RESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Mucosa Intestinal/metabolismo , 5'-Nucleotidase/metabolismo , Células CACO-2 , Humanos , HidróliseRESUMO
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.
Assuntos
Plasmodium falciparum/crescimento & desenvolvimento , Protoporfirinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD34/metabolismo , Transporte Biológico , Diferenciação Celular , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Células Eritroides/citologia , Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Glutationa/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Parasitos/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de GABA/química , Receptores de GABA/genética , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismoAssuntos
Ductos Biliares/anatomia & histologia , Ductos Biliares/anormalidades , Ductos Biliares/cirurgia , Ductos Biliares Intra-Hepáticos/anatomia & histologia , Ductos Biliares Intra-Hepáticos/cirurgia , Ducto Colédoco/anormalidades , Ducto Colédoco/anatomia & histologia , Ducto Colédoco/cirurgia , Humanos , Complicações IntraoperatóriasRESUMO
Sonography seems in patients with acute renal failure a reliable method for diagnosing urinary tract obstruction, giving the kidney size and localizing the kidney for percutaneous drainage. Percutaneous nephrostomy is actually the best temporary derivation for patients with obstructive anuria before definitive treatment. 18 cases of obstructive anuria are reviewed. Urinary lithiasis is the main aetiology (13/18). 11 of our patient had undergone percutaneous nephrostomy as a first step of their treatment.
Assuntos
Anuria/cirurgia , Drenagem/métodos , Nefrostomia Percutânea , Obstrução Ureteral/cirurgia , Doença Aguda , Adolescente , Adulto , Idoso , Anuria/diagnóstico por imagem , Anuria/etiologia , Criança , Pré-Escolar , Feminino , Humanos , Nefropatias/complicações , Nefropatias/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Radiografia , Estudos Retrospectivos , Ultrassonografia , Obstrução Ureteral/etiologiaRESUMO
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.
Assuntos
Doença Iatrogênica , Estreitamento Uretral/etiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Cistoscopia/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Procedimentos Cirúrgicos Operatórios/métodos , Estreitamento Uretral/cirurgia , Cateterismo Urinário/efeitos adversosRESUMO
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.
Assuntos
Antígenos de Superfície/química , Eritrócitos/química , Sistema do Grupo Sanguíneo de Kell/química , Proteínas de Membrana/química , Caseína Quinase II , Caseína Quinases , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dissulfetos/química , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Membrana Eritrocítica/química , Humanos , Fosforilação , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
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.
Assuntos
Eritroblastos/fisiologia , Receptores de Superfície Celular/genética , Animais , Sequência de Bases , Medula Óssea/fisiologia , Clonagem Molecular/métodos , DNA/genética , DNA/isolamento & purificação , Eritropoetina/metabolismo , Éxons , Feminino , Feto , Biblioteca Gênica , Humanos , Íntrons , Fígado/fisiologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Placenta/fisiologia , Reação em Cadeia da Polimerase , Gravidez , Regiões Promotoras Genéticas , Receptores da Eritropoetina , Homologia de Sequência do Ácido Nucleico , Baço/fisiologia , TATA BoxRESUMO
Rh proteins are membrane proteins encoded by genes at the blood group RH locus. They are of paramount importance in transfusion medicine, but their function is still unknown. Biochemical and biophysical studies of these proteins are scarce since only minute amounts of the very hydrophobic Rh proteins, can be purified from human erythrocytes. Recently, a human monoclonal antibody (LOR-15C9) was described as having the unique property to recognize the Rh30 protein carrying the major blood group D specificity (RhD protein), either in a membrane detergent extract or when blotted on a membrane. In this report, we describe one-step purification of the RhD protein from detergent extracts of red cell membranes, based on immunoaffinity chromatography carried out with immobilized LOR-15C9 IgG. The technique yielded RhD protein with high purity which was devoid of other associated proteins (RhAG, CD47, LW and GPB) that comprise the Rh complex in the erythrocyte membrane. By contrast immunoprecipitation performed with the same antibody led to co-isolation of both RhD and RhAG.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Membrana Eritrocítica/química , Proteínas de Membrana/isolamento & purificação , Sistema do Grupo Sanguíneo Rh-Hr , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Humanos , Testes de Precipitina , UltracentrifugaçãoRESUMO
A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.
Assuntos
Clonagem Molecular , Expressão Gênica , Glicoforinas/genética , Sialoglicoproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 4 , Códon , Éxons , Variação Genética , Glicoforinas/deficiência , Humanos , Íntrons , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Antígenos Nucleares , DNA Helicases , Sistema do Grupo Sanguíneo de Kell/genética , Cloranfenicol O-Acetiltransferase , Proteínas de Ligação a DNA , Células Precursoras Eritroides , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Células K562 , Autoantígeno Ku , Luciferases , Tecido Linfoide , Masculino , Proteínas Nucleares , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Testículo , Distribuição Tecidual , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Anticorpos/metabolismo , Antígenos de Bactérias , Antígenos de Superfície/metabolismo , Eritrócitos/metabolismo , Sistema do Grupo Sanguíneo de Kell/imunologia , Fragmentos de Peptídeos/imunologia , Western Blotting , Membrana Eritrocítica/metabolismo , Humanos , Imuno-Histoquímica , Testes de PrecipitinaRESUMO
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).
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Antígenos de Grupos Sanguíneos/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Membrana Eritrocítica/química , Biblioteca Gênica , Sistema do Grupo Sanguíneo de Kell , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Músculo Esquelético , Ligação Proteica , Distribuição TecidualRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Sistema do Grupo Sanguíneo de Kell/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Proteínas de Transporte/química , Humanos , Sistema do Grupo Sanguíneo de Kell/química , Proteínas de Membrana/química , Dados de Sequência Molecular , Ácido PalmíticoRESUMO
Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Anticorpos Monoclonais/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Proteínas de Transporte/imunologia , Proteínas de Membrana/imunologia , Espectrina/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Detergentes/farmacologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Membrana Eritrocítica/química , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/imunologia , Humanos , Isoantígenos/química , Isoantígenos/imunologia , Isoantígenos/isolamento & purificação , Sistema do Grupo Sanguíneo de Kell/química , Sistema do Grupo Sanguíneo de Kell/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross-reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross-reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell-Kx complex from RBC membrane extract by immunochromatography.