Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Covalent cross-linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29)

[125I]Monoiodinated vasoactive intestinal peptide (125I-VIP) was cross-linked with human colonic adenocarcinoma cells (HT29 cells) grown as a monolayer using dithiobis(succinimidylpropionate) as cross-linking reagent. The cross-linked polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A major polypeptide of Mr = 67 000 was characterized and it behaved like a high-affinity binding site for VIP according to the following data. The concentration of native VIP (0.5 nM) giving half-maximum inhibition of 125I-VIP covalent cross-linking with this polypeptide was very similar to that giving half-maximum displacement of 125I-VIP on HT 29 cells (0.6 nM). Glucagon or insulin was unable to inhibit the labelling of the Mr-67 000 component. In our experimental conditions neither specific 125I-VIP binding nor covalent labelling was observed with monolayers of Madin Darby canine kidney epithelial cells (MDCK cells) or African green monkey kidney fibroblasts (Vero cells) while the Mr-67 000 polypeptide was also characterized with human rectal adenocarcinoma cells (HRT 18 cells), known to possess the VIP receptor. Preincubation of HT 29 cells with native VIP at 37 degrees C, before 125I-VIP binding and subsequent cross-linking reaction, decreased the labelling of the Mr-67 000 polypeptide up to 80%. Assuming one molecule of 125I-VIP cross-linked per polypeptide, we have characterized, for the first time, a major polypeptide of Mr = 64 000, which belongs to the high-affinity VIP binding site of an intestinal human cell line.     

Differential regulation of HLA-DR mRNAs and cell surface antigens by interferon

Human interferons-alpha, -beta and -gamma enhance HLA-DR mRNAs in all the human lymphoblastoid and melanoma cell lines studied. The increase concerns both alpha and beta chain mRNAs. Moreover, we show that immune interferon-gamma preferentially enhances class II MHC mRNA. This effect of IFN-gamma on the synthesis of alpha and beta HLA-DR chains has been also analysed by immunoprecipitation. It is abolished by a monoclonal antibody directed against human IFN-gamma. The effect of interferon on the cell surface level of HLA-DR molecules does not always correspond to the enhancement of HLA-DR mRNA. Our experiments suggest that this discrepancy between the enhancement of HLA-DR mRNA and cell surface antigen might be due to a constitutively high level of the corresponding antigens on several of the human cells studied.     

Evidence for an ectophosvitin kinase activity that phosphorylates a 123 kDa endogenous substrate on a human colonic adenocarcinoma cell (HT 29)

When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kinase activities. We have characterized one of them as an ectophosvitin kinase which phosphorylated specifically a 123 kDa polypeptide and whose expression or accessibility varied according to cell density.     

Scanning and transmission electron microscopy related to immunochemical analysis of grass pollen

The exine stereostructure (scanning electron microscopy) and ultrastructure (transmission electron microscopy) of pollen of seven grass species, is related to the allergens extracted from these pollen grains. The heterogeneity of the allergens was studied by the immunoprint technique and revealed by labelling the binding of grass pollen sensitive patients IgE antibodies. Using patient sera recognizing a very restricted number of allergens, we showed that a group of pollen had a great number of allergens in common (Dactylis, Agrostis, Festuca, Lolium, Holcus) and, in decreasing cross reactivities order, we found Avena and, finally, Zea mays. The tectum stereostructure shows presence of insulae in all pollen grains except in Zea mays which has small isolated spinules. These insulae are separated by very wide and deep interinsular spaces in Avena sativa with connections between insulae. In the remaining species, no connections were seen between the insulae. These observations were in good correlation with the immunological cross-reactivity of the allergens present in the pollen. In all species, there are microperforations in the bottom of the interinsular spaces, which are the opening of the tectal microchannels.     

Dehydroepiandrosterone regulation of the hepatic glucocorticoid receptor in the zucker rat. The obesity research program

Dehydroepiandrosterone (DHEA) decreases the activity of hepatic tyrosine aminotransferase (TAT), a glucocorticoid-inducible enzyme, in the obese, hypercorticosteronemic Zucker rat. To investigate the mechanism of this antiglucocorticoid action, the effect of exogenous DHEA on hepatic glucocorticoid receptor (GC) number and affinity was quantitated. Food supplementation with DHEA (0.6% w/w) for 1 or 7 days had no effect on either receptor number or affinity in obese Zucker rats. After 28 days, however, DHEA treatment resulted in a nearly 40% decrease in cytosolic hepatic receptor content (B_max; fmol/mg cytosolic protein) without any change in affinity (K_d) in both lean and obese rats. DHEA treatment for 28 days also resulted in an increased liver size and cytosolic protein content. When the hepatic GC receptor content was normalized based on the change in liver size and protein content, the apparent number of GC binding sites per liver was not affected by DHEA treatment. This observation suggests that DHEA's effect on GC receptor content may not be a specific action and that downregulation of the GC receptor is not the mechanism of DHEA action on GC induced TAT activity. This is supported by the effect of DHEA on obese rat TAT activity in the same experiment where the greatest inhibition occurred after only 1 day of treatment. From these experiments it is concluded that although long-term DHEA treatment may decrease the relative concentration of GC receptors in rat liver, this change is not the mechanism through which DHEA mediates its acute antiglucocorticoid action.