ABSTRACT
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.
Subject(s)
Down Syndrome/diagnosis , Pregnancy/blood , Prenatal Diagnosis , Biomarkers , DNA/blood , Female , HumansABSTRACT
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.
Subject(s)
Biomarkers, Tumor , HSP70 Heat-Shock Proteins , Insulin-Secreting Cells , Membrane Proteins , Animals , Humans , Mice , Apoptosis/drug effects , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Line , Cell Survival/drug effects , Cell Survival/genetics , Electrophoresis, Gel, Two-Dimensional , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , In Situ Nick-End Labeling , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Isoelectric Focusing , Membrane Proteins/genetics , Membrane Proteins/metabolism , Palmitic Acids/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , RNA, Small Interfering , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tumor Protein, Translationally-Controlled 1ABSTRACT
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.
Subject(s)
Animal Testing Alternatives/methods , Drug Evaluation, Preclinical/methods , Toxicity Tests/methods , Adolescent , Aged , Animal Testing Alternatives/trends , Animals , Cells, Cultured , Coculture Techniques , Cytochrome P-450 Enzyme System/metabolism , Dogs , Drug Evaluation, Preclinical/trends , Hep G2 Cells , Humans , Liver/cytology , Male , Mesenchymal Stem Cells/drug effects , Mice , Pharmaceutical Preparations/metabolism , Prodrugs/pharmacology , Prodrugs/standards , Rats , Species Specificity , Spheroids, CellularABSTRACT
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.
Subject(s)
Cation Transport Proteins/physiology , Escherichia coli Proteins/physiology , Quaternary Ammonium Compounds/metabolism , Saccharomyces cerevisiae Proteins/physiology , Ammonia/metabolism , Biological Transport , Blood Proteins/chemistry , Blood Proteins/physiology , Cation Transport Proteins/chemistry , Escherichia coli Proteins/chemistry , Gene Expression Regulation , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , Models, Molecular , Nucleotidyltransferases/physiology , PII Nitrogen Regulatory Proteins/physiology , Protein Conformation , Rh-Hr Blood-Group System/chemistry , Rh-Hr Blood-Group System/physiology , Saccharomyces cerevisiae Proteins/chemistry , Signal Transduction , Species Specificity , Structure-Activity Relationship , Substrate SpecificityABSTRACT
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.
Subject(s)
Fetal Blood , Pregnancy/blood , Rho(D) Immune Globulin/blood , DNA/blood , Female , Fetus/metabolism , Forecasting , Gene Dosage , Genotype , Humans , Male , Pregnancy, Multiple , Rho(D) Immune Globulin/genetics , Sensitivity and Specificity , Sex Characteristics , TwinsABSTRACT
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.
Subject(s)
Rh-Hr Blood-Group System/physiology , Animals , Female , Humans , Infant, Newborn , Pregnancy , Rh-Hr Blood-Group System/geneticsABSTRACT
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.
Subject(s)
Kidney/metabolism , Membrane Glycoproteins/physiology , Quaternary Ammonium Compounds/metabolism , Rh-Hr Blood-Group System/physiology , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Biological Transport , Carrier Proteins , Genetic Complementation Test , Humans , Ion Transport , Membrane Glycoproteins/genetics , Molecular Sequence Data , Organ Specificity , Rh-Hr Blood-Group System/chemistry , Rh-Hr Blood-Group System/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Species SpecificityABSTRACT
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.
Subject(s)
Mutagenesis, Site-Directed , Rh-Hr Blood-Group System/genetics , Epitopes/chemistry , Epitopes/genetics , Flow Cytometry , Gene Expression , Humans , K562 Cells , Phenotype , Protein Structure, Tertiary , TransfectionABSTRACT
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.
Subject(s)
Rh-Hr Blood-Group System/immunology , Antigens/chemistry , Antigens/immunology , Epitope Mapping , Epitopes/chemistry , Epitopes/immunology , Humans , Rh-Hr Blood-Group System/chemistry , Serologic TestsABSTRACT
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.
Subject(s)
Blood Group Antigens/analysis , Fetal Blood/immunology , Prenatal Diagnosis/methods , Blood Group Antigens/genetics , Female , Genetic Testing , Humans , Infant, Newborn , Polymerase Chain Reaction/methods , Pregnancy , Rh Isoimmunization/diagnosis , Vitamin K Deficiency Bleeding/immunologyABSTRACT
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)
Subject(s)
Blood Proteins , Rh-Hr Blood-Group System/genetics , Rh-Hr Blood-Group System/physiology , Animals , Chromosome Mapping , Chromosomes, Human, Pair 1 , Erythrocyte Membrane/physiology , Humans , Membrane Glycoproteins/genetics , Models, Molecular , Polymorphism, Genetic , Protein Structure, Secondary , Rh-Hr Blood-Group System/chemistry , Terminology as TopicABSTRACT
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.
Subject(s)
Blood Transfusion , Protein Engineering , Recombinant Proteins/pharmacology , Animals , Blood Grouping and Crossmatching , Humans , Recombinant Proteins/therapeutic useABSTRACT
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)
Subject(s)
Black People/genetics , Glycoproteins/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Pseudogenes , Recombinant Fusion Proteins , Rh-Hr Blood-Group System , Amino Acid Sequence , Anemia, Hemolytic, Congenital/genetics , Base Sequence , Blood Donors , Ethnicity/genetics , Exons , Female , Ghana , Glycoproteins/chemistry , Humans , Huntingtin Protein , Infant, Newborn , Introns , Louisiana , Molecular Sequence Data , Mutation, Missense , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Phenotype , Polymerase Chain Reaction , Pregnancy , Pseudogenes/genetics , Repetitive Sequences, Nucleic Acid , Risk Factors , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , South Africa , Transcription, Genetic , ZimbabweABSTRACT
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.
Subject(s)
Rh-Hr Blood-Group System/chemistry , Rh-Hr Blood-Group System/genetics , Epitopes/chemistry , Epitopes/genetics , Humans , K562 Cells , Leukemia, Erythroblastic, Acute , Mutagenesis, Site-Directed , Protein Conformation , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Rh-Hr Blood-Group System/genetics , Antibodies, Monoclonal , Epitope Mapping/methods , Epitopes/chemistry , Humans , K562 Cells/metabolism , Mutagenesis, Site-Directed , Phenotype , Protein Structure, Tertiary , RNA, Messenger/metabolism , Rh-Hr Blood-Group System/immunologyABSTRACT
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.
Subject(s)
Black People/genetics , Rh-Hr Blood-Group System/genetics , Alleles , Blood Donors , Codon , England , Humans , Hybrid Cells , Netherlands , Phenotype , Polymorphism, Genetic , Reverse Transcriptase Polymerase Chain Reaction , South AfricaABSTRACT
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.
Subject(s)
Cytarabine/pharmacology , Immunosuppressive Agents/pharmacology , K562 Cells/metabolism , Rh-Hr Blood-Group System/metabolism , Blotting, Northern , Erythrocyte Membrane/metabolism , Hemoglobins/metabolism , Humans , K562 Cells/drug effects , Membrane Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
The scale of the application of molecular biological techniques to modern medicine and research in the biological sciences is vast, and in many instances has captured widespread public appeal. The intention of this review is to summarise the impact of molecular techniques on Transfusion Medicine ranging from diagnostic testing (platelet, granulocyte and red cell genotyping; microbiological testing), stable gene integration into haematopoeitic stem cells (gene therapy), production of blood products in transgenic animals and cell lines, and the inhibition of gene expression using synthetic antisense oligodeoxynucleotides. All of these techniques involve the manipulation of genes, be it from the relatively simple examination of different alleles to the technically demanding ability to express mammalian genes in culture and other animals.
Subject(s)
Blood Transfusion , Molecular Biology/methods , Animals , Antigens, Human Platelet/genetics , Biological Products/biosynthesis , Blood/microbiology , Blood/virology , Bone Marrow Transplantation , Cloning, Molecular , DNA, Complementary/genetics , DNA, Recombinant/genetics , Factor VIII/therapeutic use , Feasibility Studies , Gene Expression Regulation/drug effects , Gene Library , Genetic Therapy , Genotype , Hemophilia A/therapy , Humans , Mammals/blood , Mammals/genetics , Mass Screening , Oligonucleotides, Antisense/pharmacology , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Recombinant Proteins/therapeutic use , Safety , Transfusion ReactionABSTRACT
Antenatal genotyping of the fetus is now in widespread use as an aid to the clinical management in cases where there is the potential of haemolytic disease of the newborn occurring. The rapid diagnosis of an antigen-negative fetus will preclude the requirement for further, potentially risky invasive procedures being performed, whilst the determination of an antigen-positive fetus allows the potential of intensifying obstetric care for this pregnancy. Molecular genotyping is a major clinical application which has led from the determination of the molecular bases of blood group antigens expressed, most of which have been defined at the level of the gene. All assays used are dependent on the Polymerase Chain Reaction amplification of fetal DNA derived from either amniotic fluid or chorionic villi. Recent work has explored the potential of utilising fetal cells found to be present in maternal peripheral blood as a source of nucleic acid for prenatal diagnosis. Using non-invasive methods will preclude exposing mother and fetus to the potential hazards of invasive methods (amniocentesis, chorionic villus sampling and cordocentesis) which include miscarriage, fetal malformations and further maternal alloimmunisation.