologic and immunohistochemical analysis of the nodules showed extensive necrosis with severe endothelial damage. The infiltrating cells were mainly macrophages, while natural killer (NK) cells were scarce. However, depletion of NK cells by anti-CD122 monoclonal antibody indicated that the rejection process required NK cell activity. The relevance of these data for the development of therapeutic approaches using cytokine-engineered NB cell lines is discussed.

Animals↗
PubMedDate not supplied

[The identification of minimal residual disease in the bone marrow and peripheral blood in neuroblastoma. The prognostic and therapeutic implications].

A highly sensitive and specific methodology to detect neuroblastoma cells in the bone marrow and peripheral blood of children with neuroblastoma is of critical importance for proper staging and treatment of these patients. In addition, patients with bone marrow infiltration at diagnosis need to undergo regular investigation to measure the effectiveness of chemotherapy (so called "in vivo" purging). Finally, the evaluation of autologous stem cells taken from bone marrow or peripheral blood is necessary to rule out or minimise the possibility of reinfusing tumor cells to the patient following myeloablative therapy. The authors provide a "state of the art" data on this complicated issue and give their preliminary results of their own experience, mainly concerning the immunocytological methods.

Biomarkers, Tumor↗