Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3

Summary The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-pb GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Evaluation of the risk in autosomal dominant diseases

A lot of traps and difficulties complicate the estimation of a genetic risk in the autosomal dominant diseases. The authors recapitulate the notions of mutation, penetrance and variability and illustrate by some examples the part of each of them, isolated or associated together. The increasing of molecular biology allows to resolve some of these problems, but generate new dangers which are analysed and illustrated.     

Transforming growth factor-beta inhibits endothelial cell proliferation

Transforming growth factor-beta (TGF-beta) is an inhibitor of the proliferation of bovine aortic endothelial cells in culture. Basal cell growth in serum-containing medium and cell proliferation stimulated by fibroblast growth factor (FGF) are inhibited by TGF-beta in a dose-dependent manner. Half-maximal inhibition occurs at an inhibitor concentration of 0.5-1.0 ng/ml. TGF-beta does not appear to be cytotoxic and cells treated with the inhibitor grow normally after removal of TGF-beta. High concentrations of FGF are ineffective in overcoming TGF-beta-induced inhibition of cell proliferation, suggesting that antagonism of growth factor-induced cell proliferation by TGF-beta is of a noncompetitive nature.     

Synthesis and secretion of the human vitamin B12-binding protein, transcobalamin II, by cultured skin fibroblasts and by bone marrow cells

Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.     

Haptoglobin groups in dementia of Alzheimer type and multi-infarct dementia

Haptoglobin (Hp) groups were determined in 65 patients with dementia of Alzheimer type (DAT) and 74 with multi-infarct dementia (MID). The increased Hp1 gene frequency among patients with DAT described by other investigators could not be confirmed in this study. The patients showed no significant difference from the controls with respect to phenotype and gene frequencies, and there were no significant differences between early and late onset cases of DAT.     

Haptoglobin groups and rheumatoid arthritis

Haptoglobin types were determined in 200 patients with rheumatoid arthritis (RA) subdivided according to sex and familial occurrence of polyarthritis. A highly significant excess of the Hp2 gene was found among patients with a family history of polyarthritis, more pronounced among males. The possible association between Hp2 and predisposition for increased immune reactivity is discussed.