Characterization of a mammalian smooth muscle cell line that has retained transcriptional and posttranscriptional potencies.

Unlike skeletal and cardiac muscle cells that differentiate irreversibly, smooth muscle cells (SMCs) retain a high degree of plasticity. During the so-called phenotypic modulation, SMCs can undergo transition between a contractile phenotype and a highly proliferative synthetic phenotype, as apparent from the extinction of numerous smooth muscle (SM) markers when they are passaged in culture. It would be very useful to have an SMC line that can be indefinitely propagated for the cellular and molecular analysis of the mechanisms that underlie the control of SM differentiation. This report describes an immortalized rabbit aorta SMC-derived cell line (U8A4) that has conserved differentiated properties through multiple subcultures. U8A4 cells can grow in the absence of serum and express the SMC markers studied, including SM alpha-actin, SM calponin, SM22alpha, SM alpha-tropomyosin (alpha-TM), SM myosin heavy chain (SM-MHC), and myocardin. U8A4 cells can activate SMC-restricted promoters like those of SM22alpha, SM calponin, and SM-MHC genes as efficiently as described previously for rat SMC lines (PAC1, A7r5, and A10). These cells can also process exogenous alpha-TM transcripts according to an SM-specific pattern. These results demonstrate that the U8A4 cell line constitutes a good alternative model to existing SMC lines that could facilitate the study of the transcriptional and posttranscriptional regulatory mechanisms underlying SMC differentiation.

[Molecular characterization and biodiversity of Mycobacterium tuberculosis in the Antilles-Guiana region and comparative analysis in a metropolitan region, Aquitaine]. / Caractérisation moléculaire et biodiversité de Mycobacterium tuberculosis dans la région Antilles-Guyane et analyse comparative à une région métropolitaine, l'Aquitaine.

Tuberculosis is a highly contagious infectious disease in recrudescence whose epidemiologic monitoring is reinforced by molecular biology. In this context, we were particularly interested in the cases of tuberculosis of French West Indies and French Guiana (FWI-FG). This study covered a period of two years (1997 and 1998) and focused on the demographical and epidemiological characteristics of the cases diagnosed by an analysis of their genotypes. Our results were confronted with a French metropolitan area (Aquitaine) with similar demographic background. Moreover, Aquitaine area has privileged links with FWI-FG region and also has a similar network for monitoring tuberculosis as ours. So we used a PCR method called spoligotyping as a first line method to optimize the alternative IS6110-RFLP method which remains cumbersome. A total of 105 strains of FWI-FG and 172 strains of Aquitaine were typed by spoligotyping and by the standard IS6110-RFLP method. The results of the first grouping by spoligotyping were analyzed in comparison with IS6110-RFLP. The results obtained showed a rate of recent transmission of tuberculosis being 34.3% in FWI-FG and 10.5% in Aquitaine. These observations underlined a high degree of polymorphism in the Aquitaine region as compared to the FWI-FG region. Thanks to the various profiles obtained by spoligotyping, we could study their distribution in the three areas and highlight common types like type 53, 50 and 42 and types found locally like the types 33 and 14 found respectively in Aquitaine and FWI as well as endemic types like type 76 found only in FG. These results are discussed in the context of the evolution of clinical isolates of tubercle bacilli with time.

AP-1 is involved in UTP-induced osteopontin expression in arterial smooth muscle cells.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.

Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL.

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.

Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

Evidence of a role for SHP-1 in platelet activation by the collagen receptor glycoprotein VI.

The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.

Thrombopoietin potentiates collagen receptor signaling in platelets through a phosphatidylinositol 3-kinase-dependent pathway.

Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cgamma2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca(++) [Ca(++)](i), aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.

[Transmission of tuberculosis in Gironde: epidemiologic investigation by genomic analysis of Mycobacterium tuberculosis]. / Transmission de la tuberculose en Gironde : approche épidémiologique par l'analyse génomique du Mycobacterium tuberculosis.

BACKGROUND: Over the past few years, epidemiologic surveys of tuberculosis have been strengthened by new biologic technology, in particularly using RFLP (Restriction Fragment Length Polymorphism). This technique, which identifies Mycobacterium tuberculosis patterns, has allowed to study thoroughly tuberculosis bacilli transmission and pathogenesis. First applied on tuberculosis epidemics in at risk groups, RFLP has now an interest in the epidemiologic molecular survey of urbans populations. The aim of this study is to identify, in a French department, the proportion of clustering cases of tuberculosis, suspected of recent contamination. METHODS: An active surveillance of tuberculosis allows to record systematically the cases of tuberculosis-disease in Gironde. All M. tuberculosis isolates from the patients reported in this surveillance system were processed through IS6110 based RFLP analysis. Patients were interviewed face to face before this analysis, using a standardised data collection instrument. RESULTS: 102 patients were included in 1997; the RFLP analysis of all available strains identifies a high degree of polymorphism with 71 unique patterns; twelve groups with clustering patterns were found, grouping two (nine clusters), three (two clusters) and seven patients (one cluster) each. Those cases suspected of recent transmission were younger (age<60 years) and lived in poorer conditions. Epidemiologic links were confirmed in only 35% of the 31 patients clustered. CONCLUSION: This community survey analysis has allowed to identify at risk groups for tuberculosis transmission and to strengthen tuberculosis control in Gironde.

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

[Survey on cases of primary tuberculosis in the department of Gironde]. / Enquête sur les cas de primo-infection tuberculeuse dans le département de la Gironde.

French official statistics do not mention primary tuberculosis and data on its prevalence are rare, despite the fact that the annual number of cases of primary tuberculosis is a clear indicator of the progression or regression of what remains an endemic disease in Europe. In order to collate information on the subject, a questionnaire was sent to 132 doctors practising in Gironde. These included pulmonologists, pediatricians and child health doctors. One hundred and one questionnaires were returned, listing a total of 18 cases of primary tuberculosis for the first half of 1997. Children were more often affected by the disease and presented a latent form. In 61% of cases, patients infected had not previously received BCG vaccination and in 56% of cases the infectious patient was identified. Furthermore, 4 of the non-vaccinated patients had been in close contact with an infectious patient and 3 patients among these should have been vaccinated since they were living in community structures. The 2 symptomatic cases reported occurred in non-vaccinated adults. This study was of limited-scope and duration but provides interesting information on the population affected by primary tuberculosis. These results underline the necessity of maintaining a high level of BCG vaccination amongst children and adults in community structures if we wish to lower the prevalence of the disease in France.

Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress-activated protein kinase pathways.

Stress-activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin-stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild-type human cPLA2 in vitro, although to different extents, but not cPLA2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42-kDa mitogen-activated protein kinase incorporated similar levels of phosphate into cPLA2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen-induced phosphorylation of cPLA2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially ( approximately 50%) blocks phosphorylation at both sites and to a similar extent in thrombin-stimulated platelets. Inhibition of phosphorylation resulted in a two- to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin- and collagen-stimulated platelets. Our data suggest that cPLA2 is a substrate for several SAPK cascades and that phosphorylation of cPLA2 augments arachidonic acid release.

[Comparison of cases of isolated pulmonary tuberculosis with cases of other localizations of tuberculosis in the course of an active surveillance (Gironde, 1995-1996)]. / Comparaison des cas de tuberculose pulmonaire isolée aux cas de tuberculose d'autre localisation dans un système de surveillance active (Gironde, 1995-1996).

PURPOSE: Data collected during the years 1995 and 1996 in the course of an epidemiological survey of tuberculosis in Gironde allowed comparison of pulmonary tuberculosis with extrapulmonary localizations, evaluation of the importance of each localization and highlighting of potential risk factors. METHODS: Patients living in Gironde who had evidence of either clinical, radiological or bacteriological expression of tuberculosis were included in the survey. Statistical comparisons were done using either Pearson's Chi 2 or Fisher's exact test. RESULTS: The survey included 292 cases subdivided into 183 cases of pulmonary tuberculosis (63%) and 109 cases in which another localization had been diagnosed (37%). Extrapulmonary localizations that were the most often encountered either alone or in association with pulmonary localization were the following: lymphadenopathy (32%), pleural (28%), genito-urinary (12%) and osteo-articular localizations (7%). The survey showed that patients in whom tuberculosis localization was extra-pulmonary were more frequently under 20 years of age or over 60 years of age (P < 0.04). These patients also presented more often with HIV-infection (P < 0.02). CONCLUSION: Extrapulmonary localizations of tuberculosis should be systematically investigated in young and elderly patients as well as in HIV-infected patients.

Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase.

The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38- and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A2. In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [3H]arachidonic acid to [3H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H2 and the conversion of prostaglandin H2 to thromboxane A2 in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A2 as these two MAPKs are capable of phosphorylating cytosolic phospholipase A2, thereby increasing its intrinsic activity.

[Educational diagnosis in asthma. Descriptive results of a questionnaire survey]. / Diagnostic éducatif de l'asthme. Résultats descriptifs d'une enquête par questionnaire.

The object of this work was to carry out an audit on an asthmatic's knowledge of their disease and of the risk factors, their usual therapy and of therapy for acute exacerbations as well as their need for information and education. The study was carried out by a survey of 327 adult asthmatics who were in consultation with specialist physicians in thoracic medicine. The information was gathered using an anonymous questionnaire completed by a nurse. The doctors looking after these patients had given their opinion on the severity of their asthma and the educational needs of their patients. The population studied had an average of 47 +/- 17 years. Forty seven per cent of the subjects were masculine. The general level of education was high. Seventy five per cent of the patients were from an urban environment. The duration of the asthma was on an average of 19 years. Half of these patients had already been in hospital for a serious attack. One third of the patients were considered by their doctors as having severe asthma. The patients were good at distinguishing the level of severity of their acute exacerbations and adapted their therapy on their own initiative in appropriate fashion based on the degree of severity of their disease. The enquiry revealed that patients take some liberties around the basic treatment that they had been prescribed. It also showed some defects or errors in their knowledge of the disease and the appropriate approach vis-à-vis the disease. One noted for example that one third of the asthmatics had already stopped the treatment before the date fixed by their doctor. In a general, patients who were surveyed were interested in their health problems with their health and hoped to be better informed both on asthma and its treatment. This study was biased towards an interest in the understanding of the education in order to manage the asthmatic population better.