Search PubMed⌕ Search

Biomedical subjects

R De Santis

Publications and source records attributed to R De Santis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Production of recombinant human GM-CSF-EPO hybrid proteins: in vitro biological characterization.

Selective lineage differentiation depends upon the combined action of several colony-stimulating factors. Here we describe 3 human granulocyte-macrophage colony-stimulating factor-erythropoietin (GM-CSF-EPO) hybrid proteins generated by recombination of the relevant cDNAs. The expression vector containing the murine cytomegalovirus (mCMV) promoter and dihydrofolate reductase (DHFR) gene was used for the expression of the hybrid genes in Chinese hamster ovary (CHO) cells. Purified hybrid proteins from CHO transfectant cultures induced proliferation of both EPO and GM-CSF dependent cell lines. The clonogenic test, performed on purified human hematopoietic precursor cells, indicates that the hybrid proteins are more efficient at inducing erythroid differentiation compared with the equimolar mixture of GM-CSF and EPO.

Amino Acid Sequence↗
PubMed1996

Expression and characterization of a mouse/human chimeric antibody specific for EGF receptor.

Murine antibodies which recognize the epidermal growth factor receptor (EGF-r) are good candidates for therapy and diagnosis of tumors overexpressing this receptor. Here we report the isolation of the variable regions from a murine monoclonal antibody anti-EGF-r (Mint5), the procedure to obtain the mouse/human chimeric antibody (chMint5) and its expression in COS, NS0 and CHO cells. The approach followed to construct chMint5 is based on the use of consensus primers specific for the ends of the variable regions. The sequence imposed by the primers did not affect the targeting potential of the antibody. In fact, the affinity of the chimeric antibody for EGF-r was nearly the same as that of the parental murine antibody. Based on previous in vitro and in vivo animal studies. Mint5 was shown to be a good candidate for the targeting of EGF-r overexpressing tumours. chMint5 is expected to be less immunogenic than murine antibody and therefore, could be useful for human treatment.

Amino Acid Sequence↗
PubMed1996

Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein.

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.

Amino Acid Sequence↗
PubMed1996

Duplex polymerase chain reaction for the simultaneous detection of the human polyomavirus BK and JC DNA.

With the aim of further simplifying the detection of human polyomaviruses BK (BKV) and JC (JCV), by reducing the time and the cost of the reactions, a duplex polymerase chain reaction (PCR) was developed for the simultaneous detection and identification of BKV and JCV DNA. A nested PCR was performed. In a first amplification reaction the same set of primers was used to amplify a sequence of either BKV or JCV genomes. Two sets of primers, each specific for BKV or JCV DNA, were used in a second amplification. The products of BKV and JCV DNA amplification were distinguishable on the basis of their different size. The sensitivity and the specificity of the duplex PCR was shown to be exactly comparable to that of the single PCR, not only with the BKV and the JCV plasmid but also for BKV and JCV DNA detection in clinical specimens.

BK Virus↗
PubMed1996

Human polyomaviruses DNA detection in peripheral blood leukocytes from immunocompetent and immunocompromised individuals.

Peripheral blood leukocytes from immunocompetent and immunocompromised individuals were analyzed for human polyomarivus BK and JC DNA presence. A nested polymerase chain reaction which amplify the transcriptional control region of the genome of both viruses was employed. The immunocompromised patients included bone marrow transplantation recipients and AIDS patients. BKV sequences were detectable in 52.8-62.5% of the individuals included in this study, whereas the percentage of individuals with JCV sequences in peripheral blood lymphocytes varied from 38.8% to 50%. The frequency of reactivations of BKV and JCV were also determined by detection of shedding in urine of viral DNA. The highest frequency of reactivations of either BKV or JCV was demonstrable in the group of bone marrow transplantation recipients, but reactivations occurred also in immunocompetent individuals. JCV sequences amplified from urine samples showed a restriction pattern similar to the archetype one, whereas sequences obtained from lymphocytes showed rearranged pattern as well as archetype pattern. Finally all JCV sequences from cerebrospinal fluid seemed to be rearranged. These observations suggest that peripheral blood lymphocytes have a fundamental role in the persistence of polyomaviruses infection and in the dissemination at least of JCV within the organism allowing that rearranged variants, better adapted to grow in brain tissue, emerge.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa.

In Ciona intestinalis a chymotrypsin-like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin-like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS-PAGE. The molecular weight of the chymotrypsin-like enzyme was estimated to be 35 kDa by gel filtration and 24 KDa by SDS-PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.

Amino Acid Sequence↗
PubMed1992

Evidence that metalloendoproteases are involved in gamete fusion of Ciona intestinalis, ascidia.

The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.

Animals↗
PubMed1991

Gamete self-discrimination in ascidians: a role for the follicle cells.

Gamete self-incompatibility in the hermaphrodite tunicate Ciona intestinalis is a useful system with which to study self-nonself recognition. We have used in vitro fertilization of oocytes isolated from the gonad of Ciona intestinalis to identify the cellular source of self-sterility elements present on the egg envelopes. Here we show for the first time that self-discrimination, which occurs on the egg vitelline coat, is established there in late oogenesis and is contributed or controlled by products of the overlying follicle cells. The acquisition of self-sterility by the oocyte is prevented by the ionophore monensin, which suggests that the follicle cell self-sterility controlling factor is a glycoprotein.

Animals↗
PubMed1991

Production of a nef-specific monoclonal antibody by the use of a synthetic peptide.

Monoclonal antibodies have been generated against a synthetic peptide of the nef protein of human immunodeficiency virus type 1 (HIV-1) in order to further characterize the biochemical and functional nature of this protein and its role in the control of HIV-1 transcriptional regulation. Earlier studies indicated nef to be a negative regulatory factor for viral transcription, whereas more recent studies report evidence against this original hypothesis. Nef is a protein of 206 amino acids of approximately 27 kD in most HIV-1 isolates; however, in some other isolates a truncated form of 124 amino acids has been described. A peptide sequence of six amino acids, corresponding to a region of the nef protein exhibiting high-sequence homology to thymosin alpha 1 protein, has been synthesized by Merrifield solid-phase methodology. This peptide is coded by a sequence located upstream to the stop codon described in some HIV-1 isolates and then is maintained in both complete and truncated forms of the nef protein. F14.11 is a nef peptide-specific monoclonal antibody (IgG2a/k) exhibiting the ability to recognize natural nef protein in either radioimmunoassay, radioimmunoprecipitation assay, or immunocytochemical analysis. Since F14.11 is able to identify nef protein in the cytoplasm of lymphocytes from HIV-infected seronegative subjects it may prove useful in monitoring the expression of nef during the silent HIV-1 infection.

Amino Acid Sequence↗
PubMed1990

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg.

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.

Amino Acid Sequence↗
PubMed1988

Partial duplication of chromosome 1q preceding the development of an L3 lymphoblastic leukemia with t(8;14), secondary to treatment for Hodgkin's disease.

We report on a case of secondary lymphoblastic leukemia L3 in a patient treated with chemo- radiotherapy for a previous Hodgkin's disease. The typical chromosome translocation t(8;14) was found in association with a translocation t(9;19) and a marker chromosome 1, dup(1q). The same marker chromosome had been detected in a few cells of peripheral blood 2 years before the onset of the acute leukemia and may represent a proliferation-associated change, responsible for the initiation of the process of leukemogenesis.

Adult↗
PubMed1987

T cell receptor-homologous mRNA from a suppressor T cell clone directs the synthesis of antigen-specific suppressive products.

The LH8-105 T cell clone, obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific mouse suppressor T lymphocytes, constitutively releases in the culture supernatant products able to induce specific suppression of the immune response to HEL. LH8-105 cells rearrange and express the genes encoding the alpha and beta chains of the antigen-specific T cell receptor and display membrane T cell receptor structures. LH8-105 mRNA specific for the alpha and beta chains of the T cell receptor were positively selected on filter-bound cDNA encoding constant regions of alpha and beta chains, eluted and translated into Xenopus laevis oocytes. Translation products were then tested in vivo for specific suppression of the anti-HEL antibody response. LH8-105 culture supernatant and translation products of LH8-105 poly(A)+ RNA or a mixture of alpha and beta chain-homologous mRNA induce HEL-specific suppression whereas translation products of LH8-105 mRNA eluted from irrelevant DNA or unrecombined mRNA eluted from alpha and beta cDNA do not exert suppressive activity. These results indicate that alpha and beta chain-homologous mRNA expressed in LH8-105 cells direct the synthesis of molecules able to induce HEL-specific suppression.

Animals↗
PubMed1985

Immunosuppression by cell-free translation products from monoclonal antigen-specific suppressor T cell mRNA.

Polypeptides synthesized in a rabbit reticulocyte lysate system directed by mRNA from the T cell line LH8-105, obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes, are able, when injected into mice, to specifically suppress the antibody response and delayed-type hypersensitivity to HEL. The suppressive activity exerted by in vitro translated proteins appears to be independent from post-translational modifications. These in vitro translated polypeptides display fine antigenic specificity in immunosuppression and bind to HEL but not to the closely related ring-necked pheasant egg-white lysozyme immunosorbents. Suppressive molecules obtained by cell-free translation of LH8-105 mRNA or by culture supernatant of LH8-105 cells display, by gel filtration, a similar molecular mass of about 82-90 kDa.

Animals↗
PubMed1985

Rearrangement and expression of the alpha- and beta-chain genes of the T-cell antigen receptor in functional murine suppressor T-cell clones.

Two different antigen-specific radiation leukemia virus (RadLV)-transformed suppressor T-cell clones, LH8.105 and LA41, exhibiting anti-lysozyme and anti-acetylcholine-receptor suppressor activity, respectively, have been examined for rearrangement and expression of genes encoding the alpha and beta chains of the T-cell receptor for antigen. LH8.105 cells express the T-cell-receptor polypeptides, as shown by specific immunoprecipitation. In both cell lines, potentially functional transcripts of alpha- and beta-chain genes are detected by RNA blot analysis. These suppressor T-cell clones exhibit alpha-chain gene rearrangements, deletion of both alleles of the constant-region (C) gene segment C beta 1, and rearrangement of the two alleles of C beta 2 when analyzed by Southern blot hybridization. Restriction analysis suggests that the DNA rearrangement is beyond the second joining-region (J) minigene of the J beta 2 cluster. These results establish that at least some mouse suppressor T-cell clones, like helper and cytotoxic T lymphocytes, rearrange and transcribe the genes coding for the alpha and beta chains of the antigen-specific T-cell receptor.

Animals↗
PubMed1985

Glycosylation changes in membrane glycoproteins after transfection of NIH 3T3 with human tumor DNA.

Glycoproteins from NIH 3T3 transformed by transfection with human tumor DNA had altered glycosylation. That is, the glycoproteins have a decrease in biantennary oligosaccharides with a concomitant increase in tri- and tetraantennary oligosaccharides. After swainsonine treatment, glycosylation of oncogene transfected NIH 3T3 was more similar to the normal counterpart. Thus, this inhibitor of glycoprotein processing is able to reverse at least one phenotypic expression of transformation, the formation of more highly branched oligosaccharides. The mechanism may be by inhibition of a specific glycosyl transferase.

Animals↗