Biomedical subjects

M Ponassi

Publications and source records attributed to M Ponassi.

12 recordsLinked to original sources

PubMed1999

Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody.

Tenascin-C exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats. Large Tenascin-C isoforms are present in almost all normal adult tissues but are upregulated in fetal, regenerating, and neoplastic tissues. Here, we report a human antibody fragment, TN11, derived from a phage library with high affinity for the spliced repeat C and demonstrate that this repeat is undetectable in normal adult tissues, barely detectable or undetectable in breast, lung and gastric carcinomas, meningioma, and low grade astrocytoma, but extremely abundant in high grade astrocytoma (grade III and glioblastoma), especially around vascular structures and proliferating cells. The antibody appears to have potential for development of a therapeutic agent for patients with high grade astrocytoma.

Alternative Splicing↗

PubMed1999

Expression of the rat homologue of the Drosophila fat tumour suppressor gene.

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the b-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.

Amino Acid Sequence↗

PubMed1997

The human homeodomain protein OTX2 binds to the human tenascin-C promoter and trans-represses its activity in transfected cells.

Homeodomain-containing proteins mediate many transcriptional processes in eukaryotes during development. Recently, mammalian homeodomain proteins involved in the anterior head formation have been discovered, but their effect on gene transcription has never been investigated. Here we report on the ability of the human homeodomain protein OTX2 to bind with high affinity to a target sequence present in the promoter of the gene encoding the human extracellular matrix protein tenascin-C and to repress its transcriptional activity in transiently transfected cells.

Animals↗

PubMed1995

The first untranslated exon of the human tenascin-C gene plays a regulatory role in gene transcription.

The transcription of the human tenascin-C (TN-C) gene is directed by a single promoter. Here we demonstrate, in transiently transfected cells, that two distinct regions of the untranslated 179 bp-long exon 1 play antagonistic roles in transcriptional regulation: bases from 1 to 20 strongly increase the transcription of the reporter gene CAT directed by the human TN-C gene promoter, while bases from 79 to 179 significantly reduce this activation.

3T3 Cells↗

PubMed1995

Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences.

This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to -2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene.

Animals↗

PubMed1995

The glucagon gene is transcribed in beta-like pancreatic cells.

In this report we demonstrate that approximately 1.1 kb of the rat glucagon gene promoter upstream of the transcriptional start site specifically directs the transcription of the reporter gene chloramphenicol acetyl transferase (CAT) (p[-1.1]GLU-CAT) in insulinoma beta-TC1 cells. On the contrary, the 350 bp closest to the transcription start site (p[-0.35]GLU-CAT) are ineffective in beta-TC1 cells. Both constructs are transcriptionally active in InR1-G9 glucagonoma cells. While protein kinase A and protein kinase C activators, acting through independent pathways, strongly increase both the transcription of p[-1.1]GLU-CAT and the accumulation of glucagon transcript in beta-TC1 cells, they are weaker activators in InR1-G9 cells. Our experiments suggest that some positive transcription control elements, necessary for the glucagon gene transcription in insulinoma beta-TC1 cells, are localized in the -350/-1100 region of the glucagon gene. Furthermore, our data indicate that glucagon gene transcription can be strongly activated through the protein kinase A pathway in some specific cellular contexts.

Animals↗

PubMed1994

Cell-cycle dependent alternative splicing of the tenascin primary transcript.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Alternative Splicing↗

PubMed1993

Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin.

We have obtained and characterized 11 monoclonal antibodies (mAbs) specific for different domains of human tenascin (TN). Five of these mAbs reacted with epitopes contained in the TN area that undergoes alternative splicing and are thus able to recognize specific TN isoforms. These mAbs are a useful tool to study the expression and distribution of TN and its different isoforms in normal and pathological tissues.

Antibodies, Monoclonal↗

PubMed1993

Steady-state levels of different tenascin mRNAs in various normal human tissues.

Northern blot analysis of TN mRNA from different human tissues shows two major bands of about 6 and 8 kb which correspond to two different mRNAs generated by alternative splicing of the primary transcript. In liver, pancreas and kidney only the 6 kb TN mRNA was detectable. The highest levels of 8 kb TN mRNA were observed in placenta and skin representing 30% and 52% of total TN mRNA, respectively. In all other tissues tested the 8 kb TN mRNA represented less than 20% of total TN mRNA.

Blotting, Northern↗

PubMed1992

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.

Cell Adhesion Molecules, Neuronal↗

PubMed1992

Tenascin and fibronectin distribution in human normal and pathological synovium.

Tenascin is a glycoprotein found mainly in the extracellular matrix of developing and malignant tissues. The distribution of this molecule in normal and pathological synovia from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) was investigated by indirect immunofluorescence utilizing specific monoclonal antibodies. The same technique was used to study total fibronectin (tFn) in synovial tissues as well as ED-A and ED-B containing fibronectin (Fn) isoforms (A-Fn, B-Fn), generated by alternative splicing of pre-mRNA. Tenascin was found in normal synovium just beneath the whole lining cell layer. However, a higher density and spreading pattern of distribution was observed in OA and RA sections. A-Fn and B-Fn isoforms were prominent and widespread throughout the normal synovial lining; in hypercellular synovial lining (in RA and OA samples), A-Fn and B-Fn were also observed spreading in the sublining, as well as tFn.

Antibodies, Monoclonal↗

PubMed1991

Procedure for the purification of the fibronectin proteolytic fragments containing the ED-B oncofetal domain.

Fibronectin (FN) is the blend of structurally different molecules (isoforms) whose makeup varies depending on the FN sources. Fibronectin polymorphism is caused by three sequences (called ED-A, ED-B, and IIICS) which may be included or excluded from the FN molecule depending on the alternative splicing patterns of a single primary transcript. The sequence ED-B, which is a complete type III repeat of 91 amino acids, presents some interesting peculiarities: it is the most conserved FN region and, in normal adult tissues, the ED-B-containing FN has an extremely restricted distribution while having a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989, J. Cell Biol. 108, 1139-1148), suggesting that the ED-B sequence may confer to the FN molecules specific biological activities required during ontogenesis and oncogenetic processes. Here we describe a detailed procedure to purify fibronectin fragments containing the ED-B sequence. These purified fragments are useful reagents in the study of the biological function(s) of the ED-B-containing FN molecules.

Amino Acid Sequence↗