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Biomedical subjects

F Blasi

Publications and source records attributed to F Blasi.

At least 127 records · Page 7Linked to original sources
PubMed1996

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity.

The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of UEF3, a novel factor involved in the regulation of the urokinase and other AP-1 controlled promoters.

Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.

Amino Acid Sequence↗
PubMed1996

Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and alpha 2MR/LRP genes.

During the process of embryo implantation, trophoblast cells invade deep into uterine stroma and play a key role in establishing fetomaternal exchange of molecules. We have studied the in vivo expression patterns of the molecules of the urokinase system, during the process of mouse embryo implantation and early placentation. The sites of synthesis of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR), plasminogen activator inhibitor type 1 (PAI-1) and alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) transcripts were determined by in situ hybridization. These genes were found to be expressed in a finely regulated pattern. High levels of uPA mRNA were found in invasive trophoblast cells, while the same cells did not appear to synthesize PAI-1. Starting from day 6.5, endothelial cells of newly forming vessels also transcribed uPA gene. uPAR and alpha 2MR/LRP were in all stages expressed by decidual tissue, and their expression domains overlapped in large areas. Immunohistochemistry with uPA and PAI-1 antibodies revealed areas of co-localization of these secreted proteins with the expression domains of uPAR and alpha 2MR/LRP, which is of great interest in view of the role of these two receptors in clearing uPA-PAI-1 complexes. In situ zymography demonstrated the presence of active uPA in the ectoplacental cone region at 7.5 and 8.5 days. Our studies outline the expression of a set of functionally related genes that is well coordinated between fetal and maternal tissues. This coordination may model other physiological and pathological invasive processes.

Animals↗
PubMed1996

The urokinase receptor and cell migration.

The receptor (u-PAR) for urokinase plasminogen activator (u-PA) is a three-domain protein, GPI-anchored to the cell surface, which focuses the enzymatic activity of u-PA, and allows the cell surface activation of plasminogen. Regulation of the activity of u-PA is also mediated by u-PAR. In fact, while active u-PA is not internalized or degraded, rather remaining active at the cell surface, serpin-inactivated u-PA is internalized, through the intervention of a second molecule, the alpha 2-macroglobulin receptor (LRP). In the process u-PAR too is internalized and possibly recycles back to the cell surface. In addition, u-PAR occupancy can also directly transduce migratory signals, like chemotaxis, that do not require the protease activity of u-PA. Occupancy of u-PAR activates tyrosine kinases, in particular of p56/p59hck, through an as yet undefined transmembrane adaptor.

Cell Movement↗
PubMed1996

New pathogens for respiratory infections.

The emergence of new pathogens and the increasing antimicrobial resistance patterns of old pathogens are contributing factors to the high epidemiologic impact of lower respiratory tract infections. Hantaviruses, Chlamydia pneumoniae, and penicillin-resistant Streptococcus pneumoniae have recently gained most of the attention of international researchers. Hantavirus pulmonary syndrome has been confirmed in 100 cases with a very high mortality (52%). Risk factors for infection are peridomestic cleaning, agricultural activities, and an increased number of small rodents like deer mouse, mainly Peromyscus maniculatus. Pulmonary capillary leak and multiorgan involvement characterise Hantavirus pulmonary syndrome. Most recent reports rank C. pneumoniae among the three most common etiologic agents in community-acquired pneumonia, but it is also involved in chronic diseases such as chronic obstructive pulmonary disease and asthma. Several recent reports indicate the striking increase of penicillin-resistant S. pneumoniae strains. These data emphasize the crucial need for new therapeutic agents and more effective prevention programs.

Agriculture↗
PubMed1996

Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms.

Recent reports suggest an association between Chlamydia pneumoniae and Helicobacter pylori bacteria and atherosclerosis. We studied 51 patients (mean age, 68.3 years) who underwent abdominal aortic aneurysm surgery. For each patient we performed a microimmunofluorescence test for immunoglobulin G (IgG), IgA, and IgM antibodies to C. pneumoniae specific antigen (TW-183). Anti-H. pylori antibodies were determined by means of an EIA-G test. Each aortic aneurysm surgical specimen was sampled into multiple sections of 0.3 cm2 each and frozen at -20 degrees C. Two samples of each aneurysm were used for a nested PCR with two sets of C. pneumoniae and two sets of H. pylori specific primers. Specimens were treated with a solution containing 20 mM Tris-HCl, Tween 20-Nonidet P-40 (0.5% [vol/vol] each), and 100 micrograms of proteinase K per ml and incubated at 60 degrees C for 1 h and at 98 degrees C for 10 min. DNA was extracted twice with phenol-chloroform-isoamylic alcohol and precipitated with sodium acetate-ethanol by standard methods. Forty-one patients were seropositive for C. pneumoniae with past-infection patterns in 32 patients (16 < or = IgG < 512; 32 < or = IgA < 256) and high antibody titers in 9 patients (IgG > or = 512). In 26 of 51 patients, C. pneumoniae DNA was detected in aortic aneurysm plaque specimens. Of these patients, 23 had a serologic past-infection pattern, 2 had an acute reinfection pattern, and 1 was seronegative. Forty-seven of 51 patients were seropositive for H. pylori. In all cases PCR showed no evidence of H. pylori presence in plaque specimens. This study provides data on a possible C. pneumoniae involvement in the pathogenesis of aortic aneurysm and additional evidence for an association between this agent and atherosclerosis. Conversely, notwithstanding a high H. pylori seroprevalence observed, our results tend to rule out the possibility of a direct involvement of H. pylori in atherosclerosis.

Aged↗
PubMed1996

Severe community-acquired pneumonia: a possible role for Chlamydia pneumoniae.

Between July 1992 and June 1993, 61 patients with severe community-acquired pneumonia were admitted to our semi-intensive care unit. For all patients chest X-ray, blood gas analysis while breathing room air, Gram stain and culture of bronchoaspirate, determination of acute and convalescent anti-body titers for Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae, blood culture when body temperature was greater than 38 degrees C, and pharyngeal swab for C. pneumoniae detection by means of an indirect immunofluorescence test were obtained. Among the patients enrolled, 15 suffered from chronic obstructive pulmonary disease, 18 had serious chronic diseases, 9 were immunodeficient and 15 had cardiovascular diseases, and only 4 had no underlying disease. Etiologic diagnosis was reached in 30 cases (49%). As expected, due to the high rate of seriously ill patients, gram-negative pathogens were identified most commonly (15%), followed by Streptococcus pneumoniae (10%) and, surprisingly, by C. pneumoniae (10%). These data, showing the possible emergence of Pseudomonas aeruginosa and C. pneumoniae, warrant further studies in order to verify whether the epidemiological pattern of severe community-acquired pneumonia is actually changing.

Aged↗
PubMed1995

Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.

Base Sequence↗
PubMed1995

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Aging↗
PubMed1995

Biosynthesis and apical localization of the urokinase receptor in polarized MDCK epithelial cells.

The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell-cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down-regulation of uPAR by the uPA-PAI-1 complex was observed only from the apical membrane.

3T3 Cells↗
PubMed1995

A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene.

The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes and contains relatively GC-rich proximal sequences. A subregion of this sequence, highly conserved between human and murine genes, contains most of the promoter activity and is specifically bound by HeLa nuclear proteins, one of which belongs to the SP1 class.

Animals↗
PubMed1995

PMA-induced down-regulation of the receptor for alpha 2-macroglobulin in human U937 cells.

Transcription and expression of the urokinase (uPA) receptor (uPAR) are strongly stimulated by PMA. As for uPAR, the expression of alpha 2-MR is regulated by PMA in U937 cells. Ligand blotting experiments with the 39 kDa receptor-associated protein RAP, a ligand for alpha 2-MR, indicated that alpha 2-MR levels first increased and then decreased after PMA treatment. FACscan as well as immunoblotting analysis with alpha 2-MR-specific antibodies showed an identical trend: alpha 2-MR levels increased within the first day of treatment with PMA, decreased at later times, and totally disappeared by three days of treatment. The effect of PMA was not due to transcriptional down-regulation, as the alpha 2-MR mRNA level did not decrease at later times. Sensitivity of U937 cells to uPA-saporin, a toxin conjugate that reguires binding to uPAR for killing activity, was also markedly decreased. These results suggest that uPAR-mediated endocytosis depends on alpha 2-MR expression.

Base Sequence↗
PubMed1995

The urokinase/urokinase-receptor system and cancer invasion.

u-PA binds with high affinity to its specific GPI-anchored receptor on the cell surface. The binding has at least two important consequences: (1) it enhances the rate of plasminogen activation on the cell surface; and (2) it focuses the u-PA proteolytic activity at the leading front of migrating cells. Several recent findings suggest that surface-bound u-PA is essential for the invasive ability of tumour cells, even if a picture is emerging indicating a concerted action with other proteases, like collagenases and cathepsin B (Kobayashi et al, 1992; Ossowski, 1992; Schmitt et al, 1992; (Danø et al, 1994). In some tumours, e.g. colon, breast and skin cancer, in situ hybridization studies have given an insight into the u-PA/u-PAR tumour biology showing a complex interplay between stromal and cancer cells Danø et al, 1994). u-PA, u-PAR, and PAI-1 tumour content are now well established prognostic factor in breast cancer. This body of knowledge could be used for theurapeutic purposes. For example, a large study with 671 patients has allowed the identification of node-negative patients which, according to their u-PA levels, would need adjuvant therapy (Foekens et al, 1992). Many other tumours, especially colorectal cancer, expect a direct clinical evaluation of u-PA, u-PAR and serpins as prognostic factors.

Animals↗
PubMed1995

alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

Animals↗
PubMed1995

Urokinase/urokinase receptor system: internalization/degradation of urokinase-serpin complexes: mechanism and regulation.

The urokinase-type plasminogen activator (uPA) is secreted as a single-chain inactive zymogen (pro-uPA). Upon its secretion, pro-uPA binds to its glycosylphosphatidylinositol-anchored specific cell receptor (uPAR). The activation of pro-uPA to the active two-chain uPA is accelerated with uPAR-bound pro-uPA and is achieved by plasmin and proteases of other classes like cathepsins G and L. uPAR-bound uPA is susceptible to inhibition by its specific inhibitors (PAI-1, PAI-2, and PN-1). uPA-PAI-1 and uPA-PN-1 complexes, but not free uPA, are readily internalized and degraded through a mechanism that involves the multiligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-associated protein (alpha 2-MR) and epithelial glycoprotein 330 (gp330). Upon uPA-inhibitor internalization, uPAR is itself endocytosed and recycled back to the cell surface. PMA-induced differentiation of myeloid cells is accompanied by inhibition of uPA-PAI-1 internalization/degradation and the down-regulation of alpha 2-MR. The regulation of uPAR and alpha 2-MR levels might be part of the differentiation program of myeloid cells.

Animals↗
PubMed1994

Differential DNA sequence specificity and regulation of HIV-1 enhancer activity by cRel-RelA transcription factor.

The cRel-RelA and NF-kappa B (p50-RelA) transcription factors bind to a kappa B-like sequence termed Rel-related proteins binding element localized in the regulatory region of the human urokinase plasminogen activator (uPA) gene. This sequence is highly conserved in murine and porcine uPA genes where it retained the ability to associate with cRel-RelA. On the other hand, NF-kappa B binding was obtained with the human and porcine elements only. Methylation interference analysis showed that NF-kappa B and cRel-RelA had identical interference patterns. Mutational analysis showed that DNA binding was highly sensitive to mutations within the decameric Rel-related proteins binding element core site. However, alterations of nucleotides flanking the decameric IgK-kappa B motif, which preferentially associated with NF-kappa B, resulted in high affinity cRel-RelA binding both in vitro and in vivo. These data demonstrate that NF-kappa B and cRel-RelA have overlapping but distinct DNA sequence specificities. Bandshift analysis with HeLa and Jurkat cell extracts or with in vitro translated proteins revealed that the SV40-, HIV-1-, and interleukin-2 receptor alpha subunit kappa B elements efficiently associated with cRel-RelA, suggesting that this heterodimer may be involved in the regulation of several genes. Cotransfection studies of HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter DNA with RelA, cRel, and p50 expression vectors were performed in COS7 and U293 cells to analyze the ability of cRel-RelA to regulate HIV-1 enhancer activity. In vivo formation of the cRel-RelA complex resulted in specific stimulation of the viral enhancer at a level comparable with that obtained with NF-kappa B. These data suggest that activation of cellular cRel-RelA may play a critical role in the regulation of HIV-1 enhancer activity.

Animals↗