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Biomedical subjects

S Barlati

Publications and source records attributed to S Barlati.

At least 55 records · Page 3Linked to original sources
PubMed1994

Urokinase-type and tissue-type plasminogen activators as growth factors of human fibroblasts.

In this study we have verified the mitogenic effect of urokinase-type (u-PA) and tissue-type plasminogen activators (t-PA) on human normal fibroblasts. We report that both PAs can induce DNA replication and cell division in serum-deprived cultured human skin fibroblasts. The activity of u-PA and t-PA is, respectively, three- and twofold more potent than that exerted by epidermal growth factor (EGF) with an activity slightly lower (50-60%) than that induced by basic fibroblast growth factor (bFGF). The u-PA and t-PA, but not plasmin, induced DNA synthesis, which could be neutralized by anti-u-PA and anti-t-PA antibodies, respectively, but was insensitive to aprotinin treatment. The addition of anti-u-PA-receptor (u-PAR) monoclonal antibodies to the assays selectively suppressed the mitogenic effect exerted by u-PA, but not that of t-PA, and the amino-terminal fragment of u-PA, containing the EGF-like domain and the kringle module, did not elicit any mitogenic activity. Anti-bFGF antibodies completely suppressed the mitogenic activity of bFGF, but did not have any effect on that of u-PA and t-PA; the activity of both PAs was inhibited by anti-fibronectin IgG concentrations ineffective on bFGF. These results indicate that PAs may be considered growth factors of human fibroblasts.

Adult↗
PubMed1993

One-lane chemical sequencing of 3'-fluorescent-labeled DNA.

A method for DNA sequencing that combines limited chemical degradation of 3'-fluorescent-labeled DNA with densitometric analysis using an automatic sequencer is presented. The DNA sequence is determined in a single electrophoretic lane by monitoring the intensities of bands representing products of cleavage at the four bases obtained by solvolysis in hot aqueous piperidine (10%) followed by treatment with hot formamide. An application of the method for the detection of point mutations is reported.

Autoanalysis↗
PubMed1993

The type III-9 repeat of human fibronectin is encoded by a single exon which is not alternatively spliced.

Type III homologies of human fibronectin are generally encoded by two exons, with the exception of the ED-A and ED-B repeats which are encoded by a single exon undergoing alternative splicing. We report that also the type III-9 homology is encoded by a single exon. Further more, RT-PCR analysis, performed on mRNA purified from fetal and adult tissues and from normal and tumor-derived cell types, showed that the III-9 region is not undergoing alternative splicing in all samples tested.

Adult↗
PubMed1993

Quantitative evaluation of mRNAs by in situ hybridization and image analysis: principles and applications.

We describe an image analysis (IA) program that has been developed for the quantitative evaluation of mRNA evidenced by in situ hybridization (ISH) with radiolabeled probes in cultured cells and tissue sections. ISH-IA allowed the detection and quantitative evaluation of mRNA expressed by heterogeneous in vitro cultured cells. This method, when combined with dot-blot analysis, allowed the evaluation of the approximate number of mRNA molecules expressed by single cells. IA permitted the evaluation of cultured cells' morphological parameters (such as cytoplasm and nucleus areas) modifications in relation to specific mRNAs expression, which can vary during cell cycle, development, aging, and in different pathologies and treatment with drugs. ISH-IA was applied for the evaluation of mRNA isoforms generated by alternative splicing in single cells. This methodology was also applied for the semiquantitative evaluation and comparison of mRNA levels expressed by different cell types in human normal and tumor tissue sections.

Alternative Splicing↗
PubMed1992

Study of fibronectin and mRNA in human laryngeal and ectocervical carcinomas by in situ hybridization and image analysis.

The expression of fibronectin (FN) mRNA was studied in histological sections of surgical biopsies from human laryngeal and ectocervical invasive carcinomas of different grading stages by in situ hybridization and image analysis. This approach made it possible to identify the cell types synthesizing FN mRNA in the tissue sections and to compare semi-quantitatively the FN mRNA levels expressed in the different specimens. The carcinoma cells synthesized low levels of FN mRNA, comparable to those detected in control epithelia and connective-tissue fibroblasts. Well-differentiated (G1) laryngeal and ectocervical carcinomas induced the synthesis of FN mRNA--to levels 7 to 13 times higher than in control connective tissues--in the stromal fibroblasts surrounding the tumors. In carcinoma samples analysed, the amount of FN mRNA detected in the stroma decreased in relation to tumor grading (from G1 to G3) and the stromal destruction. FN mRNA was not detectable in the endothelial cells of venules while it was present in large amounts in those surrounding the capillaries present in the stroma. These data indicate that FN, usually observed around carcinomas, is produced by stromal fibroblasts, which are induced to express FN mRNA, presumably in response to diffusible factors produced by the tumor cells, and/or by endothelial cells of the infiltrating capillary vessels. The induction of FN mRNA, inversely proportional to the tumor grading, may be useful in evaluating the invasion potential of the tumor.

DNA Probes↗
PubMed1992

Exclusion of stromelysin-1, stromelysin-2, interstitial collagenase and fibronectin genes as the mutant loci in a family with recessive epidermolysis bullosa dystrophica and a form of cerebellar ataxia.

The interstitial collagenase gene (CLG), one of the main candidates in severe generalized recessive epidermolysis bullosa dystrophica (SGREBD), is closely linked to the stromelysin-1 (STMY1) and stromelysin-2 (STMY2) genes. These three loci map on chromosome 11 (q21-q22.3), where they constitute a cluster of genes coding for metalloproteinases involved in the degradation of the extracellular matrix (ECM). A recessive form of cerebellar ataxia of post-puberal onset (CLA1) has also been assigned to chromosome 11 (q14-q21). Since useful restriction fragment length polymorphisms (RFLPs) for the CLG gene are not available, we have studied the inheritance of the marker TaqI RFLP of the STMY1 gene in a North Italian family with a child affected by SGREBD, and his two sisters showing cerebellar ataxia (CA) of post-puberal onset. We have also studied the MspI RFLP of the fibronectin gene (FN1), which is located on chromosome 2q34-q36, and which codes for non-collagenous matrix proteins. Since we did not observe the segregation of the pathological phenotypes with STMY1 and FN1 RFLPs, we excluded the involvement of these genes in both the SGREBD and CA present in this family. The exclusion of the STMY1 gene indicates that the mutation causing SGREBD cannot be located in the CLG and/or STMY2 genes because of their proximity to the STMY1 locus. These data also indicate that the CA form here reported is not attributable to alterations in regions close to the collagenase cluster on chromosome 11.

Blotting, Southern↗
PubMed1992

Histological human papillomavirus induced lesions: typization by molecular hybridization techniques.

The viral typization in Human Papillomavirus (HPV) infections of the lower female genital tract is relevant both from the epidemiological and the clinical point of view. We have tested DNA from specimens obtained by guided biopsies on neoplastic and benign lesions, using single probes of the different virus types (6, 11, 16 and 18) by Dot-blot and Southern blot analysis. According to previous studies HPV 16 and 18 have been detected in neoplastic lesions, while 6 and 11 were more frequently found associated to condylomata. Negative specimens and intermediate were 36% of the total. In our experience both methods have shown same sensibility.

Adenocarcinoma↗
PubMed1991

Tyrosine phosphorylation of human urokinase-type plasminogen activator.

Immunoblotting analysis of purified human urokinase plasminogen activator (u-PA), gives a positive signal when reacted with anti-phosphotyrosine monoclonal antibodies (MoAb anti-P-Tyr); competition with o-phospho-DL-tyrosine (P-Tyr) but not o-phospho-DL-threonine or serine (P-Treo, P-Ser) completely suppresses this signal. Either the 55 kDa u-PA form and the lower Mw form (33 kDa) derived from the 55 kDa u-PA are Tyr-phosphorylated also the u-PA secreted in the culture media of human fibrosarcoma cells (HT-1080) is phosphorylated in tyrosine as well as u-PA present in tissue extracts of tumors induced in nude mice by HT-1080 cells. These data show that urine purified human u-PA and u-PA produced by human fibrosarcoma cells, in vitro and in vivo, are phosphorylated in tyrosine; furthermore our data show that u-PA is the major Tyr-phosphorylated protein present in these human tumor cells.

Animals↗
PubMed1991

Phenotypic correction of the defective fibronectin extracellular matrix of Ehlers-Danlos syndrome fibroblasts.

In vitro cultured skin fibroblasts derived from Ehlers-Danlos Syndrome (EDS) type I to VIII patients lack fibronectin-containing extracellular matrix (FN-ECM) which can be restored when EDS cells are cocultivated over a feeder of control fibroblasts. Further analysis, focused on EDS types III and IV cells, showed that partial matrix correction in EDS type III cells can be obtained by their cultivation over a feeder of EDS type IV fibroblasts, but not vice versa. An apparently normal FN-ECM can be restored in EDS types III and IV cells also by the addition of cellular--but not plasma--FN. These biological features might be used for a better understanding of ECM assembly and for the characterization of the different EDS cell types.

Cells, Cultured↗
PubMed1991

Altered fibronectin mRNA splicing in skin fibroblasts from Ehlers-Danlos syndrome patients: in situ hybridization analysis.

The expression of fibronectin (FN) mRNA isoforms generated by alternative splicing of the EDA region was studied by dot-blot and in situ hybridization, using specific FN cDNA probes, in skin fibroblasts from controls and Ehlers-Danlos Syndrome (EDS) patients (types III, IV, VII and non classified). An Image Analysis program was used for the quantitative evaluation and comparison of FN mRNAs levels in the different cell strains. The in situ hybridization analysis showed that FN mRNAs are homogeneously expressed in all cells of each fibroblast strain analyzed. While in control fibroblasts about 70% of FN mRNA isoforms contain the EDA region (EDA+ FN mRNAs), in EDS fibroblasts this fraction is reduced up to about 30%. This indicates that in the EDS fibroblasts analyzed a deregulation of the alternative splicing processes acting at the EDA region takes place.

Autoradiography↗
PubMed1990

Establishment and characterization of a leukemic murine cell line derived from MCF 247 MuLV-induced T-cell lymphoma.

Understanding of the leukemic evolution of human non-Hodgkin's lymphomas is hindered by the lack of appropriate animal models. For this purpose, a highly leukemic cell line NQ22, derived from a MCF 247 murine leukemia virus (MuLV)-induced murine T-cell lymphoma, was established, and its preliminary characterization is described. The NQ22 cell line is easily transplantable subcutaneously (s.c.) into syngeneic AKR mice exhibiting early peripheral blood invasion and widespread dissemination with a leukemic pattern of infiltration. Such peculiar in vivo behavior is a stable phenotypic feature, probably determined genetically. Biological and differentiation characteristics of the NQ22 cell line were analyzed and compared to those of other non-leukemic T-lymphoma lines. In addition, no evidence of possible involvement of plasminogen activator (PA) enzymes and of their inhibitors (PAI) in the spreading ability of NQ22 cells was observed.

Animals↗
PubMed1990

Analysis of fibronectin, plasminogen activators and plasminogen in tear fluid as markers of corneal damage and repair.

Corneal damage of various origins initiates a series of processes which lead to repair but also tend to perpetuate the damage; healing thus depends on the prevalence of repair over progression processes. The plasminogen/plasmin system has an important impact on this process, particularly by degrading the extracellular matrix components with resulting interference of the repair processes. This paper presents the immunoblotting analysis of fibronectin, tissue and urokinase-type plasminogen activators and plasminogen/plasmin in the tear fluid of control subjects and patients affected by various ocular pathologies (corneal ulcers, thermal or chemical burns, herpetic keratitis). A significant modification was noted in the protein profiles of fibronectin, tissue and urokinase-type plasminogen activators and plasminogen/plasmin in the cases of corneal ulcers and thermal or chemical burns relative to the pattern observed in the control subjects, while in cases of herpetic keratitis, only plasminogen/plasmin showed slight variations. The altered protein patterns gradually normalized during therapeutic treatment and, at remission, coincided with those of the control subjects.

Adult↗
PubMed1990

Study of fibronectin expression in tumour cells by dot-blot and in situ hybridization: quantitative evaluation by image analysis.

An Image Analysis program was used for the quantitative evaluation and comparison of the fibronectin (FN) mRNA detected by dot-blot and in situ hybridization in different cell lines. These techniques were applied for the evaluation of FN mRNA synthesized by human normal fibroblasts (Flow 7000) and by four tumour-derived cell lines (HeLa, epithelioid carcinoma; 8387, fibrosarcoma; RD, rhabdomyosarcoma; SK Hep-1, hepatocarcinoma). Dot-blot analysis showed that the cell types analysed synthesize different levels of FN mRNA. Flow 7000 are the highest producers while HeLa the lowest. In situ hybridization confirmed these results and furthermore showed that while Flow 7000, 8387 and HeLa cells synthesized homogeneous levels of FN mRNA, RD and SK Hep-1 could be subdivided into two populations expressing high or low levels of FN mRNA. The combined analysis of dot-blot, in situ hybridization and Image Analysis allowed the quantitation of the number of FN mRNA molecules expressed by single cells. This approach is therefore an invaluable tool when evaluating mRNA expression in heterogeneous cell populations like tumour-derived cell lines, during cell cycle or in histological tissue sections.

Cell Line↗