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C M Redman

Publications and source records attributed to C M Redman.

At least 37 records · Page 2Linked to original sources
PubMed1995

The Kell blood group system.

The Kell blood group system is complex containing over 20 different antigens. Some of the Kell antigens may be organized in 5 sets of paired alleles with opposing high and low incidence antigens while others are independently expressed. Molecular cloning established that Kell antigens are carried on a 93kDa, type II, membrane glycoprotein. The Kell gene (KEL) is located at 7q 32-36 and spans about 21,5 kb. The coding sequence is organized in 19 exons. The promoter region does not contain TATA sequences but has possible transcription binding sites for GATA-1 and Sp1. Kell protein shares a putative enzymatic active amino acid sequence with a large family of zinc endopeptidases and has closest structural and sequence homology with neutral endopeptidase 24,11 (a.k.a. enkephalinase, CALLA) and endothelin converting enzyme (ECE-1). The molecular basis of several important Kell antigens has been determined and all are due to base substitutions causing single amino acid changes. The K1/K2 polymorphism is due to a C to T substitution in exon 6, encoding a threonine to methionine change. This mutation disrupts an N-glycosylation site. Two PCR-based methods, including use of allele-specific primers, have been developed which may be used to determine fetal K1/K2 genotypes. These tests can potentially identify those pregnancies at risk for hemolytic disease of the newborn. The allelic relationship of Kpa, Kpb and Kpc was confirmed, since single base substitutions in the same codon encode 3 different amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

K12 is located on the Kell blood group protein in proximity to K/k and Jsa/Jsb.

K12 is a high-prevalence antigen, with no known antithetical partner, that is associated with the Kell blood group system. We report studies on the 5th propositus (MS) with the K:-12 phenotype and the second case to show that the K12 is inherited. The anti-K12 in his serum did not destroy antigen-positive incompatible red cells transfused on at least 3 occasions over 6 years. Red cell membranes from MS possessed the Kell protein that was indistinguishable from control membranes. K12 antigen was shown by immunoprecipitation to be on a protein with an apparent molecular mass of 93,000 and by monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay to be on the Kell protein in proximity to K/k and Jsa/Jsb antigens. These data remove the K:-12 phenotype from its current Kell-related (or para Kell) status and elevates the K12 antigen to a bona fide member of the Kell blood group system.

Antibodies, Monoclonal↗
PubMed1994

Topology of Kell blood group protein and the expression of multiple antigens by transfected cells.

Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS-1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.

Animals↗
PubMed1994

Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen.

The cysteines involved in joining the 2 half-molecules of fibrinogen and also those located on either side of the alpha-helical coiled-coil region, were substituted, by site-directed mutagenesis, with serine. Fibrinogen assembly and secretion were determined in transiently transfected COS cells. Our studies indicate that in order to assemble the 2 half-molecules into a dimer, it is not sufficient to only have the disulfide linkages which keep the 2 half-molecules intact. The disulfide rings which flank the coil-coiled region also play important roles in dimer assembly. Intact interchain disulfide linkages at the NH2-terminal end of the coiled-coil region are essential for assembly of the 2 half-molecules. Disruption of these disulfide rings leads to the formation and secretion of half-molecules. Disruption of the interchain disulfide rings at the COOH-terminal end of the coiled-coil region allows dimer formation, but the 6-chain molecule which is assembled is not secreted. Disruption of both disulfide rings at either end of the coiled-coil region disallows assembly of half-molecules and of dimeric fibrinogen.

Animals↗
PubMed1993

Symmetrical disulfide bonds are not necessary for assembly and secretion of human fibrinogen.

Human fibrinogen is a dimer, and each half-molecule is composed of three different polypeptides (A alpha, B beta, and gamma). The two half-molecules are joined together at the amino-terminal ends by three symmetrical disulfide bonds between adjacent A alpha chains, at position 28, and between adjacent gamma chains at gamma 8 and 9. To determine the role of these disulfide bonds in fibrinogen assembly and secretion, site-directed mutagenesis was used to change cysteines of A alpha 28 and gamma 8 and 9 to serine, the mutant chains were coexpressed with normal fibrinogen chains in COS cells, and assembly and secretion of fibrinogen were measured. Elimination of the symmetrical disulfide bonds did not affect assembly of the chains and dimeric fibrinogen was secreted. Analysis by plasmin digestion indicated that the secreted mutant fibrinogens have a similar structure to normal fibrinogen. Our results indicate that other domains of fibrinogen participate in dimer formation and that the three symmetrical disulfide bonds are not crucial for assembly and secretion of fibrinogen.

Animals↗
PubMed1993

The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells.

The Kell blood group is one of the major antigenic systems in human red blood cells. To determine the location of the Kell gene on human chromosomes, panels containing genomic DNA of human-hamster somatic cell hybrids were hybridized with radiolabeled cDNA probe specific for the Kell locus. Only the samples containing DNA from chromosome 7 gave positive hybridization signals. In situ hybridization analysis, using genomic clones isolated with the cDNA, localized the KEL gene to 7q33. Northern blot analysis of poly(A)+ RNA from human brain, kidney, lung, fetal and adult liver, and bone marrow showed that Kell transcripts were only present in fetal liver and bone marrow. This indicates that the Kell protein, which carries the Kell antigens, may only be expressed in erythroid tissues.

Animals↗
PubMed1992

Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits.

Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.

Amino Acid Sequence↗
PubMed1992

Identification of B beta chain domains involved in human fibrinogen assembly.

Fibrinogen chains are assembled in a stepwise manner in the rough endoplasmic reticulum prior to secretion of the final six-chain dimeric molecule. Previous studies indicated that the synthesis of B beta may be a rate-limiting factor in the assembly of human fibrinogen. To determine the domains of B beta which interact with the other two component chains of fibrinogen, deletion mutants of B beta were transiently co-expressed, together with A alpha and gamma chains, in COS cells, and fibrinogen assembly and secretion were measured. Deletion of the COOH-terminal half of the B beta chain (amino acids 208-461) did not affect assembly and secretion. Assembly of A alpha, gamma, and B beta also occurred when the first NH2-terminal 72 amino acids of B beta were deleted, but not when 93 amino acids were deleted. This indicates that the B beta domain between amino acids 73 and 93 is necessary for the assembly of the three fibrinogen chains. This domain marks the start of the alpha-helical "coiled-coil" region of fibrinogen.

Amino Acid Sequence↗
PubMed1992

Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes.

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.

Albumins↗
PubMed1991

Molecular cloning and primary structure of Kell blood group protein.

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.

Amino Acid Sequence↗
PubMed1991

Assembly and secretion of recombinant human fibrinogen.

Expression vectors containing full-length cDNAs for each of the human fibrinogen chains were constructed. COS-1 cells were transfected with single vectors, mixtures of two, or with all three vectors and stable cell lines selected. Cells transfected with single vectors, or with mixtures of any two vectors, expressed the appropriate fibrinogen chains but did not secrete them. COS cells transfected with three vectors expressed all of the chains and secreted fibrinogen. COS cells transfected with three vectors contained, intracellularly, a mixture of fibrinogen-related proteins. The four main intracellular products were nascent fibrinogen, an A alpha.gamma complex, free A alpha chains, and free gamma chains. This is a similar pattern to that noted in Hep G2 cells. The intracellular forms of fibrinogen were sensitive to endoglycosidase H, indicating that they reside in a pre-Golgi compartment. Secreted fibrinogen was endoglycosidase H-insensitive, suggesting that the secreted glycoprotein moieties were processed in the normal manner. When mixed with plasma fibrinogen, radiolabeled recombinant fibrinogen was incorporated into a thrombin-induced clot. These studies demonstrate that COS cells transfected with all three fibrinogen chain cDNAs are capable of assembling and secreting a functional fibrinogen molecule.

Cell Line↗
PubMed1991

Diagnostic tests for choreoacanthocytosis.

Two patients with striatal atrophy and a clinical syndrome consistent with choreoacanthocytosis had normal dried blood smears but their red cells demonstrated an abnormal sensitivity to various conditions known to promote discocyte-echinocyte transformation. Dilution in normal saline, in vitro aging, and contact with glass caused a great proportion of these patients' red cells to develop multiple spiny or rounded projections. Under identical conditions, such shape changes did not occur in normal patients or in those with Huntington's disease. Scanning electron microscopy showed that the age-induced increase in acanthocytic-appearing cells could be reversed with chlorpromazine. These data suggest that the red cells from these patients with striatal degeneration are deficient in their ability to preserve normal shape in the face of echinocytic stress and that this observation has diagnostic and, possibly, pathophysiologic significance.

Acanthocytes↗
PubMed1990

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen.

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.

Blotting, Northern↗
PubMed1989

Expression and secretion of chicken apolipoprotein AI in transfected COS cells.

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.

Acylation↗
PubMed1989

Effect of phosphatidylserine on the shape of McLeod red cell acanthocytes.

The rare McLeod blood group phenotype is characterized by weak Kell antigens, lack of the common Kx antigen, and acanthocytic morphology. Previous studies that did not detect membrane or cytoskeletal protein abnormalities suggested a lipid disturbance. In normal red cells, dimyristoyl phosphatidylserine (DMPS) is transported across the membrane by an enzymatic process and accumulates in the inner leaflet of the membrane bilayer causing discocyte to stomatocyte shape changes. Scanning electron microscopy of McLeod red cells shows a mixture comprised of 15% discocytes, 51% with irregular surfaces, and 34% acanthocytes. On incubation with various concentrations of DMPS at 37 degrees C for periods up to two hours, McLeod red cells transported DMPS across the membrane and caused irregularly shaped and acanthocytic McLeod red cells to attain normal discocyte shape and later to become stomatocytes. Chlorpromazine, which at 0 degrees C preferentially partitions into the inner monolayer of the membrane, had a similar effect on the shape of McLeod red cells. This suggests that in McLeod cells acanthocytosis is due to a lack of lipid in the inner leaflet of the membrane bilayer but that the imbalance is not caused by defective transport of phosphatidylserine across the membrane.

Acanthocytes↗