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Biomedical subjects

M Zani

Publications and source records attributed to M Zani.

13 recordsLinked to original sources

BRCA1 and BRCA2 genetic testing in Italian breast and/or ovarian cancer families: mutation spectrum and prevalence and analysis of mutation prediction models.

BACKGROUND: Breast cancer is an extremely complex disease, characterized by a progressive multistep process caused by interactions of both genetic and non-genetic factors. A combination of BRCA1 and BRCA2 gene mutations appears responsible for about 20%-30% of the cases with breast cancer familial history. The prevalence of BRCA1/2 pathogenic mutations largely varies within different populations; in particular, the rate of mutations in Italian breast and/or ovarian cancer families is rather controversial and ranges from 8% to 37%. PATIENTS AND METHODS: Of the 152 breast/ovarian cancer families counseled in our centre, 99 were selected for BRCA1/2 mutation screening according to our minimal criteria. The entire coding sequences and each intron/exon boundary of BRCA1/2 genes were screened by direct sequencing (PTT limited to BRCA1 exon 11). For each proband, the a priori probability of carrying a pathogenic BRCA1/2 germline mutation was calculated by means of different mutation prediction models (BRCApro, IC and Myriad Table) in order to evaluate their performances. RESULTS: Our analysis resulted in the identification of 25 and 52 variants in the BRCA1 and BRCA2 genes, respectively. Seventeen of them represent novel variants, including four deleterious truncating mutations in the BRCA2 gene (472insA, E33X, C1630X and IVS6+1G>C). Twenty-seven of the 99 probands harbored BRCA1 (n = 15) and BRCA2 (n = 12) pathogenic germline mutations, indicating an overall detection rate of 27.3% and increasing by more than 15% the spectrum of mutations in the Italian population. Furthermore, we found the lowest detection rate (19.4%) in pure hereditary breast cancer family subset. All of the prediction models showed praises and faults, with the IC software being extremely sensitive but poorly specific, compared to BRCApro. In particular all models accumulated most false-negative prediction in the HBC subset. Interestingly preliminary results of a study addressing the presence of genomic rearrangements in HBC probands with BRCApro or IC prediction scores >/=95%, provided evidence for additional mutations undetectable with our conventional screening for point mutations. CONCLUSIONS: Altogether our results suggest that HBC families, the largest pool in our series, represent an heterogeneous group where the apparently faulty performances of the prediction models might be at least partially explained by the presence of additional kinds of BRCA1/2 alteration (such as genomic rearrangements) or by mutations on different breast cancer related genes.

Adult↗

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

Amino Acid Sequence↗

EGF regulates a complex pattern of gene expression and represses smooth muscle differentiation during the neurotypic conversion of the neural-crest-derived TC-1S cell line.

EGF, known to sustain CNS neuronal progenitors, also promotes a neurotypic response in the thymic neural-crest-derived TC-1S cell line. We report here the use of TC-1S cells as a model to identify the genetic programs regulated during the neurotypic response induced by EGF and to isolate 23 EGF-responsive genes. Among them 5 represent novel cDNAs, while 18 are known genes, whose regulation by EGF is associated with the mitogenic or differentiating effects of the growth factor. The repression of smooth muscle alpha-actin and SM22alpha genes by EGF and their increase by TGFbeta suggest that the TC-1S line includes neural crest multipotent cells whose smooth muscle differentiation is repressed upon EGF treatment and stimulated by TGFbeta. Therefore, we identified a complex pattern of EGF-target genes and propose EGF as a novel signal able to recruit postmigratory neural-crest-derived cells along proliferation and cell lineage choice pathways.

Animals↗

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

Blotting, Northern↗

Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status.

HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours.

Antineoplastic Agents↗

HMGI(Y) and HMGI-C genes are expressed in neuroblastoma cell lines and tumors and affect retinoic acid responsiveness.

HMGI-C and HMGI(Y) are architectural DNA-binding proteins that participate in the conformational regulation of active chromatin. Their pattern of expression in embryonal and adult tissues, the analysis of the "pygmy" phenotype induced by the inactivation of the HMGI-C gene, and their frequent qualitative or quantitative alteration in experimental and human tumors indicate their pivotal role in the control of cell growth, differentiation, and tumorigenesis in several tissues representative of the epithelial, mesenchymal, and hematopoietic lineages. In contrast, very little information is available on their expression and function in neural cells. Here, we investigated the expression of the HMGI(Y) and HMGI-C genes in neuroblastoma (NB), a tumor arising from an alteration of the normal differentiation of neural crest-derived cells and in embryonal and adult adrenal tissue. Although HMGI(Y) is constitutively expressed in the embryonal and adult adrenal gland and in all of the NB cell lines and ex vivo tumors examined, its regulation appears to be associated to growth inhibition and differentiation because we observed that HMGI(Y) expression is reduced by retinoic acid (RA) in several NB cell lines that are induced to differentiate into postmitotic neurons, whereas it is up-regulated by RA in cells that fail to differentiate. Furthermore, the decrease of HMGI(Y) expression observed in RA-induced growth arrest and differentiation is abrogated in cells that have been made insensitive to this drug by NMYC overexpression. In contrast, HMGI-C expression is down-regulated during the development of the adrenal gland, completely absent in the adult individual, and only detectable in a subset of ex vivo NB tumors and in RA-resistant NB cell lines. We provide evidence of a causal link between HMGI-C expression and resistance to the growth arrest induced by RA in NB cell lines because exogenous HMGI-C expression in HMGI-C-negative and RA-sensitive cells is sufficient to convert them into RA-resistant cells. Therefore, we suggest that HMGI-C and HMGI(Y) may participate in growth- and differentiation-related tumor progression events of neuroectodermal derivatives.

Adrenal Glands↗

2-Aminopurine unravels a role for pRB in the regulation of gene expression by transforming growth factor beta.

Transforming growth factor type beta (TGFbeta) is a pleiotropic factor that regulates different cellular activities including cell growth, differentiation, and extracellular matrix deposition. All the known effects of TGFbeta appear to be mediated by its interaction with cell surface receptors that possess a serine/threonine kinase activity. However, the intracellular signals that follow receptor activation and lead to the different cellular responses to TGFbeta are still largely unknown. On the basis of the different sensitivity to the protein kinase inhibitor 2-aminopurine and the phosphatase inhibitor okadaic acid, we identified two distinct pathways through which TGFbeta activates a genomic response. Consistently, 2-aminopurine prevented and okadaic acid potentiated the induction of JE by TGFbeta. The induction of PAI-1 and junB was instead potentiated by 2-aminopurine, after a transient inhibition and was unaffected by okadaic acid. The superinducing effect of 2-aminopurine required the presence of a functional RB protein since it was abolished in SV40 large T antigen-transfected cells, absent in the BT549 and Saos-2 RB-defective cell lines, and restored in BT549 and Saos-2 cells after reintroduction of pRB. The effects of 2-aminopurine on the TGFbeta inducible junB expression occur in all the cell lines examined suggesting that junB, and possibly other genes, can be regulated by TGFbeta through a distinct pRB-dependent pathway.

2-Aminopurine↗

[The Internet and clinical medicine. An introduction to the biomedical information services available in electronic form on the "net of all nets"].

Clinical users expectations from medical informatics are evolving due to the wide availability of biomedical information services on the Internet. Thanks to hypertexts and advanced browsing tools users with no informatical expertise can travel on the Internet and easily gain access to textual databases. With a multimedia computer other kinds of information can be grabbed: images, sounds and audiovisual documents. Basic Internet services (electronic mail, discussion lists, file transfer protocol, terminal emulation) can be accessed from a wide range of hardware equipment. However, the real power of a world-scale computer network like the Internet will be unleashed only when its global connectivity will be linked to the powerful retrieval ability of existing clinical and related databases. While biomedical journals editors and other medical information providers are in the way to offer electronic versions of their paper-based products, at the leading edge of this world-scale process is the USA National Library of Medicine, with the Internet-compatible version of its Grateful Med software which is expected to be launched during 1996.

Computer Communication Networks↗

The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake.

Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatoza. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.

Animals↗

The interaction between exogenous DNA and sperm cells.

Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.

Animals↗

HTLV-V: a new human retrovirus isolated in a Tac-negative T cell lymphoma/leukemia.

A new human retrovirus was isolated from a continuous cell line derived from a patient with CD4+ Tac- cutaneous T cell lymphoma/leukemia. This virus is related to but distinct from human T cell leukemia/lymphoma virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus (HIV-1). With the use of a fragment of provirus cloned from one patient with T cell leukemia, closely related sequences were found in DNA of the cell line and of tumor cells from seven other patients with the same disease; these sequences were only distantly related to HTLV-I. The phenotype of the cells and the clinical course of the disease were clearly distinguishable from leukemia associated with HTLV-I. All patients and the wife of one patient showed a weak serological cross-reactivity with both HTLV-I and HIV-1 antigens. None of the patients proved to be at any apparent risk for HIV-1 infection. The name proposed for this virus is HTLV-V, and the date indicate that it may be a primary etiological factor in the major group of cutaneous T cell lymphomas/leukemias, including the sporadic lymphomas known as mycoses fungoides.

Antigens, Viral↗

HTLV-I is endemic in southern Italy: detection of the first infectious cluster in a white population.

Human T-cell leukemia virus (HTLV-I) infection is observed among black and Japanese populations in well-delimited endemic spots in association with a high incidence of adult T-cell leukemia (ATL). We present evidence of HTLV-I infection in two ATL patients from southeastern Italy who have not travelled and who have no known relations abroad, and in 8% of non-leukemic controls from the same area. Thus, populations exhibiting HTLV-I infection appear more widespread than supposed up to now.

Antibodies, Viral↗