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Biomedical subjects

F Bernardi

Publications and source records attributed to F Bernardi.

At least 19 recordsLinked to original sources
PubMed1995

Inhibition of viral aphid transmission by the N-terminus of the maize dwarf mosaic virus coat protein.

Since removal of the exposed N-terminus of the coat protein of some potyviruses abolishes aphid transmission, the role of this coat protein region of maize dwarf mosaic potyvirus (MDMV) in aphid transmission was investigated. The viral cDNA encoding this region was cloned and expressed as a fusion protein in bacteria. The resulting purified N-terminus of the coat protein was used in controlled aphid transmission experiments in competition with MDMV. The results show that this region inhibits aphid transmission of MDMV, indicating a direct involvement of the N-terminal region of the coat protein in aphid transmission.

Amino Acid Sequence↗
PubMed1995

Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy.

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

Base Sequence↗
PubMed1995

Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis.

Between December 1990 and January 1994, bone marrow (BM) samples from 151 patients with multiple myeloma (MM), including 117 patients evaluated at diagnosis, were collected for cytogenetic analysis. A total of 129 patients had assessable metaphases (100 patients at diagnosis). Cytogenetic studies were performed on BM cells after longterm cultures (6 days) with stimulation of cultures by granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF plus interleukin (IL)-6, IL-3 plus IL-6, or GM-CSF plus IL-3 plus IL-6 to improve myeloma cell growth, and 91 patients had an additional unstimulated culture. Sixty-six patients (51%) had cytogenetic abnormalities, including 47 of 100 patients at diagnosis (47%) and 17 of 24 patients at relapse (71%; P = .04). The aberration rate increased with stage (P = .007), BM plasmacytosis (P = .003), beta 2 microglobulin level (P = .001), C-reactive protein (CRP) level (P = .001), and Ki-67 (P = .007). The abnormality detection rate was higher in stimulated than unstimulated cultures, and the difference was statistically significant (P < .01). Hyperdiploidy was observed in 39 patients (30% of patients with an assessable karyotype) and hypodiploidy in 19 patients (15%). Among numeric changes, gains predominantly involved chromosomes 3, 5, 7, 9, 11, 15, 19 and losses, chromosomes 8, 13, 14, and X. The most frequent loss was loss of chromosome 13, observed in 22 patients (15%), including 18 patients at diagnosis (12%). We observed frequent structural changes of chromosomes 1 (15%) and 14 (10%) but also a 5% incidence of 19q13 abnormality and two patients with translocation t(1;16)(p11;p11). By using the proportional hazard univariate model, patients with abnormal karyotypes were demonstrated to have 2.5-fold greater chance of death than patients with normal karyotypes (P < .014). Despite a multivariate approach with the same model, the respective roles of karyotype abnormality, age, stage, and beta 2 microglobulin level could not be clearly ascertained. From these results we conclude that cytogenetic analysis using stimulation of cultures by cytokine(s) may be a promising method to identify about 50% of cytogenetic abnormalities in patients with newly diagnosed MM. Cytogenetic analysis may help to define a high-risk population that would benefit from intensive therapeutic approaches.

Adult↗
PubMed1995

Molecular bases of CRM+ factor X deficiency: a frequent mutation (Ser334Pro) in the catalytic domain and a substitution (Glu102Lys) in the second EGF-like domain.

The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in asymptomatic subjects with FX deficiency characterized by the presence of dysfunctional molecules in plasma, as demonstrated by the discrepancy between clotting activity and antigen level. A missense mutation (Ser334Pro) in the catalytic domain was found in three unrelated families in both the homozygous and the heterozygous conditions, and also in the compound heterozygous form with the substitution of Lys for 102 Glu. None of the mutations was detected in 40 unrelated subjects from the same geographic area. The Ser334Pro mutation affects a serine protease region characterized by extensive variation in the coagulation factors but conserved in mammalian factor X molecules. The Glu102Lys mutation affects a residue of the second EGF-like module also conserved in protein C. Both mutated residues are surface-exposed and found in protein regions suggested to be involved in macromolecular interactions which are impaired in the dysfunctional molecules.

Base Sequence↗
PubMed1995

Monoclonal gammopathy of undetermined significance: chromosome changes are a common finding within bone marrow plasma cells.

We used two indirect approaches [image analysis (Feulgen staining) and fluorescence in situ hybridization (FISH)] to study bone marrow plasma cells (BMPC) in 28 patients fulfilling criteria for MGUS. 61% of patients were found to be aneuploid after image analysis: three were hypodiploid and 14 were hyperdiploid. 12/14 hyperdiploid patients also revealed abnormalities after FISH: 12-72% of BMPC exhibited trisomy for at least one of chromosomes 3, 7, 9 and 11. These latter chromosomes are the four chromosomes most frequently implicated (in the shape of trisomy) in MM, confirming the tight relationship between both conditions. After a median follow-up of 19 months (12-41 months) no patient developed overt MM. Also, we failed to find any relationship between currently available biological parameters and DNA findings. As literature data give a transformation rate of 20-30% after a follow-up of 20-35 years, it is worth presuming that some aneuploid patients will evolve to MM, whereas others (also with aneuploid bone marrow plasma cells) will never develop cancer. Our findings indicate that numeric abnormalities, as they are shared both by MGUS and MM patients, are certainly an additional or a prerequisite event, but are not related to an overt disease. They also emphasize the importance of cytogenetic study in the pathophysiology of MGUS.

Adult↗
PubMed1995

CD19 and immunophenotype of bone marrow plasma cells in monoclonal gammopathy of undetermined significance.

AIMS: To determine whether a particular phenotype or antigen is preferentially related to monoclonal gammopathies of undetermined significance (MGUS). METHODS: Bone marrow specimens from 56 patients with MGUS were stained immunocytochemically (ABC peroxidase) for CD38, CD56, CD9, CD10, CD19, CD20, CD22, and MB2. Specimens from patients recently diagnosed with multiple myeloma and reactive bone marrow samples were studied in parallel. RESULTS: CD38 was expressed on all plasma cells from all MGUS samples tested, while 36% were positive for CD56, CD9 and MB2 were both expressed strongly; CD20 was moderately expressed, and staining for CD10 and CD22 was uncommon. For these five B cell antigens there was no clear difference between their expression in MGUS and in multiple myeloma. A great difference was found for CD19: in MGUS this antigen was expressed on 2-91% of plasma cells (mean 35%) and 77% patients had > 10% positive plasma cells; in multiple myeloma its expression was low and only 12% patients had > 10% positive plasma cells. When these results were converted to numbers of CD19 positive plasma cells per 100 nucleated bone marrow cells, reactive bone marrow and MGUS specimens had a similar number of positive plasma cells. There was no correlation between expression of any of the antigens tested. CONCLUSIONS: Many of the so-called pre-B, B or activation antigens are present on plasma cells from MGUS specimens, and expression of CD9, CD10, CD20, CD22, MB2, and CD38 in MGUS was very similar to that in multiple myeloma. CD56 was frequently expressed in MGUS. In this series CD19 was highly expressed in MGUS but not in multiple myeloma. Plasma cells bearing this antigen could represent the non-neoplastic process and determination of its expression could be useful for the diagnosis of MGUS.

Antigens, CD↗
PubMed1995

Protein S mRNA in patients with protein S deficiency.

A protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) for free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.

Alleles↗
PubMed1995

Swyer-James syndrome: bronchoalveolar lavage findings in two patients.

Swyer-James syndrome (SJS) is a rare constrictive bronchiolitis that appears to be the result of acute bronchiolitis in infancy or early childhood. In the present study the cytological and immunophenotypical profile of bronchoalveolar lavage (BAL) was studied in two patients with SJS who showed a different spectrum of clinical outcome. The total BAL yield was markedly increased in the patient with chronic cough and acute episodes of dyspnoea but not in the patient with decreased exercise tolerance and longer duration of disease. In the two patients, the differential cell counts in percentage were characterized by a significant increase of neutrophils and a slight increase of lymphocytes. The analysis of lymphocyte subsets showed a significant increase of CD8+ cells (T-suppressor-cytotoxic) in both cases, resulting in a decreased CD4/CD8 ratio. In addition, an increase of cells bearing a B-phenotype (CD19+ cells) was noted in the fluid recovered from the patient with chronic cough and acute episodes of dyspnoea. In conclusion, our data suggest that SJS is an active process with inflammatory characteristics. Further studies are needed to explain the mechanism leading to the expansion in situ of immunocompetent cells.

Adolescent↗
PubMed1994

Combined surgery and chemotherapy in primary gastric non-Hodgkin's lymphoma: a retrospective study in sixty-six patients.

Sixty-six consecutive patients with primary gastric non-Hodgkin's lymphoma are reported. All patients underwent surgery which consisted of radical resection in 23 patients (36%) and partial or palliative excision in the remaining 43 cases (36 and 7 respectively). Three patients died before starting chemotherapy, two refused the treatment and 61 completed the postoperative chemotherapeutic programme. We analysed this group of patients in order to assess the efficacy of chemotherapy following surgery. Chemotherapy included either CVP or the original protocols from our institution. Excluding patients who underwent radical resection, postoperative chemotherapy induced complete remission in 87% of the remaining 39 patients. After a median follow-up of 84 months (range 6-216), the 10-year cause-specific survival was 90% with a stable curve plateau after about 25 months. The survival was only influenced by response to therapy (p < 0.0001). The disease-free survival for patients who were not radically resected was 93%. We encountered only two relapses after 15 and 32 months. One of these was local and the other systemic. Our results indicate that chemotherapy following surgery induces long-term remission and survival in primary gastric lymphoma and in particular improves remission and survival, in stage II. In our opinion, surgery may also be fundamental for the treatment of gastric lymphoma in the majority of cases.

Adolescent↗
PubMed1993

D8S7 is consistently deleted in inverted duplications of the short arm of chromosome 8 (inv dup 8p).

Ten patients with inverted duplication of 8p (inv dup 8p) were studied with cytogenetic, biochemical and molecular techniques. The duplication for the region 8p12-p22 was always associated with a deletion of the locus D8S7 (mapped in 8p23.1) as demonstrated with the probe pSW50 by both in situ hybridization and Southern blot. Restriction fragment length polymorphisms detected by probes pSW50 (1 case) and by pG2LPL35 (locus LPL) (two cases) were informative as to a maternal origin of the anomaly. The activity of glutathione reductase, whose gene maps in the duplicated region at 8p21.1, was increased in all patients. The recognizable phenotype of inv dup 8p includes neonatal hypotonia, prominent forehead, large mouth with everted lower lip, abnormally shaped large ears, brain malformations and severe mental retardation. Our findings indicate that the chromosome rearrangement is homogeneous at least for the presence of the deletion and support the hypothesis of a common mechanism of origin.

Abnormalities, Multiple↗
PubMed1993

Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency.

A protein S gene polymorphism, detectable by restriction analysis of amplified exonic sequences, was investigated in a family with members affected by protein S deficiency, deep vein thrombosis and ictus. The clinical laboratory findings as well as RFLP analysis were consistent with the presence of a type WP III protein S deficiency clearly marked by a polymorphic allele, thus enabling us to determine the carrier status in several subjects. The RFLP analysis, extended to platelet mRNA after reverse transcription and amplification, demonstrated that the mRNA produced by the putative defective gene was present in a subject affected by thrombosis.

Adult↗
PubMed1992

Species specific protein--DNA interactions may determine the chromatin units of genes in S.cerevisiae and in S.pombe.

Yeast genes, such as URA3, are chromatin units characterized by positioned nucleosomes and flanking nuclease sensitive regions (NSRs). To investigate the structural determinants at the chromatin level in vivo, the URA3 gene was dissected into three parts (U5', Umid and U3'), and the chromatin structures of the individual parts were analysed after insertion into minichromosomes and after chromatin assembly in vivo in Saccharomyces cerevisiae. While nucleosome positions were altered on Umid, the 5'-end and the 3'-end of URA3 maintained their native structures (a positioned nucleosome and a NSR each) independent of the site or orientation of insertion. This suggests that the chromatin unit of the native URA3 gene is dominated by strong protein boundaries at the 5'- and 3'-ends. In an alternative approach, we investigated whether nucleosome positions or NSRs were maintained when the whole URA3 gene was placed on a shuttle vector and assembled into chromatin by Schizosaccharomyces pombe providing different proteins, but the same nucleosomal spacing. In a complementary exchange experiment, the ade6 gene of S.pombe was shuttled to S.cerevisiae. In spite of a general conservation of histone proteins and nucleosome core structures, neither nucleosome positions nor NSRs were maintained in the heterologous background. The results demonstrate that chromatin structures are species specific and that the structural boundaries of yeast genes may be dominated by strong species specific protein-DNA interactions.

Chromatin↗
PubMed1992

Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7).

The 3' portion of the coagulation factor VII gene, containing the activation and serine protease domains, was investigated in four subjects with factor VII deficiency by temperature gradient gel electrophoresis and sequencing of polymerase chain reaction (PCR) products. Molecules displaying an altered melting behaviour were detected in three subjects, and direct sequencing showed two mutations. A G-to-T transversion causing a missense mutation, Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases. This mutation, which in the homozygous form causes a severe reduction in protease activity (4%), was found in two patients from different Italian regions. A G-to-A transition, which gives rise to a missense mutation, Arg-304 to Gln, and is associated with the factor VII padua variant, was found in the heterozygous form in a subject also affected by von Willebrand disease. Two polymorphic alleles, which differ in one repeat monomer element, were precisely mapped in a region spanning the exon-intron 7 border of the factor VII gene and studied in families with factor VII or X deficiency.

Base Sequence↗