Extracellular ATP hydrolysis in Caco-2 human intestinal cell line.

Extracellular nucleotides and nucleosides activate signaling pathways that play major roles in the physiology and pathophysiology of the gastrointestinal tract. Ectonucleotidases hydrolyze extracellular nucleotides and thus regulate ligand exposure to purinergic receptors. In this study, we investigated the expression, localization and activities of ectonucleotidases using Caco-2 cells, a model of human intestinal epithelial cells. In addition, by studying ATP release and the rates of extracellular ATP (eATP) hydrolysis, we analyzed the contribution of these processes to the regulation of eATP in these cells. Results show that Caco-2 cells regulate the metabolism of eATP and by-products by ecto-nucleoside triphosphate diphosphohydrolase-1 and -2, a neutral ecto-phosphatase and ecto-5'-nucleotidase. All these ectoenzymes were kinetically characterized using intact cells, and their presence confirmed by denatured and native gels, western blot and cytoimmunofluorescence techniques. In addition, regulation of eATP was studied by monitoring the dynamic balance between intracellular ATP release and ectoATPase activity. Following mechanical and hypotonic stimuli, Caco-2 cells triggered a strong but transient release of intracellular ATP, with almost no energy cost, leading to a steep increase of eATP concentration, which was later reduced by ectoATPase activity. A data-driven algorithm allowed quantifying and predicting the rates of ATP release and ATP consumption contributing to the dynamic accumulation of ATP at the cell surface.

TSPO ligands stimulate ZnPPIX transport and ROS accumulation leading to the inhibition of P. falciparum growth in human blood.

After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS.

Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells.

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5' distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

Structure and expression of the mouse homologue of the XK gene.

The human Kx blood group antigen is carried by a 37,000 M(r) apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43,000 M(r) Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140,000 M(r) species was detected instead of the 43,000 M(r) protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93,000 M(r).

Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.

Kell and Kx, two disulfide-linked proteins of the human erythrocyte membrane are phosphorylated in vivo.

Kell and Kx are two quantitatively minor proteins from the human erythrocyte membrane which carry blood groups antigens and are thought to be a metalloprotease and a membrane transporter, respectively. In the red cell membrane, these proteins form a complex stabilized by disulfide bond(s). Phosphorylation status of these proteins was studied, in the presence or absence of effectors of several kinases, either on intact cells incubated with [32P]-orthophosphate or on ghosts incubated with [gamma-32P]ATP. Purification of Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity allowed to establish that (i) neither protein is phosphorylated on tyrosine; (ii) the Kell protein is a putative substrate for Casein Kinase II (CKII) and Casein Kinase I (CKI) but not for protein kinase C (PKC), whereas Kx protein is phosphorylated by CKII and PKC but not by CKI; (iii) Protein Kinase A neither phosphorylates the Kell nor the Kx proteins.

Immunochemical analysis of the Kx protein from human red cells of different Kell phenotypes using antibodies raised against synthetic peptides.

The Kx protein is an erythrocyte membrane polypeptide which is deficient in rare individuals suffering from the McLeod syndrome. The gene encoding this protein has been recently cloned and the Kx protein independently purified as a covalent complex with the Kell blood group protein. To further study the Kx membrane protein, antisera raised in rabbits against six synthetic peptides derived from the primary sequence of this protein were characterized. All antisera but two precipitated the recombinant Kx protein synthesized in coupled transcription-translation in vitro. Three antisera reacted on immunoblots with the 37 kD Kx protein present in the purified Kell-Kx complex and in SDS red cell membrane lysates from variants with different Kell blood group phenotypes, including Ko, which lack the Kell protein of 93 kD. However, no reactivity was found with McLeod preparations lacking Kx protein, thus clearly indicating that these antibodies have a Kx specificity. Unexpectedly, the relative amount of Kx protein in Ko cells was found to be lower than in red cells with common Kell phenotypes, suggesting that the absence of the Kell protein may alter the amount of Kx in the membrane.

Cloning of the gene encoding the human erythropoietin receptor.

The genomic and complementary DNAs of the human erythropoietin receptor (hEpo-R) have been isolated and characterized from a genomic placental library and from two cDNA libraries prepared from bone marrow and fetal liver. The five different partial cDNAs isolated were aberrant in the predicted reading frames as compared with the Epo-R protein sequence, because all retained insert sequences that may represent splicing intermediates (three clones), cloning artifact (one clone), or a new sequence at a splice junction (one clone) of the gene. The cDNAs were used to isolate several genomic clones encompassing the complete hEpo-R gene. This gene, which encodes a 508-amino acid polypeptide chain of predicted M(r) 55,000, is organized into eight exons spread over 6 kb of DNA and exhibited a high degree of sequence homology (81.6% in the coding region) and structural organization with its murine counterpart. Primer extension analysis indicated that the transcription initiation site is located 141 bp upstream of the initiation codon. Sequence homology 320 bp upstream of the cap site was significantly lower (60%) and diverged completely further upstream as compared with the murine gene. Similarly, the human and murine sequences were largely divergent downstream of the stop codon, indicating that a strong conservation during evolution was restricted to the coding sequence of the Epo-R protein. The 320-bp region upstream of the cap site does not contain the typical TATA or CAAT boxes present in many tissue-specific genes, but does include potential binding sites for the ubiquitous Sp1 and the erythroid-specific GATA-1 trans-activating factors. These boxes are well conserved in sequence and position relative to the cap site within the promoter region of the human and murine genes, but the CACCC boxes present in the murine gene are absent in the human gene.

[Iatrogenic urethral strictures of the male urethra]. / Les rétrecissements iatrogènes de l'urètre masculin.

The iatrogenic aetiology of urethral stricture appears to be increasing in frequency. Transurethral catheterization and endourethral manipulation are the principal aetiologic factors. Prevention is based essentially upon a greater respect of the urethra when an endoscopic exploration is necessary and the use of suprapubic catheterization whenever bladder drainage is necessary.