PubMed Health⌕ Search

Biomedical subjects

S Rusconi

Publications and source records attributed to S Rusconi.

At least 73 records · Page 4Linked to original sources
PubMed1996

Magnetic selection of transiently transfected cells.

An efficient and rapid method for selecting transiently transfected cells is described. A plasmid encoding for a neural cell-specific surface marker is co-transfected into mammalian cells along with the gene of interest. After uptake and expression of these two plasmids, the transfected cells are immuno-adsorbed to magnetic beads pre-coated with antibodies against the surface marker. These immuno-complexes are then isolated by means of a strong magnet. In a single round of magnetic selection, we were able to enrich the cell population more than 7-fold for a co-transfected reporter. These specifically selected cells can now be used for either further cultivation or for immediate analysis. This method has been shown to be effective on HeLa and on CV-1 cells and is expected to give similar results on any other transfectable, non-neuronal cell lines.

Animals↗
PubMed1996

Viral load, viral phenotype modification, zidovudine susceptibility and reverse transcriptase mutations during the first 6 months of zidovudine monotherapy in HIV-1-infected people.

We studied 14 zidovudine-naive, HIV-1-infected patients attending an infectious diseases clinic in Milan during zidovudine therapy for 6 months. We monitored CD4 cell counts, immune complex-dissociated p24 antigen, viral phenotype and viral load in plasma. The virus infecting a subset of patients was examined for zidovudine susceptibility and zidovudine resistance-associated mutations. A significant correlation was established between the increase in the CD4 cell count and the decrease in viral load (Spearman's coefficients < -0.5). Patients who were p24 antigen positive had a higher viral load (P < 0.005 at baseline and after 6 months of therapy). Patients with non-syncytium-inducing (NSI) virus had higher CD4 cell counts over time than those with syncytium-inducing (SI) virus. We also examined the viral load in relation to viral phenotype. The median viral load in patients with NSI virus was higher than in SI controls at baseline, but not after 3 and 6 months of therapy. Sequential isolates of HIV-1 were obtained from nine patients and tested for resistance to zidovudine by monitoring the drug susceptibility and the reverse transcriptase-encoding sequence. Amino acid changes at codons 70 and 215 were present in some but not all isolates with zidovudine-resistant phenotype in vitro. It was possible to perform a correlation between zidovudine susceptibility and zidovudine-associated pol gene mutations only at the 6-month time point (Spearman's coefficient = 0.076). SI phenotype was associated with the development of a decreased zidovudine susceptibility. A correlation between zidovudine-associated pol gene mutations and SI phenotype was detected at the 6-month time point.

Acquired Immunodeficiency Syndrome↗
PubMed1995

A transcriptional repressor obtained by alternative translation of a trinucleotide repeat.

Triplet nucleotide repeats are ubiquitous and rapidly evolving sequences in eucaryotic genomes. They are sporadically found in coding regions of transcription regulators where they become translated in different homopolymeric aminoacid (HPAA) stretches, depending on the local frame. Poly(CAG) yields three different HPAAs (poly Gln, Ser or Ala). Current sequence databases indicate a clear bias in the size and frequency of these HPAAs according to the rule: (Gln)n > (Ser)n >> (Ala)n. Aiming to understand the reasons of this bias, we changed the translational reading frame of the highly polymorphic CAG-repeat that normally encodes poly-Gln in the N-terminal portion of the rat glucocorticoid receptor (GR). The GR mutant in which the CAG repeat is translated to poly-Ala (called GR[Ala]) is incapable of transactivation, but maintains competence for hormone binding, nuclear translocation and specific DNA binding. We show that GR desactivation is obtained only when a very precise threshold length of the repeat is reached. GR[Ala] displays a strong negative dominance when tested for transcriptional activation in vivo and may become useful for selective competition of receptor dependent activities in tissue culture cells and transgenic animals. We discuss the implications of our findings for the understanding of the evolutionary behaviour of trinucleotide repeats in coding sequences.

Amino Acid Sequence↗
PubMed1995

In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Ephemerally expressed wild-type and mutant steroid hormone receptors are equally able to influence expression of transient or resident templates.

We have tested transiently expressed mutant and chimeric glucocorticoid receptors (GR) for their ability to influence transcription of either a co-transfected or a stably integrated reporter gene. To the latter purpose we have generated a cell line harbouring 2 chromosomally anchored copies of the well-characterized mouse mammary tumor virus (MMTV) promoter/enhancer region fused to the bacterial beta-galactosidase gene (LacZ). We were particularly interested in verifying whether some earlier characterized dominant negative GR mutants would still act the same way on chromosomal targets. We show that trans-regulation (activation/-repression) of the chromosomally anchored reporter is qualitatively and quantitatively indistinguishable from trans-regulation obtained with transient co-transfection. In parallel, we also tested ephemerally expressed wild-type progesterone receptor (PR) and androgen receptor (AR) for their capacity of acting on either transient or resident MMTV reporter templates. Also in this case we show that activation of chromosomally anchored or transiently co-transfected reporter by both these steroid hormone receptors is qualitatively and quantitatively indistinguishable. These results outline that newly expressed trans-effectors may exert their specific function independently of the precise structural organization of their responsive genes.

Animals↗
PubMed1994

Transcriptional activation modulated by homopolymeric glutamine and proline stretches.

Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Amino Acid Sequence↗
PubMed1994

Active, interactive, and inactive steroid receptor mutants.

In transient co-transfection assays, there is extensive cross-interaction between glucocorticoid receptor (GR) domains. For example, mutation of the conserved Ile residue at position 484 (rat GR map) to cysteine allows a net separation of transactivation and DNA binding. We also observed that the ligand binding domain plays a key role in cooperative transactivation. Furthermore, some carboxy-located mutations markedly alter the response of GR to agonists and antagonists. Finally, different reading frames of the CAG repeat that normally produces an amino-located poly-Gln repeat profoundly affect GR transactivation without altering DNA or ligand binding. This trans-dominant negative phenotype, seen when the CAG repeat yields a poly-Ala stretch, may turn out to be an excellent tool for functional analysis of GR in transgenic organisms.

Amino Acid Sequence↗
PubMed1994

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Adenoviridae↗
PubMed1994

Serum 2'-5'-oligoadenylate synthetase levels and clinical response to interferon-beta therapy in women with genital human papillomavirus infection.

To investigate the activity of parenterally administered interferon-beta (IFN-beta) in women with genital human papillomavirus (HPV) infection, serum 2'-5'-oligoadenylate synthetase (2'-5'OAS), an enzyme induced by IFNs, was measured before, during, and after therapy. Serum levels of 2'-5'OAS significantly increased during therapy (P = .005). The patients classified as not responding to treatment at 3 months of follow-up had baseline levels of 2'-5'OAS significantly higher than those who responded to treatment (P = .0011). Differences between the IFN-beta-treated group and untreated controls appeared at day 6 (P = .0019). 2'-5'OAS is a useful parameter in monitoring parenterally administered IFN-beta. The data suggest that women with genital HPV infection and elevated baseline levels of 2'-5'OAS are unlikely to respond to therapy with IFN-beta.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 replication in cytokine-stimulated monocytes/macrophages by combination therapy.

Combination regimens against human immunodeficiency virus type 1 (HIV-1) were studied in granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated monocyte/macrophage cultures. Regimens included those that inhibited the same target (reverse transcriptase) or multiple targets. Treatment conditions assessed efficacy during prophylaxis and ongoing infection. Drugs included zidovudine, didanosine, nevirapine, foscarnet, pyridinone, the protease inhibitor RO31-8959 (also known as saquinavir), interferon-alpha A, the Tat inhibitor RO24-7429, and N-butyl-deoxynojirimycin. Two-, three-, and four-drug combinations were tested. Drugs were tested at individually inhibitory concentrations of IC99, IC95, IC75, and IC50. All prophylactic regimens prevented HIV-1 replication at IC99. As drug concentrations were reduced, differences among the regimens became apparent. Regimens that acted at both single and multiple targets were effective in prophylactic settings and less so in acute infection. In ongoing infections, only modest reductions in viral replication were seen, even at IC99.

Antiviral Agents↗
PubMed1994

Alternating versus continuous drug regimens in combination chemotherapy of human immunodeficiency virus type 1 infection in vitro.

We compared the in vitro efficacies of two-, three-, and four-drug combinations given continuously or in alternating regimens against a clinical isolate of human immunodeficiency virus type 1. In H9 cells and peripheral blood mononuclear cells, at the drug concentrations used in this study, there was greater suppression of human immunodeficiency virus type 1 infection as the number of drugs in the regimen was increased from one to four simultaneously administered agents. Although alternating drug regimens were effective, they were not better than continuous administration of either single drugs or combinations of agents and were less effective than giving all drugs of an alternating regimen simultaneously.

Antiviral Agents↗
PubMed1994

A conserved carboxy-terminal subdomain is important for ligand interpretation and transactivation by nuclear receptors.

Nuclear receptors share a highly conserved region located at the very carboxy-terminal part of the ligand-binding domain. Site-directed mutagenesis of conserved hydrophobic residues in this region was reported to create mouse and human glucocorticoid receptors (GRs) and estrogen receptors that cannot transactivate but apparently maintain all the other functions. We constructed analogous mutations in the rat GR to compare the mechanism of deficiency to our recently generated trans-dominant-negative mutant. We found that in the rat GR these carboxy-terminal mutations do not generate trans-dominant-negative receptors. We show that these GR mutants fail to bind dexamethasone properly, and hence receptor transformation and subsequent functions are abolished. Furthermore, we report the identification of a GR mutant that is strongly responsive to the antagonist RU486 but is silent with the agonist dexamethasone. We demonstrate that the reversal of the responsiveness is restricted to GR, since analogous mutations in related receptors do not generate similar phenotypes. Contrary to the case of the progesterone receptor, we show that carboxyl-end truncated GR mutants are not activated by RU486. We conclude that sequence conservation of this subdomain does not necessarily imply functional conservation. Chimeric constructs with GAL4 revealed the importance of protein-protein interactions to exert ligand discrimination, which is mediated by the carboxy-terminal subdomain.

Alanine↗
PubMed1994

Tracheoesophageal fistula in an HIV-1-positive man due to dual infection of Candida albicans and cytomegalovirus.

We report a 35-year-old HIV-1-positive man who presented with severe dyspnea and a nonproductive cough. Three fiberoptic bronchoscopic examination revealed an infiltrating and vegetating tracheal mass that was diagnosed as necrotizing candidiasis of the trachea. The lesion resulted in the formation of a tracheoesophageal fistula that eventually led to the death of the patient. Postmortem examination showed cytomegalovirus vasculitis in the esophageal wall.

AIDS-Related Opportunistic Infections↗
PubMed1994

Mycobacterial infections in AIDS: an overview of epidemiology, clinical manifestations, therapy and prophylaxis.

One of the most frequent complications of AIDS is Mycobacterial infections. The incidence of tuberculosis has dramatically increased in all countries as a result of the HIV epidemic. Lately, it has been found that the natural history of new Mycobacterium tuberculosis infection is accelerated by HIV disease. In a wide number of cases the emergence of Mycobacterium tuberculosis nosocomial outbreaks of drug-sensitive and drug-resistant strains has been reported in HIV infected patients. The inadequate efforts to provide complete therapy to this kind of patient has caused the emergence of multidrug-resistant tuberculosis, that is responsible for the increased mortality rate in AIDS patients. A renewed interest in mycobacterial infections has also been kindled by the occurrence of Mycobacterium avium infections in patients with acquired immunodeficiency syndrome. The role of Mycobacterium avium as a pathogen is actually confusing and controversial for clinicians who care for AIDS patients. Disseminated Mycobacterium avium infections occur in a high population of HIV infected patients with low CD4+ cell count. Recent studies reported that rifabutin significantly reduced the incidence of Mycobacterium avium bacteremia, although, new macrolides such as clarithromycin and azithromycin are also effective in the treatment of the infection. Therefore, because of the emergence of macrolides resistance, the use of combination therapy is highly recommended in the Mycobacterium avium infection management.

AIDS-Related Opportunistic Infections↗
PubMed1993

Zinc finger mutations that alter domain interactions in the glucocorticoid receptor.

The DNA binding domain of steroid receptors coincides with the cysteine-rich region encompassing the two conserved zinc fingers. In the case of the glucocorticoid receptor (GR), a weak transactivation function has been described to be adjacent or partly overlapping to the DNA binding domain, whereas stronger trans-acting functions are encoded by the amino and the carboxy domain. In this report we describe the phenotype produced by stochastic mutations of the zinc finger region. The mutants were obtained either by selected rearrangements of the rat GR cDNA, or by semi-random nucleotide substitutions. All the identified permissive rearrangements were confined to a region downstream from the first zinc finger (duplications starting between residue 474 and 492). In general, the phenotype of point mutations is compatible with established structural data. Nevertheless, we found two unexpected phenotypes. First, we noticed that the double mutant His451 Asn/Ser459Gly is stronger than the wild-type sequence in DNA binding. Secondly, substitution of the conserved Lys461 results in an abnormal behavior of the mutated GR. In particular, the mutant Lys461Tyr (61Y) displays about the same transactivation when tested in form of a minimal GR fragment (amino acids 407/556) as when tested in the amino-prolongued GR fragment (amino acids 3-556, which contains the major transactivation domain of the GR). This is in contrast with the behavior of the other mutants in which the residue 461 is intact. In these cases, transactivation capacity is normally increased more than 30-fold from GR407-556 to GR3-556. These results are discussed in terms of possible cross-talk among the DNA binding domain and other functions residing in the amino domain of the GR.

Amino Acid Sequence↗
PubMed1993

Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanogaster ecdysteroid receptor (dEcR).

We have cloned a cDNA sequence coding for a Chironomus tentans steroid hormone receptor homologue which exhibits extensive amino acid sequence co-linearity with the ecdysteroid receptor of Drosophila melanogaster (dEcR; cell 67, 59-77). The DNA-binding domain has 95% and the hormone-binding domain 75% amino acid sequence identity with the cloned dEcR. The gene for this C. tentans protein is located on chromosome II, region 17C, as determined by in situ hybridization to polytene chromosomes of salivary glands. On Northern blots cDNA probes of the cloned gene hybridize to polyadenylated RNA of ca 4.2 kb. The expression of the cloned gene seems to be developmentally regulated and correlates to changes in ecdysteroid titer. Transfection of this C. tentans protein into D. melanogaster Schneider's line 2 cells leads to transcriptional interference with endogenous dEcR on an ecdysteroid-regulated promoter.

Amino Acid Sequence↗
PubMed1993

Persistent repression of a functional allele can be responsible for galactosyltransferase deficiency in Tn syndrome.

A human hematopoietic disorder designated as Tn syndrome or permanent mixed-field polyagglutinability has been ascribed to a stem cell mutation leading to a specific deficiency of UDP-Gal:GalNAc alpha 1-O-Ser/Thr beta 1-3 galactosyltransferase (beta 3 Gal-T) activity in affected cells. To test for the possibility that an allele of the beta 3Gal-T gene might be repressed instead of mutated, we have investigated whether 5-azacytidine or sodium n-butyrate, both inducers of gene expression, would reactivate expression of beta 3Gal-T in cloned enzyme-deficient T cells derived from a patient affected by the Tn syndrome. Flow cytometry revealed that a single treatment induced de novo expression of the Thomsen-Friedenreich antigen (Gal beta 1-3GalNAc-R), the product of beta 3Gal-T activity. In addition, a sialylated epitope on CD43 (leukosialin), which is present on normal but not on beta 3Gal-T-deficient T cells, was also reexpressed. Although no beta 3Gal-T activity was detectable in untreated Tn syndrome T cells, after exposure to 5-azaC,beta 3Gal-T activity reached nearly normal values. Both agents failed to reactivate beta 3Gal-T in Jurkat T leukemic cells, which also lack beta 3Gal-T activity. These data demonstrate that Tn syndrome T cells contain an intact beta 3Gal-T gene copy and that the enzyme deficiency in this patient is due to a persistent and complete but reversible repression of a functional allele. In contrast, the cause of beta 3Gal-T deficiency appears to be different in Jurkat T cells.

Alleles↗