Partial expression of RHc on the RHD polypeptide.

BACKGROUND: In the human Rh blood group system, c is, after D, the most immunogenic antigen. STUDY DESIGN AND METHODS: The background of a new partial c phenotype (D(c)), identified on the RBCs of two unrelated white persons, was studied. This was done by analyzing the reactivity of the RBCs from the donors with anti-c reagents, by performing sequence analysis, and by carrying out transduction studies. RESULTS: Serologic results suggested the existence of a new partial c phenotype. Genomic DNA and cDNA analysis revealed a normal RHCe allele, a normal RHD allele, and an RHD allele that carried two point mutations: 307T>C and 329T>C (the latter known to be associated with the DVII, Tar-positive phenotype). No normal RHc allele was found. Thus, it was most likely that c is encoded by the mutated RHD allele (phenotype DD(c)CCee). Indeed, subsequent transduction of K562 erythroleukemic cells with an RHD cDNA carrying the 307T>C point mutation (leading to S103P) resulted in the expression of c. CONCLUSION: In the human Rh system, P103 is involved in the expression of c. Moreover, c can be expressed in vivo on the D polypeptide.

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer.

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans.

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.

[Prenatal typing of Rh- and Kell- blood group system antigens]. / Prenatale typering van de bloedgroepantigenen van het Rh- en het Kell-systeem.

Rhesus (Rh) and Kell blood group immunisations are the most frequent causes of haemolytic disease of the newborn. Recently, the molecular bases of the Rh and Kell antigens have been elucidated. Subsequently, specific polymerase chain reactions (PCRs) could be developed to determine the RhD, RhC/Rhc and RhE/Rhe genotypes as well as the KI genotype (from the Kell blood group) with genomic DNA. The tests were applied to genomically determine the foetal Rh and Kell blood groups with DNA obtained from amniotic fluid cells. The genotypes obtained were compared with the Rh phenotypes established by cord blood red cell serology. The PCRs to determine the RhD, Rhc, RhE and Rhe and KI genotypes were found to be reliable. The test for RhC however, resulted in false-positive C genotypes. Indeed, more than half of the subsequently tested C-negative Negroid donors were false-positive with the DNA test. Thus, except for RhC, it is possible to reliably determine the Rh and KI genotypes of a foetus with DNA isolated from amniotic fluid cells. Amniocentesis, however, carries a risk for the pregnancy and therefore the tests will only be justified in pregnant women in whom an antibody has been detected and the father of the foetus is heterozygous for the specific antigen. Recently foetal RhD genotypes were determined in foetal DNA circulating in the plasma of RhD-negative pregnant women. This could eventually lead to the introduction of assays with which the foetal blood group can be determined without any risk to the foetus.

Determinants in the cytoplasmic domain of P-selectin required for sorting to secretory granules.

P-selectin is a granule membrane protein of platelets and endothelial cells that is expressed at the plasma membrane after cell activation. To determine which residues in its cytoplasmic tail are important for sorting to storage granules during biosynthesis, we expressed P-selectin mutants in AtT-20, a murine cell line with secretory granules that contain the hormone corticotropin ('ACTH'). Immunofluorescence microscopy of permeabilized cells revealed that wild-type P-selectin and mutants with alanine substitutions at 14 different positions in the cytoplasmic tail were concentrated in the tips of the cellular processes, which contain the majority of corticotropin granules. However, targeting to the cell tips was greatly decreased for Tyr777-->Ala, Tyr777-->Phe, Gly778-->Ala, Phe780-->Ala and Leu768/Asn769-->Ala/Ala mutants. The reduced presence of these mutants in corticotropin granules was confirmed by immunoelectron microscopy. Stimulation of AtT-20 transfectants with 8-Br-cAMP resulted in a significant increase in membrane expression of wild-type P-selectin, but in only a marginal increase in the surface expression of the five mutants. Antibody binding studies with intact and permeabilized cells demonstrated that the percentage of P-selectin that is expressed on the surface of the cells was considerably higher for these mutants than for wild-type P-selectin (6%), ranging from approximately 20% for the Gly778 and Phe780 mutants to 63% for the Leu768/Asn769 mutant. Taken together, these results indicate that Tyr777, Gly778 and Phe780 form part of an atypical tyrosine-based motif, which also requires the presence Leu768 and/or Asn769 to mediate sorting of P-selectin to secretory granules.