RESUMEN
Down syndrome is the most common cause of mental retardation and has an incidence of between 1:600 and 1:800 pregnancies. It is the condition for which prenatal diagnosis is requested the most and most developed countries have adopted a screening program based around maternal plasma/serum testing and ultrasound. Advances have been made recently to eliminate invasive testing for genetic diagnosis of this condition based on the analysis of free fetal DNA in maternal plasma. But, routine noninvasive prenatal diagnosis for trisomy 21 still appears to be years away. Screening based on assessment of various biomarkers present in maternal plasma represents a front-line test to assess the risk of the mother carrying an aneuploid fetus. Recent comparative proteomics techniques have resulted in studies that have assessed maternal plasma from mothers carrying normal and trisomy 21 fetuses and various gestational ages. Over 100 biomarker candidates have been described, but little consensus has emerged. This may be due to a number of compounded factors, but interesting to note that other neurological disorders have overlapping biomarkers. This article describes these developments and how these biomarkers could contribute to future screening in an emerging era where next-generation sequencing of free fetal DNA will be established in prenatal diagnostics, which appears imminent.
Asunto(s)
Síndrome de Down/diagnóstico , Embarazo/sangre , Diagnóstico Prenatal , Biomarcadores , ADN/sangre , Femenino , HumanosRESUMEN
AIMS/HYPOTHESIS: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out. METHODS: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells. RESULTS: TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death. CONCLUSIONS/INTERPRETATION: Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.
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Biomarcadores de Tumor , Proteínas HSP70 de Choque Térmico , Células Secretoras de Insulina , Proteínas de la Membrana , Animales , Humanos , Ratones , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Electroforesis en Gel Bidimensional , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Etiquetado Corte-Fin in Situ , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Focalización Isoeléctrica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ácidos Palmíticos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Interferente Pequeño , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Prior to introduction to the clinic, pharmaceuticals must undergo rigorous toxicity testing to ensure their safety. Traditionally, this has been achieved using in vivo animal models. However, besides ethical reasons, there is a continual drive to reduce the number of animals used for this purpose due to concerns such as the lack of concordance seen between animal models and toxic effects in humans. Adequate testing to ensure any toxic metabolites are detected can be further complicated if the agent is administered in a prodrug form, requiring a source of cytochrome P450 enzymes for metabolism. A number of sources of metabolic enzymes have been utilised in in vitro models, including cell lines, primary human tissue and liver extracts such as S9. This review examines current and new in vitro models for toxicity testing, including a new model developed within the authors' laboratory utilising HepG2 liver spheroids within a co-culture system to examine the effects of chemotherapeutic agents on other cell types.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Toxicidad/métodos , Adolescente , Anciano , Alternativas a las Pruebas en Animales/tendencias , Animales , Células Cultivadas , Técnicas de Cocultivo , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Evaluación Preclínica de Medicamentos/tendencias , Células Hep G2 , Humanos , Hígado/citología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Preparaciones Farmacéuticas/metabolismo , Profármacos/farmacología , Profármacos/normas , Ratas , Especificidad de la Especie , Esferoides CelularesRESUMEN
OBJECTIVE: To test the hypothesis that free fetal DNA (ffDNA) circulating in maternal plasma originates mainly from the placenta we studied ffDNA levels in anembryonic pregnancies. METHODS: Maternal blood samples were collected from 15 normal first-trimester pregnancies in which fetal sex was subsequently determined and nine patients with a diagnosis of anembryonic gestation (AG). The Y chromosome DYS14 gene was quantified by real-time quantitative PCR (RT-PCR) for the determination of fetal sex in both plasma and chorionic tissue samples. Fetal sex in chorionic tissue samples was also determined using quantitative fluorescence PCR (QF-PCR). RESULTS: The correct sex result was obtained from maternal plasma in all. Four AG pregnancies were female (DYS14 negative) results. In five of the AG cases, the chorionic tissue was found to be male (by both QF-PCR and RT-PCR which agreed) and positive male signal was found in maternal plasma by RT-PCR. There was no statistical difference between median free fetal DNA concentration in plasma between the AG male cases (148.3 GE/mL) and controls (145.8 GE/mL). CONCLUSION: Since ffDNA levels are normal in pregnancies without a fetus, the data support the hypothesis that the trophoblastic cells are the major source ffDNA in maternal plasma.
Asunto(s)
Aneuploidia , Cromosomas Humanos Y/genética , ADN/sangre , Diagnóstico Prenatal , Trofoblastos/química , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Embarazo , Primer Trimestre del Embarazo , Análisis para Determinación del Sexo/métodosRESUMEN
The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.
Asunto(s)
Proteínas de Transporte de Catión/fisiología , Proteínas de Escherichia coli/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Amoníaco/metabolismo , Transporte Biológico , Proteínas Sanguíneas/química , Proteínas Sanguíneas/fisiología , Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Regulación de la Expresión Génica , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Modelos Moleculares , Nucleotidiltransferasas/fisiología , Proteínas PII Reguladoras del Nitrógeno/fisiología , Conformación Proteica , Sistema del Grupo Sanguíneo Rh-Hr/química , Sistema del Grupo Sanguíneo Rh-Hr/fisiología , Proteínas de Saccharomyces cerevisiae/química , Transducción de Señal , Especificidad de la Especie , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell-free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D- genomes. STUDY DESIGN AND METHODS: Real-time PCR assays were designed to distinguish RHD from RHDpsi (possessed by the majority of D- black Africans). Plasma-derived DNA from 137 D- women was subjected to real-time PCR to detect fetal RHD and Y chromosome-associated SRY sequences. The accuracy of RHD genotyping from maternal plasma was investigated by comparing results with those obtained by conventional RHD genotyping from fetal tissue or serologic tests on the infant's RBCs. The quantity of fetal DNA in maternal plasma was investigated in 94 pregnancies. RESULTS: Fetal D status was predicted with 100-percent accuracy from maternal plasma. The number of copies of fetal DNA in maternal plasma was found to increase with gestation. CONCLUSION: Combination of the sensitivity of real-time PCR with an improved RHD typing assay to distinguish RHD from RHDpsi enables highly accurate prediction of fetal D status from maternal plasma. This has resulted in the implementation of a clinical noninvasive fetal RHD genotyping service.
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Sangre Fetal , Embarazo/sangre , Globulina Inmune rho(D)/sangre , ADN/sangre , Femenino , Feto/metabolismo , Predicción , Dosificación de Gen , Genotipo , Humanos , Masculino , Embarazo Múltiple , Globulina Inmune rho(D)/genética , Sensibilidad y Especificidad , Caracteres Sexuales , GemelosRESUMEN
Rh molecular biology has made many advances since the first Rh cDNA was cloned in 1990. This review summarizes the current knowledge concerning the molecular basis of Rh antigenicity, D-epitope expression, and the structures of the Rh genes and proteins. Although many recent reviews have appeared regarding these subjects, advances in Rh protein function that have been published within the last 12 months have had a fundamental impact on the future direction of Rh research. In November 2000, an article described the role of Rh proteins in ammonium transport, which has remained undescribed in vertebrates, except for non-specific transport via K+ channels. The recent identification of nonerythroid Rh proteins, their expression in diverse tissues, and notably polarized epithelial and endothelial cells will be of broad functional significance and will greatly increase our understanding of the role of Rh in ammonium transport and the biology of ammonium metabolism as a whole. The advances in Rh molecular genetics have enabled the development of diagnostic tests in the clinic. At present, this is largely confined to the prenatal diagnosis of fetal blood group status in alloimmunized pregnancies, but could be extended to the noninvasive prenatal testing of all D-negative pregnant women and eventually, perhaps, to all patient and donor blood.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/fisiología , Animales , Femenino , Humanos , Recién Nacido , Embarazo , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
Occasionally, an original research paper has an unusually significant impact on a particular research field. Such a paper, published recently in Nature Genetics, describes the uncovering of the functional role of the Rh protein family--the proteins that express the Rh blood group antigens. Marini et al. (1) demonstrate how two human Rh glycoproteins can correct ammonium transport deficiency in mutant yeast cells. Rh proteins are therefore ammonium transporters--a role that, in vertebrates, has remained previously uncharacterized. These data herald a new era in Rh protein research, beyond their role as blood group antigens, and into the characterization of ammonium transport mechanisms, notably in the kidney.
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Riñón/metabolismo , Glicoproteínas de Membrana/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr/fisiología , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras , Prueba de Complementación Genética , Humanos , Transporte Iónico , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Sistema del Grupo Sanguíneo Rh-Hr/química , Sistema del Grupo Sanguíneo Rh-Hr/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Especificidad de la EspecieRESUMEN
Previous attempts to define the molecular configuration of D epitopes has been confined to the analysis of the serological profile and Rh D molecular structure of partial D phenotypes. There are numerous drawbacks in this approach, most fundamental of which is that with the exception of RoHar, partial D phenotypes are defined by the loss of D epitope expression, and is thus difficult to directly correlate a specific amino acid to a particular D epitope. Furthermore, most partial D phenotypes are associated with multiple amino acid changes in the mutant Rh protein species associated with partial D expression. In our study we have applied site directed mutagenesis to introduce RhD amino acids in a stepwise manner to a Rh cE cDNA. This cDNA was introduced into K562 cells using retroviral mediated gene delivery, and D epitope expression analysed by flow cytometry. Our study provides evidence for at least six different epitope clusters on the external face of the Rh D protein. The relative predicted positions of these epitope clusters has resulted in us presenting a model for the hypothetical arrangement of external Rh D protein loops.
Asunto(s)
Mutagénesis Sitio-Dirigida , Sistema del Grupo Sanguíneo Rh-Hr/genética , Epítopos/química , Epítopos/genética , Citometría de Flujo , Expresión Génica , Humanos , Células K562 , Fenotipo , Estructura Terciaria de Proteína , TransfecciónRESUMEN
Analyses of the reactions of monoclonal anti-D with Rh D variant red cells have shown that there are at least 24 different epitopes of the Rh D antigen. Similar studies Of Rh E variant red cells with monoclonal anti-E indicate that there are at least 4 epitopes of the Rh E antigen. The relation of these serologically defined epitopes to the structure of the Rh proteins is reviewed. Most epitopes are discontinuous, with critical residues present in different loops of the proteins.
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Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Antígenos/química , Antígenos/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Humanos , Sistema del Grupo Sanguíneo Rh-Hr/química , Pruebas SerológicasRESUMEN
BACKGROUND AND OBJECTIVES: The prenatal determination of fetal blood group status by molecular techniques has been used in the clinical management of alloimmunised pregnancies for seven years, in particular for the definition of fetal Rh D, c and E, K, Fya and Jka status. This has arisen in response to the definition of the molecular bases of human blood group polymorphism. MATERIALS AND METHODS: PCR-based amplification assays have been designed to define fetal blood group status, where the source of template DNA is normally derived from amniotic fluid or chorionic villus. Recently, non-invasive methods have been explored to obtain fetal DNA from maternal peripheral blood. RESULTS: PCR-based tests are now available to screen for all fetal medicine significant blood group antigens. The Rh system is the most complex, and assays to define Rh genotype have been modified in response to our increased understanding of the molecular biology of this blood group system. CONCLUSION: Prenatal diagnosis of fetal blood group status is now in widespread use in the clinical management of HDN. Non-invasive testing, if applied in the clinical setting may invoke a dramatic increase in the numbers of pregnancies that may be analysed prenatally.
Asunto(s)
Antígenos de Grupos Sanguíneos/análisis , Sangre Fetal/inmunología , Diagnóstico Prenatal/métodos , Antígenos de Grupos Sanguíneos/genética , Femenino , Pruebas Genéticas , Humanos , Recién Nacido , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Isoinmunización Rh/diagnóstico , Sangrado por Deficiencia de Vitamina K/inmunologíaRESUMEN
The Rh blood group system is one of the most polymorphic and immunogenic systems known in humans. In the past decade, intense investigation has yielded considerable knowledge of the molecular background of this system. The genes encoding 2 distinct Rh proteins that carry C or c together with either E or e antigens, and the D antigen, have been cloned, and the molecular bases of many of the antigens and of the phenotypes have been determined. A related protein, the Rh glycoprotein is essential for assembly of the Rh protein complex in the erythrocyte membrane and for expression of Rh antigens. The purpose of this review is to provide an overview of several aspects of the Rh blood group system, including the confusing terminology, progress in molecular understanding, and how this developing knowledge can be used in the clinical setting. Extensive documentation is provided to enable the interested reader to obtain further information. (Blood. 2000;95:375-387)
Asunto(s)
Proteínas Sanguíneas , Sistema del Grupo Sanguíneo Rh-Hr/genética , Sistema del Grupo Sanguíneo Rh-Hr/fisiología , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Membrana Eritrocítica/fisiología , Humanos , Glicoproteínas de Membrana/genética , Modelos Moleculares , Polimorfismo Genético , Estructura Secundaria de Proteína , Sistema del Grupo Sanguíneo Rh-Hr/química , Terminología como AsuntoRESUMEN
Recombinant technology in transfusion medicine has really only just begun to have large-scale impact. The preparation of blood products, determination of blood group phenotype, detection of blood group specific antibodies does not currently employ DNA-based methods for their preparation or detection. The detection of bloodborne viruses, production of blood grouping reagents and diagnosis of HLA polymorphism all include recombinant DNA-based technologies and are beginning to impact on routine laboratory life in Transfusion medicine. This review analyses the current use of recombinant technology in transfusion medicine, and indicates where there is likely to be significant development of this methodology (particularly in molecular diagnostics) oven the next decade or so. The impact of molecular medicine in the field of transfusion has already begun. Recent licensing of thrombopoietin for clinical use may have a profound effect on the very high current demand for platelet transfusions. Gene therapy protocols for the treatment of haemophilias and other coagulation disorders, and the production of recombinant blood products may reshape the demand for clotting factors from human plasma. I also consider the potential impact of the exciting technologies of DNA arraying and nucleic acid therapeutics in the fields of molecular diagnostics and the possible treatment of leukemia respectively.
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Transfusión Sanguínea , Ingeniería de Proteínas , Proteínas Recombinantes/farmacología , Animales , Tipificación y Pruebas Cruzadas Sanguíneas , Humanos , Proteínas Recombinantes/uso terapéuticoRESUMEN
Antigens of the Rh blood group system are encoded by 2 homologous genes, RHD and RHCE, that produce 2 red cell membrane proteins. The D-negative phenotype is considered to result, almost invariably, from homozygosity for a complete deletion of RHD. The basis of all PCR tests for predicting fetal D phenotype from DNA obtained from amniocytes or maternal plasma is detection of the presence of RHD. These tests are used in order to ascertain the risk of hemolytic disease of the newborn. We have identified an RHD pseudogene (RHD psi) in Rh D-negative Africans. RHDpsi contains a 37 base pair (bp) insert in exon 4, which may introduce a stop codon at position 210. The insert is a sequence duplication across the boundary of intron 3 and exon 4. RHDpsi contains another stop codon in exon 6. The frequency of RHDpsi in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% had RHDpsi, 15% had the RHD-CE-D hybrid gene associated with the VS+ V- phenotype, and only 18% completely lacked RHD. RHDpsi is present in about 24% of D-negative African Americans and 17% of D-negative South Africans of mixed race. No RHD transcript could be detected in D-negative individuals with RHDpsi, probably as a result of nonsense-mediated mRNA decay. Existing PCR-based methods for predicting D phenotype from DNA are not suitable for testing Africans or any population containing a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp insert in exon 4 of RHDpsi. (Blood. 2000; 95:12-18)
Asunto(s)
Población Negra/genética , Glicoproteínas/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Seudogenes , Proteínas Recombinantes de Fusión , Sistema del Grupo Sanguíneo Rh-Hr , Secuencia de Aminoácidos , Anemia Hemolítica Congénita/genética , Secuencia de Bases , Donantes de Sangre , Etnicidad/genética , Exones , Femenino , Ghana , Glicoproteínas/química , Humanos , Proteína Huntingtina , Recién Nacido , Intrones , Louisiana , Datos de Secuencia Molecular , Mutación Missense , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Fenotipo , Reacción en Cadena de la Polimerasa , Embarazo , Seudogenes/genética , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Riesgo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Sudáfrica , Transcripción Genética , ZimbabweRESUMEN
The Rh D antigen is the most clinically important protein blood group antigen of the erythrocyte. It is expressed as a collection of at least 37 different epitopes. The external domains of the Rh D protein involved in epitope presentation have been predicted based on the analysis of variant Rh D protein structures inferred from their cDNA sequences and their D epitope expression. This analysis can never be absolute because (1) most partial D phenotypes involve multiple amino acid changes in the Rh D protein and (2) deficiency for 1 or more epitopes may be due to gross structural alteration in the variant Rh D protein structure. We report here the amino acid requirements for the majority of D epitopes. They have been defined by generating a series of novel Rh mutant constructs by mutagenesis using an Rh cE cDNA as template and mutagenic oligonucleotide primers. When transfected into K562 cells, the D epitope expression of the derived mutant clones was then assessed by flow cytometry. The introduction of 9 externally predicted Rh D-specific amino acids on the Rh cE protein was sufficient to express 80% of all tested D epitopes, whereas other clones expressed none. We concluded from our data that the D epitope expression is consistent with at least 6 different epitope clusters localized on external regions of the Rh D protein, most involving overlapping regions within external loops 3, 4, and 6.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/química , Sistema del Grupo Sanguíneo Rh-Hr/genética , Epítopos/química , Epítopos/genética , Humanos , Células K562 , Leucemia Eritroblástica Aguda , Mutagénesis Sitio-Dirigida , Conformación Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: The antigens of the human Rh system are of great clinical significance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic structures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to have 12 membrane-spanning segments giving rise to six exofacial domains. STUDY DESIGN AND METHODS: By site-directed mutagenesis using the method of inverse polymerase chain reaction, cE and D cDNA mutant constructs were generated with changes to the RHD-specific residues 350, 353, and 354 in the predicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBabe puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin-resistant K562 clones were screened by flow cytometric analysis using a panel of monoclonal antibodies with specificities to ep (epitope) D1 through epD9. RESULTS: De novo expression of epD3 and epD9 was generated in the K562 cell lines expressing the mutated cE polypeptide (cE-Asp350His, Gly353Trp, Ala354Asn). Expression of c and E was unaffected. Conversely, the cells expressing the mutated D polypeptide demonstrated loss of expression of epD1, epD2, epD3, epD4, and epD9. CONCLUSION: The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut cell line has an identical molecular and serologic profile as the red cell D(IVb) phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/genética , Anticuerpos Monoclonales , Mapeo Epitopo/métodos , Epítopos/química , Humanos , Células K562/metabolismo , Mutagénesis Sitio-Dirigida , Fenotipo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr/inmunologíaRESUMEN
K562 cells undergoing differentiation induced by 1-beta-D-arabino-furanosyl-cytosine (ara-C) were examined as a model for studying the biosynthesis and regulation of Rh and other blood group active membrane proteins. Untreated and ara-C-induced K562 cells were analysed for the expression of these proteins using monoclonal antibodies in combination with flow cytometry. The major membrane proteins glycophorins A and C remained unaltered upon induction by ara-C. The display of LFA-3 (CD58) and DAF (CD55) by uninduced K562 was one order of magnitude lower than that of the glycophorins; following ara-C treatment there was a 50% rise in LFA-3 but a modest decrease in the level of DAF expression. The expression by untreated K562 cells of Rh, Lutheran and Kell proteins as well as the Rh D antigen was low, whereas that of CD44 and band 3 protein was negligible. Following induction by ara-C the levels of Rh and Kell proteins rose up to 7- and 3.5-fold respectively, and there was an increase in RhD-antigen expression. In contrast, ara-C induction of K562 cells failed to augment their display of Lutheran, CD44 and band 3 proteins. Analysis of Rh transcripts following the purification and RT-PCR analysis of K562 mRNA showed that uninduced K562 cells contain two distinct mRNAs corresponding to Rh Ce (1.8 kb) and Rh D (3.5 kb). The apparent concentration of each mRNA increased following induction with ara-C. K562 plasma membranes also contained Rh polypeptides as determined by immunoblot analysis using anti-Rh polypeptide rabbit polyclonal sera raised to Rh synthetic peptides. A novel hybrid Rh transcript corresponding to exons 1-4 of RHD and exons 5-10 of RHCE has been cloned and sequenced from ara-C induced K562 cells, and may have arisen by general recombination between the RHD and RHCE genes.
Asunto(s)
Citarabina/farmacología , Inmunosupresores/farmacología , Células K562/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr/metabolismo , Northern Blotting , Membrana Eritrocítica/metabolismo , Hemoglobinas/metabolismo , Humanos , Células K562/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood. STUDY DESIGN AND METHODS: Red cells from 100 black South Africans and 43 black persons from Amsterdam, the Netherlands, were typed serologically for various Rh system antigens. Allele-specific polymerase chain reaction and sequencing of polymerase chain reaction products were used to analyze C733G (Leu245Val) and G1006T (Gly336Cys) polymorphisms in exons 5 and 7 of RHCE and the presence of a D-CE hybrid exon 3. RESULTS: The respective frequencies of all VS+ and of VS+ V-(r's) phenotypes were 43 percent and 9 percent in the South Africans and 49 percent and 12 percent in the Dutch donors. All VS+ donors had G733 (Val245), but six with G733 were VS- (4 V+w, 2 V-). The four VS- V+w donors with G733 appeared to have a CE-D hybrid exon 5. T1006 (Cys336) was present in 12 percent and 16 percent of donors from the two populations. With only a few exceptions, T1006, a D-CE hybrid exon 3, and a C410T (Ala137Val) substitution were associated with a VS+ V-phenotype ((C)ces or r's haplotype). Two VS+ V-individuals, with the probable genotype, (C)ces/(C)ces), were homozygous for G733 and for T1006. CONCLUSIONS: It is likely that anti-VS and anti-V recognize the conformational changes created by Val245, but that anti-V is sensitive to additional conformational changes created by Cys336.