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Biomedical subjects

M Giacca

Publications and source records attributed to M Giacca.

At least 91 records · Page 5Linked to original sources
PubMed1996

Rapid retrovirus titration using competitive polymerase chain reaction.

A quantitative polymerase chain reaction (PCR) procedure has been developed for rapid retrovirus titration. This procedure, which is based on the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantification of viral RNA genomes in retrovirus-producing cell clone supernatants and of proviral DNA molecules formed at 24 h after infection of different reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in complete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the producer cell clone supernatants is a poor predictor of the actual efficiency of infection. Repeated competitive PCR experiments for provirus copy number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, suggesting stable integration into the host genome. The developed procedure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the efficiency of infection of any target cell type, thus overcoming the problem of the dependency of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony formation of a reference cell line.

3T3 Cells↗
PubMed1996

Genetic factors in dilated cardiomyopathy.

Recent studies have demonstrated that genetic factors are likely to play a major role in the pathogenesis of idiopathic dilated cardiomyopathy (IDC). In clinical surveys, a familial trait has been demonstrated in 20 to 30% of idiopathic dilated cardiomyopathy patients (familial dilated cardiomyopathy). Molecular genetic studies have confirmed the clinical hypothesis of genetic heterogeneity in familial dilated cardiomyopathy, and are currently producing relevant advances in the understanding of this disease. The autosomal dominant form is considered to be the most frequent form of inherited idiopathic dilated cardiomyopathy. After the exclusion of a large series of candidate genes, the first familial dilated cardiomyopathy gene has been mapped to the long arm of chromosome 9. A second locus has been found on chromosome 1. Moreover, in two large families, characterized by a peculiar form of conduction delays and later development of myocardial dysfunction, the disease loci have been mapped to chromosome 1 and 3, respectively. The identification of the disease genes is in progress. In families with X-linked dilated cardiomyopathy, the disease gene has been identified as the dystrophin gene. The 5' end of the gene appears to be the critical region for the development of dilated cardiomyopathy without clinical evidence of muscle dystrophy. Furthermore, other cytoskeletal proteins, such as adhalin, could be involved in the pathogenesis of familial dilated cardiomyopathy. In familial right ventricular cardiomyopathy (or arrhythmogenic right ventricular dysplasia) characterized by isolated or prevalent right ventricular involvement, three different disease loci have been identified so far: two localized on the long arm of chromosome 14 and one on chromosome 1. The disease genes are still unknown and are currently under investigation. The study of the genetic factors at the molecular level is starting to elucidate the pathogenetic mechanisms of idiopathic dilated cardiomyopathy. These findings will also have relevant clinical and therapeutic implications.

Cardiomyopathy, Dilated↗
PubMed1996

Detection of c-erbB-2 amplification in transitional cell bladder carcinoma using competitive PCR technique.

PURPOSE: We used competitive PCR to verify retrospectively the prognostic significance of c-erbB-2 oncogene amplification in transitional cell bladder carcinomas as a predictive index of patient survival with a maximum follow-up of nine years, and to investigate the variations of c-erbB-2 amplification during bladder carcinoma recurrence and/or progression from superficial to more invasive states. MATERIALS AND METHODS: Oncogene amplification was determined by an accurate and sensitive procedure based on competitive PCR. Measurements were performed in DNA extracted from fresh cancers or from formalin-fixed, paraffin-embedded tumor samples. RESULTS: The overall mean incidence of c-erbB-2 oncogene amplification was 26% (24/92), with a significant relationship with tumor grade (p < 0.0001). We did not find any statistical difference in survival probability between subjects with (20%) or without (30%) oncogene amplification. During tumor progression we observed a limited increase of tumors carrying oncogene amplification (2 of 20) whereas the mean degree of amplification was not affected. CONCLUSIONS: c-erbB-2 amplification seems to be a genetic event related to the degree of bladder tumor differentiation. However the presence and/or the degree of this genetic alteration do not seem predictive of tumor progression, recurrence and survival probability, at least in patients with advanced transitional cell bladder carcinoma. These data seem to indicate that the amplification of c-erbB-2 in bladder carcinoma could be considered as an epiphenomenon, present in a subset of tumors but apparently not related to the clinical outcome.

Aged↗
PubMed1995

Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction.

BACKGROUND/AIMS: The determination of HCV-RNA concentration in liver samples is likely to provide interesting insights for the study of disease progression and for evaluation of the efficacy of anti-viral therapy. METHODS: A procedure was developed for the precise quantification of HCV-RNA in liver biopsies, based on the competitive reverse transcription-polymerase chain reaction technology. This competitive assay consists of the co-amplification of the target RNA with known amounts of a competitor RNA molecule containing the same sequence as the target plus an insertion in the middle, allowing resolution of the two amplification products by gel electrophoresis. RESULTS: The amounts of HCV-genomic-RNA and beta-actin mRNA (the latter being used as an internal standard to overcome the problem of reproducibility of quantitative RNA extraction) were evaluated in liver biopsies of 15 patients affected by hepatitis C virus-positive chronic liver disease at the time of diagnosis. All the patients underwent alpha-interferon therapy for 6 months and were subsequently followed for at least 1 further year after the end of treatment. Viral RNA concentration (which ranged from 2 to 2.7 x 10(5) HCV-RNA molecules per 10(6) beta-actin molecules) directly correlated with the efficacy of treatment, indicating that low levels of viral replication in the liver are associated with a poor response to therapy. CONCLUSIONS: This study suggests that the determination of viral load in the liver is an important prognostic tool for the prediction of the efficacy of alpha-interferon therapy.

Adult↗
PubMed1995

Molecular genetics of dilated cardiomyopathies.

The application of molecular genetics in cardiology is currently producing important results in the study of the pathogenetic mechanisms underlying cardiomyopathies. Recent clinical surveys have indicated that genetic factors play a major pathogenetic role in idiopathic dilated cardiomyopathy (IDC). Familial IDC is frequent (20-30%) and is probably a heterogeneous entity, as suggested by the clinical variability and the different pattern of inheritance in the affected families. Molecular genetic studies have demonstrated the existence of heterogeneity also at the genetic level. In a series of families with X-linked IDC, the disease gene has been identified as the dystrophin gene. In familial right ventricular cardiomyopathy (or right ventricular dysplasia), a new nosological entity characterized by isolated right ventricular involvement that can mimic IDC, the disease gene has been localized in the long arm of chromosome 14. In families with matrilineal transmission, the cardiomyopathy could be linked to mitochondrial DNA alterations. Autosomal dominant familial IDC, considered to be the most frequent form, is currently under active investigation. Our preliminary data have excluded a large series of candidate genes, among which are the cardiac beta-myosin heavy chain and several other genes encoding for cardiac contractile proteins, genes of the HLA region, and about 60 genes involved in the immune regulation.

Cardiomyopathy, Dilated↗
PubMed1995

Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1.

The human transcription factor USF, purified from HeLa cells, and its recombinant 43-kDa component bind to the long terminal repeat (LTR) of human immunodeficiency virus type 1. The proteins footprint over nucleotides from position -173 to -157 upstream of the transcription start site, generating strong DNAse I hypersensitivity sites at the 3' sides on both strands. As detected by methylation protection studies, the factor forms symmetric contacts with the guanines of the palindromic CACGTG core of the recognized sequence. Its binding ability is abolished by the mutation of this core sequence and is strongly reduced by the cytosine methylation of the central CpG dinucleotide. Upon binding, both recombinant and purified USFs bend the LTR DNA template. The role of USF in the control of transcription initiation from the LTR was tested by in vitro transcription assays. Upon addition of the protein, transcription from constructs containing an intact binding site is increased, while the responsiveness in constructs with a mutated sequence is abolished. Furthermore, the addition of a decoy plasmid which contains multiple repeats of the target sequence results in downregulation of transcription from the LTR. These results suggest that USF is a positive regulator of LTR-mediated transcriptional activation.

Base Sequence↗
PubMed1995

Linkage of familial dilated cardiomyopathy to chromosome 9. Heart Muscle Disease Study Group.

Idiopathic dilated cardiomyopathy is a heart muscle disease of unknown etiology, characterized by impaired myocardial contractility and ventricular dilatation. The disorder is an important cause of morbidity and mortality and represents the chief indication for heart transplantation. Familial transmission is often recognized (familial dilated cardiomyopathy, or FDC), mostly with autosomal dominant inheritance. In order to understand the molecular genetic basis of the disease, a large six-generation kindred with autosomal dominant FDC was studied for linkage analysis. A genome-wide search was undertaken after a large series of candidate genes were excluded and was then extended to two other families with autosomal dominant pattern of transmission and identical clinical features. Coinheritance of the disease gene was excluded for > 95% of the genome, after 251 polymorphic markers were analyzed. Linkage was found for chromosome 9q13-q22, with a maximum multipoint lod score of 4.2. There was no evidence of heterogeneity. The FDC locus was placed in the interval between loci D9S153 and D9S152. Several candidate genes for causing dilated cardiomyopathy map in this region.

Cardiomyopathy, Dilated↗
PubMed1995

Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template.

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.

Base Sequence↗
PubMed1994

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Animals↗
PubMed1994

The quest for a human ori.

Attempts at identifying DNA replication origins in human cells have been performed with a variety of molecular genetic and biochemical approaches, with often controversial results. The combination of bromodeoxyuridine labelling, immunopurification of newly synthesized labelled DNA, measurement of the relative abundance of markers in this DNA by quantitative competitive PCR, has allowed the identification within 450 bp of the start-site of DNA replication located at the human lamin B2 gene. The origin is located near the non-transcribed spacer between two highly transcribed genes and shows evidence of a number of specific protein-DNA interactions, the most prominent of which disappears when the cells are differentiated into a non-proliferating state.

Chromosome Mapping↗
PubMed1994

Low frequency of detection by nested polymerase chain reaction of enterovirus ribonucleic acid in endomyocardial tissue of patients with idiopathic dilated cardiomyopathy.

OBJECTIVES: The purpose of this study was to determine the prevalence of enteroviral infection in the myocardium of patients with idiopathic dilated cardiomyopathy by using a highly sensitive and specific detection technique. BACKGROUND: Recent molecular studies have suggested that enteroviral persistence (in particular, coxsackieviruses type B) may underlie idiopathic myocarditis and dilated cardiomyopathy. METHODS: The method used to detect enterovirus-specific ribonucleic acids (RNAs) is based on reverse transcription and nested polymerase chain reaction amplification with four pairs of primers from the conserved 5' noncoding region of the enteroviral genome. Several members of the Enterovirus genus are detectable by this assay (coxsackieviruses B1 to B6; polioviruses 1 to 3; echoviruses 9, 19 and 31), with a sensitivity threshold close to the detection of a single molecule of viral RNA in 1 mg of tissue sample. Endomyocardial tissue samples from 84 subjects were analyzed (77 samples obtained from left endomyocardial biopsies, 7 from explanted hearts). The subjects comprised 63 study patients (53 with dilated cardiomyopathy, 3 with idiopathic myocarditis, 1 with right ventricular dysplasia, 1 with restrictive cardiomyopathy, 1 with eosinophilic myocarditis, 1 with primary ventricular fibrillation and 3 with myocarditis of known etiology) and 21 control subjects with other diseases. RESULTS: Positive signals were obtained only in samples from six study patients (four with dilated cardiomyopathy, one with right ventricular dysplasia and one with myocarditis). Samples from control subjects, uninfected rat myocardium and cultured cell lines yielded systematically negative results. Moreover, the nucleotide sequence analysis of the amplification products from patients with positive samples raised doubts about the true positivity of these samples. CONCLUSIONS: This study suggests that the persistence of enteroviral RNA in dilated cardiomyopathy is not a major cause of the disease and that a careful analysis of polymerase chain reaction amplification products is essential in any study in which this technique is pushed to high sensitivity thresholds.

Adult↗
PubMed1994

Stimulation of the adenovirus major late promoter in vitro by transcription factor USF is enhanced by the adenovirus DNA binding protein.

Previous studies have shown that the sequence-independent adenovirus DNA binding protein (DBP) increases transcription from several promoters, notably from the adenovirus major late promoter (MLP) and the adeno-associated virus P5 promoter, both of which contain a USF/MLTF binding site. In order to study this mechanism, we have investigated the effects of DBP on the binding of USF/MLTF to MLP and on transcription from MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the binding affinity of USF43 to the promoter three- to fourfold without changing the footprint pattern. In contrast, the binding of the TATA box binding protein to the promoter is not influenced by DBP. No protein-protein interactions between DBP and USF43 could be observed in the absence of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcription system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not have an effect on basal transcription. Our results suggest that transcription enhancement by DBP is based on a specific increase in the binding of a transcription factor to a promoter through subtle changes in DNA structure, similar to the mechanism by which DBP stimulates the initiation of DNA replication.

Adenoviridae↗
PubMed1994

Absence of linkage between idiopathic dilated cardiomyopathy and candidate genes involved in the immune function in a large Italian pedigree.

Idiopathic dilated cardiomyopathy (IDC) is a heart disease of unknown aetiology characterised by impaired ventricular function usually associated with dilatation of the cardiac chambers. In order to test the hypothesis of an immunological cause for the disease at the genetic level, we performed linkage analysis between the putative disease locus and some of the potential candidate genes involved in the immune response or coding for the targets for autoantibodies in a large multigeneration family (63 members) from southern Italy with autosomal dominant transmission of the disease. Twenty-nine polymorphic markers on 18 different chromosomal locations were investigated, including markers linked to the genes coding for the HLA antigens, the immunoglobulin heavy and light chains, the receptors for the immunoglobulin Fc fragments, the subunits of the T cell receptor and the associated CD3, CD4, CD8, and CD45 antigens, interleukins 1, 3, 4, 5, 6, 9, and 11, the interleukin 1 and 2 receptors, and the genes coding for the beta 1 adrenoreceptor, the adenine nucleotide translocator-1, and the cardiac alpha and beta myosin heavy chains. No evidence for genetic linkage to IDC was found at any of these candidate loci. These results indicate that the still unidentified IDC gene maps outside several loci involved in the regulation of immune reactivity.

Adult↗
PubMed1994

Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction.

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.

Base Sequence↗
PubMed1994

Molecular genetics of dilated cardiomyopathy.

The recent advances in molecular biology and genetic engineering are producing relevant results in cardiology, in particular in the field of cardiomyopathies. Molecular genetics has been used for the detection of viral genomes persisting in myocardial tissue of patients with idiopathic dilated cardiomyopathy (IDC). Very recent data, based on highly sensitive and specific technologies, suggest, however, that enterovirus persistence is not a major cause of the disease. Another application of molecular genetics is the study of the familial form of IDC, using linkage analysis as a tool for the identification of the disease gene. According to this method, the X-linked form of familial IDC has been mapped within the dystrophin gene in a series of families, and preliminary reports suggest that the mutation (or mutations) concerns the muscle-promoter region. Linkage studies on the autosomal dominant form of IDC are in progress. A possible approach is the identification of linkage between the disease and an array of "candidate genes". Preliminary data have ruled out the involvement of a series of relevant candidates genes, among which the cardiac beta-myosin heavy chain gene. An alternative approach for linkage studies is to perform a random screening of the genome, in which a large number of anonymous markers are selected and tested. In conclusion, molecular genetics is starting to provide fundamental data about the pathogenetic mechanisms of IDC. The relevance of these findings is also crucial for clinical and therapeutic implications.

Cardiomyopathy, Dilated↗