Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells.

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.

Toxicity testing: the search for an in vitro alternative to animal testing.

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.

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins.

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.

A new chapter in Rh research: Rh proteins are ammonium transporters.

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.

Recombinant technology in transfusion medicine.

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.

Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte.

Endo-N-acetyl-beta-D-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-beta-D-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fya) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein alpha and the absence of such an oligosaccharide from sialoglycoprotein delta. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-beta-D-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-beta-D-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins alpha and delta carry little or no blood group A or B activity. Endo F alone, or in combination with endo-beta-D-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fya activity. The diffuse Fya component of Mr 38,500-90,000 was sharpened to a band of Mr 26,000. Either endo-beta-D-galactosidase or neuraminidase treatment reduced the Mr of the Fya component(s) but did not significantly sharpen the bands, suggesting that the Fya component contains between 40-50% by mass of N-glycosyl-linked oligosaccharides.

Human erythrocyte antigen expression: its molecular bases.

This review summarises the considerable body of information now available on the molecular bases of human blood group-antigen expression. The elucidation of this information has only been possible since the identification, purification, and subsequent cDNA cloning of the mRNAs which encode the blood group active proteins. The surface components which are responsible for antigen expression are divided into two types: carbohydrate and protein. All carbohydrate structures are attached covalently to either glycolipids or glycoproteins, and are synthesised in the Golgi apparatus of erythropoietic cells (or in other cell lines in secreted fluids). As a consequence, the molecular bases of carbohydrate antigens lie in polymorphic variation seen in the genes which synthesise these carbohydrate structures (i.e. glycosyltransferase enzymes). The structural differences in the ABO, Hh and Lewis transferase genes, which alter the substrate specificities of these glycosyltransferases and hence generate the different antigens, have been defined. The molecular bases underlying the P blood glycosyltransferases are unknown. Polymorphism in the remaining 19 blood group systems is defined by amino acid sequence changes in erythrocyte membrane proteins, which are generated by sequence variation at the DNA level (largely by point mutation). Blood group active erythrocyte membrane proteins can be categorised broadly into six functional groups: (1) membrane transporters or channels: Rh, Diego, Colton, Kidd, KX; (2) Membrane bound enzymes: Kell and Cartwright; (3) Structural or assembly proteins: Gerbich and MNSs; (4) Chemokine receptors: Duffy; (5) Cell adhesion molecules: Lutheran, LW, Xg, Indian; (6) Complement regulatory proteins: Cromer, Knops. The Chido/Rodgers blood group system is defined by polymorphic variation in C4 of the complement cascade, and is adsorbed passively onto the surface of erythrocytes. This system is not considered here. Only two remaining blood group systems defy molecular identity: Dombrock and Scianna. The potential clinical applications of such a rapid accumulation of data on the molecular bases of blood group antigens is discussed.

Molecular biology of partial D phenotypes.

We have examined all DVI variant phenotypes submitted to the workshop by a combination of RT-PCR, multiplex RHD PCR and immunoblotting with Rh antipeptide sera. Our findings suggest that all DVI phenotypes arise through hybrid RHD-RHCE-RHD genes. Genomic DNA derived from all DVI samples were shown to be RHD intron 4 negative when analysed with an RHD intron 4/exon 10 multiplex assay. We assume therefore that all DVI phenotypes involve gene conversion events involving at least exons 4 and 5 of the RHD gene. Analysis of a novel D and E variant phenotype individual (ISBT49) by RT-PCR has allowed the identification of a hybrid Rh gene composed of exons 1-4 RHD: 5 RHCE/D and 6-10 RHD. We propose that the partial D & E phenotype observed arises through D & E expression on the hybrid RHD-RHCE-RHD protein: as no transcripts encoding Rh E could be found.

A structural model for 30 Rh D epitopes based on serological and DNA sequence data from partial D phenotypes.

Both cDNA RHD sequences and reactivity with monoclonal anti-D have been reported in a number of partial D phenotypes, where parts (some epitopes) of the normal D antigen are missing, and anti-D of restricted specificity may be made in response to challenge with normal D positive blood. This paper analyses these reports together and proposes a model for the structure which comprise the epitopes of the Rh D antigen. Some epitopes are proposed to be comprised of continuous peptide sequence within one extracellular loop, whereas others require interactions between two or the extracellular peptide loops.

Maternal plasma biomarkers for down syndrome: present and future.

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.

Molecular genetic methods: principles and feasibility in transfusion medicine.

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.

Antenatal genotyping of the blood groups of the fetus.

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.

Molecular biology of the Rh blood group system.

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.

The Rh blood group system: a review.

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)

Kell typing by allele-specific PCR (ASP).

The Kell blood group system is important in transfusion medicine, and the Kell antigen (K1) is probably second in importance to Rh D as an immunogen in alloimmunized pregnancies which cause haemolytic disease of the newborn. The K/k (K1/K2) blood group polymorphism has been recently defined. A point mutation changes Thr193 (k) to Met193 (K) in the Kell glycoprotein. The mutation which creates K destroys a consensus N-glycan addition site. We describe a simple PCR test for K blood group typing. The test is based on the use of an allele-specific K-primer. We have shown the test to give results in complete concordance with serologically defined Kell blood group status using 65 genomic DNA samples derived from both amniocytes and peripheral blood lymphocytes. The test is suitable for the prenatal determination of Kell type.

Site directed mutagenesis of the human Rh D antigen: molecular basis of D epitopes.

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.

Epitopes on Rh proteins.

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.

Prenatal determination of fetal blood group status.

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.

Point mutation in the glycophorin C gene results in the expression of the blood group antigen Dha.

The blood group Duch (Dha) antigen is located on glycophorin C (GPC). Total RNA prepared from the reticulocyte fraction of two Dh(a+) individuals were used in the synthesis of first-strand cDNA. The first-strand cDNA served as templates for the amplification of GPC-related DNA by polymerase chain reaction (PCR). The expected PCR product consisted of 412 base pairs. On sequencing the PCR-amplified DNA, a base change (cytosine----thymidine) at nucleotide 40 of the GPC cDNA was detected. Thus, the variant GPC (GPC.Dha) on Dh(a+) red cells has a substitution of leucine by phenylalanine at amino acid residue 14.