Biomedical subjects

P Gane

Publications and source records attributed to P Gane.

At least 19 recordsLinked to original sources

PubMed2006

Generation of mice with inactivated Rh or Rhag genes.

Mice carrying inactivated Rh and Rhag genes were generated by insertional targeting. KO animals exhibited normal growth, development and fertility and both types were indistinguishable at a gross phenotypic level from their wild type littermates. Preliminary analysis revealed that red cells from Rh-/- mice lack Rh protein and have a moderate decrease of Rhag protein, whereas those from Rhag-/- mice have a total absence of Rhag and Rh proteins. Studies are in progress to delineate the antigenic, biochemical and functional abnormalities of red cells from these animals as well as the impact on hematological parameters and erythropoiesis.

Animals↗

PubMed2006

Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.

Animals↗

PubMed2006

Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis.

We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.

Amino Acid Substitution↗

PubMed2006

Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.

To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and Rhag-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag-/- and AQP1-/-. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions.

Ammonia↗

PubMed2004

Ensembl 2004.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organize biology around the sequences of large genomes. It is a comprehensive and integrated source of annotation of large genome sequences, available via interactive website, web services or flat files. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. The facilities of the system range from sequence analysis to data storage and visualization and installations exist around the world both in companies and at academic sites. With a total of nine genome sequences available from Ensembl and more genomes to follow, recent developments have focused mainly on closer integration between genomes and external data.

Animals↗

PubMed2003

A new murine monoclonal antibody against Kx protein.

Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross-reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross-reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell-Kx complex from RBC membrane extract by immunochromatography.

Amino Acid Transport Systems, Neutral↗

PubMed2002

[Flow cytometry in immunohematology].

Flow cytometry is an objective, sensitive and quantitative technique which allows rapid and simultaneous analysis of several parameters on a great number of cells. Hence, flow cytometry is particularly suitable for the analysis of complex cell populations, rare events and quantitative studies. In immunohematology, flow cytometry is a very powerful approach to the study of mixed red cell populations (hematopoietic chimerism, transfusion or bone marrow transplantation), the detection of low frequency cell populations (reticulocytes, fetomaternal hemorrhage) and the quantitative analysis of red blood cell antigens.

Blood Transfusion↗

PubMed2002

UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney.

UT-B1 is the facilitated urea transporter of red blood cells (RBCs) and endothelial cells of descending vasa recta in the kidney. Immunoblotting with a polyclonal antibody against the C-ter sequence of rat UT-B1 revealed UT-B1 as both nonglycosylated (29 kDa) and N-glycosylated (47.5 and 33 kDa) proteins in RBC membranes, kidney medulla, brain, and bladder in rat. In testis, UT-B1 was expressed only as a nonglycosylated protein of 47.5 kDa. Immunocytochemistry confirmed that the location of UT-B1 is restricted to descending vasa recta. In brain, UT-B1 protein was found in astrocytes and ependymal cells. Cell bodies and perivascular end feet of astrocytes were labeled in brain cortex, whereas astrocyte cell processes were labeled in corpus callosum. Flow cytometry analysis of RBCs revealed a good cross-reactivity of the antibody with mouse and human UT-B1. UT-B1 protein expression in rat kidney medulla was downregulated greatly by long-term [deamino-Cys(1),D-Arg(8)]vasopressin infusion and moderately by furosemide treatment. This study discloses an uneven distribution of UT-B1 protein within astrocytes and the regulation of renal UT-B1 protein by antidiuretic hormone.

Amino Acid Sequence↗

PubMed2001

Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29.

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.

Adenine↗

PubMed2001

The nonexpression of CD36 on reticulocytes and mature red blood cells does not modify the clinical course of patients with sickle cell anemia.

It is thought that an increase in the adhesion of circulating reticulocytes to the vascular endothelium may initiate the vascular occlusion underlying the painful crises and organ failures typical of sickle cell disease (SCD). At least 2 receptors, usually present on reticulocytes, seem to be involved in this adhesion process: glycoprotein CD36 (glycoprotein IV) and integrin alpha(4)beta(1) (very late activation antigen--4). Recently, a high frequency of the platelet CD36--deficient phenotype was reported in black Africans. The frequency of this deficiency was similar in subjects with and without SCD. The role of CD36 in vaso-occlusion was then investigated by comparing the clinical course in 2 groups of black Africans homozygous for hemoglobin S, with and without CD36 deficiency, but similar in age, sex, geographical origin, number of alpha-globin genes, and beta-globin gene haplotype. Flow cytometry showed that CD36 was absent from the circulating red blood cells and reticulocytes of platelet CD36--deficient individuals but present on those from patients with normal platelet CD36 expression, and that alpha(4)beta(1) integrin levels were similar on the reticulocytes of the 2 groups. Neither clinical severity, as evaluated by the frequency and characteristics of vaso-occlusive events, nor biological data differed significantly in the 2 groups of patients. Finally, although CD36 has been suggested to play a critical role in the pathogenesis of vaso-occlusion, this study, despite including only a small number of patients, supports the idea that the modulation of expression of a single type of adhesion molecule is insufficient to counteract the pathological process leading to vaso-occlusion in SCD patients. (Blood. 2001;98:966-971)

Adolescent↗

PubMed2001

Characterization of the laminin binding domains of the Lutheran blood group glycoprotein.

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding.

Amino Acid Motifs↗

PubMed2001

Flow cytometric analysis of the association between blood group-related proteins and the detergent-insoluble material of K562 cells and erythroid precursors.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2000

Binding sites of leukocyte beta 2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein.

The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LW(ab) block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca(2+) and Mg(2+). Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-4 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-2, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) on the CFG face and Trp(77) on the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.

Amino Acid Sequence↗

PubMed2000

A study of the coregulation and tissue specificity of XG and MIC2 gene expression in eukaryotic cells.

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism coregulated with the polymorphism of the XG blood group gene. As a preliminary study of this phenomenon, human XG and CD99 recombinant proteins were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins were expressed independently and at a similar level in single and double transfectants. Immunoprecipitation and Western blot analysis, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of association or complex formation between XG and CD99 proteins could be proven, either on transfected RAG cells or on human erythrocytes. These results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttranscriptional effect. Studies on the tissue specificity of XG expression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts revealed the existence of an XG mRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By Northern blot analysis, XG transcripts were detected in erythroid tissues and several nonerythroid tissues. (Blood. 2000;95:1819-1826)

12E7 Antigen↗

PubMed2000

Quantitative analysis of XG blood group and CD99 antigens on human red cells.

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism co-regulated with the Xga blood group polymorphism. The co-expression of X-linked MIC2 and XG genes is presumably controlled at the transcriptional level by a single XGR locus in the pseudoautosomal region of sexual chromosomes. This locus is composed of two alleles, XGR(low) and XGR(high), which determine low or high CD99 levels (CD99-L, CD99-H) and the Xg(a-)/ Xg(a+) status. To test this hypothesis, the phenotypic relationship between Xga and CD99 antigens on human RBCs was investigated by quantitative flow cytometry using NBL-1 (anti-Xga) and 12E7 (anti-CD99) monoclonal antibodies and semi-quantitative estimate of membrane proteins and RNA by Western blot and Northern blot, respectively. The antibody binding capacity of RBCs, which is an estimation of the antigen density, was determined for 118 blood donors including 60 males and 58 females. Xg(a+) RBCs, which all belong to the group of CD99-H expressors, carry 159+/-13 and 960+/-50 copies of Xga and CD99 molecules/cell, respectively. Xg(a-) RBCs have no Xga antigen, but are subdivided into CD99-H (all male) and CD99-L expressors carrying 747+/-28 and 200+/-22 CD99 copies/cell, respectively, with identical CD99 levels between CD99-L males and females. However, among males, the CD99 expression was higher in Xg(a+) than in Xg(a-)/CD99-H individuals (P<0.01). In addition, CD99-H expressors in Xg(a+) males could be clearly subdivided into two categories, high and super high expressors, which are presumably heterozygous and homozygous for the XGR(high) allele, which fits the above hypothesis. This was not the case for Xg(a+) females where CD99-H subcategories were not found. Quantitative differences were confirmed by Western blot analysis of red cell membrane preparations from individuals of different Xga and CD99 phenotypes and by Northern blot analysis showing that the reticulocytes from CD99-L individuals expressed a reduced level of MIC2 transcripts compared to CD99-H donors. These findings further support the hypothesis of a single genetic control of CD99 and Xga expression by the XGR locus.

12E7 Antigen↗

PubMed2000

Analysis of deletions in three McLeod patients: exclusion of the XS locus from the Xp21.1-Xp21.2 region.

The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene defects and may include large deletions in the Xp21 region. Analysis of three unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient Bi) and from the XK to CYBB loci (patient Lh). All three patients normally expressed the Lutheran (Lu) red cell antigens, thus excluding the interval between the RPGR and DMD genes as site of the XS locus, previously mapped to the Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood group gene on chromosome 19.

Alleles↗

PubMed1999

Characterization of a mouse liminin receptor gene homologous to the human blood group Lutheran gene.

The human Lutheran (Lu) blood group antigens are carried by two glycoproteins (gps) that belong to the immunoglobulin (Ig) superfamily. These gps represent adhesion molecules that function as the unique erythroid receptors for laminin. We report here the cloning and functional expression of the orthologous mouse Lu mRNA as well as the genomic organization of the mouse Lu gene. The deduced human and mouse Lu gps share 72.5% identity and similar organization of the Ig-like domains. As in the human, the mouse Lu gene is organized in 15 exons. The proximal promoter showed consensus CACC-binding sites whereas the distal promoter exhibits a GATA-1-binding site and multiple E boxes. Like the human gene, the mouse Lu gene is also widely expressed among tissues but is transcribed as a unique 2.4-kb mRNA species. Expression of the mouse Lu mRNA is upregulated upon dimethyl sulfoxide-induced erythroid differentiation of murine erythroleukemia cells (MEL). During mouse embryonic development, the Lu transcript is detected as early as day 7 of gestation. Analysis of transfected human erythroleukemia K562 cells indicated that the adhesive properties of the Lu gps to laminin are conserved between human and mouse.

Amino Acid Sequence↗

PubMed1999

Time-course expression of polypeptides carrying blood group antigens during human erythroid differentiation.

The time course expression of blood group antigens was examined by flow cytometry using a two-phase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71++) and glycophorin A (GPA+). Four main categories of blood group markers were identified: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epo-independent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wrb (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epo-dependent phase 2 and (iv) those like Jk3 and Lub which were expressed in late phase 2. Regarding blood group molecules exhibiting adhesive properties (LW/ICAM-4, Oka and Lu) the most significant event was a sharp decrease of Oka (neurothelin) expression with the concomitant loss of ICAMs expression during the later stage of differentiation. These studies suggest that Oka, ICAMs and LW might contribute to the adhesive interactions involved in the formation of erythroblastic islands and attachment to stroma cells and the extracellular matrix. We also noted an asynchronous expression of the proteins that compose the core of the Rh complex, since Rh50 glycoprotein was expressed earlier than Rh(D, CE) proteins.

Antigens, CD↗