Absence of fetal cell microchimerism in cutaneous lesions of lupus erythematosus.
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Adult↗
Biomedical subjects
L Mery
Publications and source records attributed to L Mery.
Detecting and eliminating erroneous gestational ages: a normal mixture model.
In perinatal research and clinical practice, gestational age is a crucial variable for measuring foetal 'growth' (birth weight for gestational age) and for estimating the risk of mortality and morbidity, yet reported gestational age values are affected by random and systematic errors due to the absence of a gold standard measure. Previous investigators have used birth weight (which is measured with greater validity and precision than is gestational age) to correct such errors, but existing methods are inadequate due to unreasonable assumptions about the distributions of birth weight and gestational age. We propose a new method for identifying and correcting implausible observations using the expectation-maximization (EM) algorithm. Using population-based data from U.S. birth certificates, we compare the resulting gestational ages, birth weight distributions at each gestational age, and gestational age-specific infant mortality based on the new method with those on the same population produced by previous published correction methods. The new method gives the best birth weight distributions for gestational age and the most realistic gestational-age-specific mortality rates, while each of the other methods has at least one significant flaw.
Algorithms↗
Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors.
The molecular basis of capacitative (or store-operated) Ca2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP3R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as alpha-hTrp4 and beta-hTrp4. The shorter variant beta-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G785-S868. Using a yeast two-hybrid assay and glutathione-S-transferase-pulldown experiments, we found that the C-terminus of alpha-hTrp4, but not of beta-hTrp4, associates in vitro with the C-terminal domain of the InsP(3) receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4-InsP3R complexes may be regulated by alternative splicing.
Alternative Splicing↗
Rat inositol 1,4,5-trisphosphate receptor isoform 2 interacts with itself in its C-terminal portion and upstream of the first transmembrane domain.
In response to stimulation at the plasma membrane, hepatocellular Ca(2+) signals are fast and precise and lead to rapid local changes in cytoplasmic free Ca(2+) concentration. These changes result from the opening of the inositol 1,4,5-trisphosphate receptor (InsP(3)R), which is a four-subunit intracellular InsP(3)-gated channel that releases Ca(2+) from the stores. To investigate the molecular mechanism underlying interactions between the InsP(3)R subunits, we cloned the predominant hepatocellular isoform, InsP(3)R isoform 2 (InsP(3)R2), and screened for interactions using the yeast two-hybrid assay. We found that the C-terminal domain of rat InsP(3)R2 interacts with itself, and that the cytoplasmic part preceding the first transmembrane domain, a region near a Ca(2+)-binding site, also interacts with itself. These interactions were confirmed by pull-down experiments. The C-terminal domain of InsP(3)R2 is also able to interact with the C-termini of rat InsP(3)R1 and InsP(3)R3. These results advance our understanding of the molecular mechanisms that underlie the oligomerization and interactions of the InsP(3)R subunits during the opening/closing of the Ca(2+) channel.
Amino Acid Sequence↗
Ion channels in health and disease. 83rd Boehringer Ingelheim Fonds International Titisee Conference.
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Animals↗
Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx.
The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.
Adenosine Triphosphate↗
Site-directed mutagenesis of conserved charged residues in the helical region of the human C5a receptor. Arg2O6 determines high-affinity binding sites of C5a receptor.
The human C5a receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg2O6, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282-->Ala) or partial loss of ligand-binding capacity (Arg175-->Ala, Arg2O6-->Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln2O6]C5aR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (<10%) of high-affinity sites (Kd approximately 2 nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of G alpha-16. The number of high-affinity sites of wild-type and [Gln2O6]C5aR in 293 cells could be up-regulated by coexpression of Gi alpha-2 and down-regulated by GTP[gamma S]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg2O6 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human C5aR by affecting the intracellular G-protein coupling.
Animals↗
The NH2-terminal region of C5aR but not that of FPR is critical for both protein transport and ligand binding.
The N-formylated tripeptide, formylmethionylleucyl-phenylalanine (fMLP), and the 74-amino-acid long human C5a anaphylatoxin activate phagocytic cells via two structurally related G protein-coupled receptors (FPR and C5aR), which are 34% identical in amino acid sequence. C5aR chimeras were constructed in which the entire NH2-terminal extracellular sequence or part of it was replaced by the counterparts from FPR or FPRH. Although the NH2-terminal region of C5aR presents an extremely high interspecies variability, substitution of the entire NH2-terminal sequence of C5aR by that of FPR or FPRH surprisingly resulted in chimeras that were apparently retained in the endoplasmic reticulum. In contrast, when the NH2-terminal domain of FPR was replaced by the corresponding region from C5aR or FPRH normal expression to the plasma membrane and high affinity binding of N-formylated peptides were observed. Thus, the NH2-terminal region of C5aR, in contradistinction to that of FPR, seems to be required either for the translocation of C5aR through the ER membrane or for correct folding and sorting of C5aR to the plasma membrane. Replacement of the first 8 residues of C5aR by the corresponding region of FPR did not alter the cellular transport and the C5a binding capacity, whereas the exchange of the first 13 residues resulted in a chimera that was readily transported to the plasma membrane but showed no capability to bind C5a. Mutations of Asp into Asn in the NH2-terminal segment of C5aR further indicated that negative charges are required to endow the receptor with a C5a binding capacity. The residues critical for binding are either involved directly by interacting with cationic residues of C5a, or indirectly by influencing the overall structure of the ligand-binding site.
Amino Acid Sequence↗
Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells.
Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.
Amino Acid Sequence↗
Evidence that the extracellular N-terminal domain of C5aR contains amino-acid residues crucial for C5a binding.
The human C5a anaphylatoxin is a cationic 74 amino-acid long glycopeptide which derives from proteolysis of the fifth component of complement. It interacts with high affinity with a receptor that belongs to the G protein-coupled receptor superfamily. Several studies have previously suggested that multiple contact points between C5a and the receptor are required to achieve high-affinity interaction. However, at the receptor level little is known about the sites of interaction with C5a. We have investigated by in vitro mutagenesis whether the N-terminal extracellular sequence of the C5a receptor, which is rich in aspartic acid residues, could play some role in C5a binding. Conversion of Asp10 into asparagine did not impair the level of expression at the plasma membrane, nor did it alter the affinity for C5a. However, we consistently observed a discrepancy between an apparent high level of surface expression and a weak capacity to bind C5a with high affinity, suggesting that many receptor molecules, although present on the cell surface, might be misfolded and unable to bind C5a. Replacement of Pro9 by an isoleucine had little effect, if any, on either the affinity or the C5a-binding capacity, whereas the conversion of Pro36 into leucine dramatically reduced the expression of high-affinity receptor at the cell surface. N-glycosylation of human C5a receptor was found to be dispensable for the function of the receptor.
Amino Acid Sequence↗
Expression cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells.
A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.
Amino Acid Sequence↗
Birth cohort effects underlying the increasing testicular cancer incidence in Canada.
PURPOSE: To examine the pattern of testicular cancer incidence by age, time period and birth cohort since 1969 in Canada. METHODS: In addition to analyses of the secular trends by age group and birth cohort separately, an age-period-cohort model and the submodels with standard Poisson assumptions were fitted to the data. RESULTS: The overall age-adjusted incidence of testicular cancer increased in Canada, from 2.8 per 100,000 males in 1969-71 to 4.2 in 1991-93. The younger age groups showed much higher absolute incidence rates in the recent period compared with those in the early period. Age-period-cohort modelling of data restricted to males aged 20-84 years suggested that the observed increase in testicular cancer could be largely attributed to a birth cohort effect. A steady increase in risk was observed among men born since 1945; those born between 1959 and 1968 were 2.0 (95% CI, 1.5-2.6) times as likely to develop testicular cancer as those born between 1904 and 1913. CONCLUSION: The risk of testicular cancer has increased over time and changing exposure to environmental factors early in life may be responsible for this.
Adolescent↗