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Connie M Westhoff

Publications and source records attributed to Connie M Westhoff.

14 recordsLinked to original sources
PubMed2007

Ammonia secretion from fish gill depends on a set of Rh glycoproteins.

Ammonia excretion from the gill in teleost fish is essential for nitrogen elimination. Although numerous physiological studies have measured ammonia excretion, the mechanism of ammonia movement through the membranes of gill epithelial cells is still unknown. Mammalian Rh glycoproteins are members of a family of proteins that mediate ammonia transport in bacteria, yeast, and plants. We identified the Rh glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the pufferfish, Takifugu rubripes. Northern blot, in situ hybridization, and immunohistochemistry revealed that the pufferfish erythroid Rh glycoprotein homologue fRhag was present in red blood cells and the hematological organs (spleen and kidney) in fish. All four pufferfish Rh glycoproteins are specifically localized in the gill and line the pillar cells, pavement cells, and the mitochondrion-rich cells. Heterologous expression in Xenopus oocytes showed that they mediate methylammonium (an analog of ammonium) transport. These results suggest that pufferfish Rh glycoproteins are involved in ammonia excretion from the gill. These findings challenge the classic view that ammonia excretion in the fish gill occurs by passive diffusion.

Amino Acid Sequence↗
PubMed2007

The structure and function of the Rh antigen complex.

The Rh system is one of the most important and complex blood group systems because of the large number of antigens and the serious complications for the fetus of a woman sensitized by transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes and with functional evidence that the Rh blood group proteins belong to an ancient family of membrane proteins involved in ammonia transport. The arrangement and configuration of the genes at the RH locus promotes genetic exchange, generating new antigens. Importantly, RH genetic testing can now be applied to clinical transfusion medicine and prenatal practice. This includes testing for RHD zygosity, confirmation or resolution of D antigen status, and detection of altered RHD and RHCE genes in individuals at risk for producing antibodies to high-incidence Rh antigens, particularly sickle cell disease (SCD) patients. The Rh proteins form a core complex that is critical to the structure of the erythrocyte membrane, and they may play a physiologic role in the sequestration of blood ammonia. The Rh family of proteins now includes non-erythroid homologs present in many other tissues, and comparative genomics reveal Rh homologs in all domains of life.

Anemia, Sickle Cell↗
PubMed2006

Molecular testing for transfusion medicine.

PURPOSE OF REVIEW: Molecular testing methods were introduced to the blood bank and transfusion medicine community more than a decade ago after cloning of the genes made genetic testing for blood groups, that is genotyping, possible. This review summarizes the progress made in the last decade in applying genotyping to prenatal practice and clinical transfusion medicine. RECENT FINDINGS: Assays that target allelic polymorphisms prevalent in all populations are reproducible and highly correlated with red blood cell phenotype. For some blood groups, assays that detect silencing mutations are also required for accurate typing, and for ABO and Rh, multiple regions of the genes must be sampled. Genotyping is a powerful adjunct to serologic testing and is superior for typing transfused patients, for D-zygosity determination, for noninvasive fetal typing, and for antigen-matching in sickle cell patients. SUMMARY: Implementation of molecular testing for transfusion medicine has been a conservative process and limited primarily to reference laboratory environments. With the development of high-throughput platforms, genotyping is poised to move into the mainstream, revolutionizing the provision of antigen-negative donor units. This will enable electronic selection of units antigen matched to recipients at multiple blood group loci, potentially eliminating alloimmunization and significantly improving transfusion outcomes.

Blood Group Incompatibility↗
PubMed2006

The RHCE allele ceCF: the molecular basis of Crawford (RH43).

BACKGROUND: The Crawford antigen (RH43) was described in 1980. It occurred in African American people, as a low-prevalence Rhesus antigen, who were also VS+. STUDY DESIGN AND METHODS: Twelve blood samples were analyzed because of inquiries into discrepant reactions in routine anti-D typing. The RHCE alleles were determined by nucleotide sequencing from genomic DNA. The D epitope profile was determined with 60 monoclonal anti-D. The population frequency was estimated in four major US regional blood centers. RESULTS: The novel RHce(W16C, Q233E, L245V) allele, dubbed ceCF, was found to be occurring in the cde haplotype as cause of the reactivity with the immunoglobulin M anti-D GAMA401. The ceCF phenotype expressed few D epitopes resembling but not matching the reaction patterns observed with other RhCE variants, like R0 (Har), ceRT, and ceSL. The frequency of the ceCF phenotype was 0.056 percent among African American persons and 0.007 percent in the general US population. CONCLUSION: The novel RHce(W16C, Q233E, L245V) allele, which is a variant of the known ce(s) allele, RHce(W16C, L245V), occurs in a haplotype with the RHD deletion and represents the molecular basis of the Crawford antigen.

Black or African American↗
PubMed2005

Characterization of ammonia transport by the kidney Rh glycoproteins RhBG and RhCG.

The erythrocyte Rh-associated glycoprotein (RhAG) was recently found to mediate transport of ammonia/ammonium when expressed in Xenopus laevis oocytes and yeast Saccharomyces cerevisiae. Nonerythroid homologs, RhBG and RhCG, are expressed in the mammalian kidney connecting segment and the collecting duct, major sites of urinary ammonia secretion. This study characterizes the transport properties of murine RhBG and RhCG by ammonium analog [14C]methylamine (MA) uptake and two-electrode voltage clamping of X. laevis oocytes. Both RhBG and RhCG mediated transport of ammonia, but differed in affinity for substrate (K(m) = 2.5 and 10 mM, respectively). The rates of RhBG- and RhCG-mediated transport were sensitive to the concentration of the protonated MA species and were stimulated by extracellular alkalosis and inhibited by acidosis, suggesting a role for H+ in the transport process. Whereas expression of RhBG or RhCG caused a small increase in plasma membrane conductance, [14C]MA uptake was not affected by depolarization of oocytes with 100 mM extracellular K+ or by clamping the membrane potential between 0 and -100 mV, indicating that RhBG- and RhCG-mediated transport was independent of the membrane potential. These results strongly suggest that RhBG and RhCG transport ammonia by an electroneutral process that involves NH4(+)/H+ exchange resulting in net NH3 translocation. The polarized localization of RhBG and RhCG in kidney tubules and the different substrate affinities may enable these proteins to participate in transepithelial ammonia secretion and to therefore play an important role in whole animal acid-base regulation.

Ammonia↗
PubMed2005

Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3).

The collecting duct is the primary site of urinary ammonia secretion; the current study determines whether apical ammonia transport in the mouse inner medullary collecting duct cell (mIMCD-3) occurs via nonionic diffusion or a transporter-mediated process and, if the latter, presents the characteristics of this apical ammonia transport. We used confluent cells on permeable support membranes and examined apical uptake of the ammonia analog [(14)C]methylammonia ([(14)C]MA). mIMCD-3 cells exhibited both diffusive and saturable, transporter-mediated, nondiffusive apical [(14)C]MA transport. Transporter-mediated [(14)C]MA uptake had a K(m) of 7.0 +/- 1.5 mM and was competitively inhibited by ammonia with a K(i) of 4.3 +/- 2.0 mM. Transport activity was stimulated by both intracellular acidification and extracellular alkalinization, and it was unaltered by changes in membrane voltage, thereby functionally identifying an apical, electroneutral NH(4)(+)/H(+) exchange activity. Transport was bidirectional, consistent with a role in ammonia secretion. In addition, transport was not altered by Na(+) or K(+) removal, not inhibited by luminal K(+), and not mediated by apical H(+)-K(+)-ATPase, Na(+)-K(+)-ATPase, or Na(+)/H(+) exchange. Finally, mIMCD-3 cells express the recently identified ammonia transporter family member Rh C glycoprotein (RhCG) at its apical membrane. These studies indicate that the renal collecting duct cell mIMCD-3 has a novel apical, electroneutral Na(+)- and K(+)-independent NH(4)(+)/H(+) exchange activity, possibly mediated by RhCG, that is likely to mediate important components of collecting duct ammonia secretion.

Acids↗
PubMed2004

Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3).

The renal collecting duct is the primary site for the ammonia secretion necessary for acid-base homeostasis. Recent studies have identified the presence of putative ammonia transporters in the collecting duct, but whether the collecting duct has transporter-mediated ammonia transport is unknown. The purpose of this study was to examine basolateral ammonia transport in the mouse collecting duct cell (mIMCD-3). To examine mIMCD-3 basolateral ammonia transport, we used cells grown to confluence on permeable support membranes and quantified basolateral uptake of the radiolabeled ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cell basolateral MA transport exhibited both diffusive and transporter-mediated components. Transporter-mediated uptake exhibited a Km for MA of 4.6 +/- 0.2 mM, exceeded diffusive uptake at MA concentrations below 7.0 +/- 1.8 mM, and was competitively inhibited by ammonia with a Ki of 2.1 +/- 0.6 mM. Transporter-mediated uptake was not altered by inhibitors of Na+-K+-ATPase, Na+-K+-2Cl(-) cotransporter, K+ channels or KCC proteins, by excess potassium, by extracellular sodium or potassium removal or by varying membrane potential, suggesting the presence of a novel, electroneutral ammonia-MA transport mechanism. Increasing the outwardly directed transmembrane H+ gradient increased transport activity by increasing Vmax. Finally, mIMCD-3 cells express mRNA and protein for the putative ammonia transporter Rh B-glycoprotein (RhBG), and they exhibit basolateral RhBG immunoreactivity. We conclude that mIMCD-3 cells express a basolateral electroneutral NH4+/H+ exchange activity that may be mediated by RhBG.

Acid-Base Equilibrium↗
PubMed2004

Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein.

The Rh blood group proteins are erythrocyte proteins important in neonatal and transfusion medicine. Recent studies have shed new light on the possible biological function of Rh proteins as members of a conserved family of proteins involved in ammonium transport. The erythrocyte Rh-associated glycoprotein (RhAG) mediates uptake of ammonium when expressed in Xenopus laevis oocytes, and functional studies indicate that RhAG might function as an NH(4)(+)-H(+)-exchanger. To further delineate the functional properties of RhAG, in this study we have expressed RhAG in both a Saccharomyces cerevisiae ammonium-transport mutant (mep1Delta mep2Delta mep3Delta) and a wild-type strain. RhAG was able to complement the transport mutant, with complementation strictly pH-dependent, requiring pH 6.2-6.5. RhAG also conferred resistance to methylamine (MA), a toxic analog of ammonium, and expression in wild-type cells revealed that resistance was correlated with efflux of MA. RhAG-mediated resistance was pH-dependent, being optimal at acid pH. The opposite pH dependence of ammonium complementation (uptake) and MA resistance (efflux) is consistent with bidirectional movement of substrate counter to the direction of the proton gradient. This report clarifies and expands previous observations of RhAG-mediated transport in yeast and supports the hypothesis that ammonium transport is coupled to the H(+) gradient and that RhAG functions as a NH(4)(+)/H(+) exchanger.

Animals↗
PubMed2004

A new hybrid RHCE gene (CeNR) is responsible for expression of a novel antigen.

BACKGROUND: The red blood cells (RBCs) of a patient, known to have the probable DC(W)(e)/D-- phenotype, typed as D(W)- and Rh32- but were unexpectedly agglutinated by an anti-D(W)/Rh32 serum. The reactivity suggested that the RBCs carried a novel antigen and that the molecular background of this DC(W)(e)/D-- phenotype might be different from those reported. The purpose of this study was to determine the molecular basis of the Rh phenotype. STUDY DESIGN AND METHODS: Samples were obtained for family studies. Standard hemagglutination methods were used. RH mRNA transcripts were isolated by reverse transcription-polymerase chain reaction and sequenced. RESULTS: Sequence analysis revealed that the probond had three different RH transcripts: a normal RHD and two different hybrid transcripts from the RHCE locus, a RHCE-D hybrid with exon 1 from RHCE associated with the D-- haplotype, and a new RHCE-D hybrid. In this new hybrid, exons 1 to 5 are RHCe-specific and exons 6 to 10 correspond to RHD. The C(W) antigen is also encoded by this hybrid gene. Family studies confirmed that the new RHCE-D hybrid is linked in cis to conventional RHD. CONCLUSION: A new RHCE-D structure is associated with altered expression of C and e antigens in this family and the generation of a novel low-prevalence antigen (CENR).

Amino Acid Sequence↗
PubMed2003

Protein 4.2 is critical to CD47-membrane skeleton attachment in human red cells.

The reduction in expression of the integral membrane protein CD47 in human red blood cells (RBCs) deficient in protein 4.2 suggests that protein 4.2 may mediate a linkage of CD47 to the membrane skeleton. We compared the fractions of membrane skeleton-attached CD47, Rh-associated glycoprotein (RhAG), Rh, and band 3 in normal and protein 4.2-deficient cells using fluorescence-imaged microdeformation. We found that CD47 attachment decreases from 55% in normal cells to 25% to 35% in 4.2-deficient cells. RhAG, which has been shown to have no significant variation in expression among the cells studied, shows a significant decrease in membrane skeleton attachment in 4.2-deficient cells from 60% to 40%. Both Rh and band 3, which have also been shown to have no change in expression, show a smaller decrease from 75% attached in normal RBCs to 55% attached in 4.2-deficient cells. In normal cells, Rh phenotype influences CD47 expression but not the level of membrane skeleton attachment of CD47. In contrast, the results indicate that protein 4.2 strongly influences CD47 levels as well as the extent of membrane skeleton attachment in the RBC, whereas protein 4.2 affects membrane skeletal attachment of RhAG, Rh, and band 3 to a lesser extent.

Actins↗
PubMed2002

Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes.

Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)- tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rh(null) cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2002

Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter.

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [(14)C]methylamine (MA), had an apparent EC(50) of 1.6 mm and a maximum uptake rate (V(max)) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na(+) gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H(+) gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC(50) = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H(+)-coupled counter-transport mechanism.

Animals↗