Heterozygous mutation in the WSXWS equivalaent motif of the growth hormone receptor in a child with poor response to growth hormone therapy.

Besides complete GH insensitivity syndrome (GHIS) described by Laron, clinical and molecular evidences have accumulated concerning partial GHIS. We studied GH receptor (GHR) gene in children who show poor response to GH treatment and detected a patient with a heterozygous mutation in exon 7 leading to the Y222H substitution. This missense mutation, located in the YGEFS motif of the GHR equivalent to the WSXWS motif highly conserved throughout all members of the cytokine receptor family, has not been described so far. Although we cannot conclude on the deleterious effect of this mutation, there are several lines of evidence suggesting that it could account for the partial GH insensitivity: (i) hormonal data including IGF-I generation test; (ii) molecular data - no other mutation was identified in the coding sequence, the father who has the same mutation is short, the brother did not inherit the mutated allele and was of normal height.

Human growth hormone receptor: cloning and expression of the full-length complementary DNA after site-directed inactivation of a cryptic bacterial promoter.

Growth hormone receptor is a cytokine-type receptor which is required for normal somatic growth and for numerous metabolic processes. Its complementary DNA (cDNA) has been isolated in various species leading to intensive studies to elucidate the mechanism of action of the growth hormone. However, serious difficulties have been reported in cloning in Escherichia coli, an intact full-length human cDNA. In this study, the cDNA is shown to contain a cryptic bacterial promoter driving inappropriate expression of a part of human growth hormone (hGH) receptor which is toxic for E. coli growth. Identification of this promoter and its inactivation by changing only one nucleotide led us to obtain stable bacterial clones containing a high copy number of full-length coding sequences. This molecular clone was used in a baculovirus/insect cell system to produce large amounts of glycosylated recombinant receptor. Binding studies with 125I-labelled hGH revealed an affinity constant of 2.8 x 10(9) M(-1), similar to that reported for the native liver receptor. This report described a general method of cloning which could be applied to similar unclonable cDNA fragments.

Expression of a recombinant human growth hormone binding protein in baculovirus/insect cell system.

An eucaryotic recombinant human growth hormone binding protein (rGHBP) was expressed in baculovirus-infected insect cells and purified by affinity chromatography from culture supernatant. This mannose-rich 34-kDa protein specifically bound human growth hormone (hGH) with the same affinity (kDa = 0.42 x 10(-9) M) than the 51.5 kDa GHBP we purified and characterised from human plasma (kDa = 1.1 x 10(-9) M). A high molecular form of the rGHBP was detected by silver-stained SDS-PAGE, Western blot (mAb 263), affinity cross-linking and Western ligand blot with 125I-hGH. Reduction experiments with beta-mercaptoethanol suggested that this form involved a disulfide bound between two rGHBPs.

Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae.

Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA. Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase. This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones. We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae. This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues.

Expression of recombinant human glutathione reductase in eukaryotic cells after DNA-mediated gene transfer.

Glutathione reductase (EC 1.6.4.2) protects tissues from oxidant stress by catalyzing the NADPH-mediated reduction of glutathione disulfide to glutathione. We describe construction of a vector for DNA-mediated gene transfer and successful transient overexpression of human glutathione reductase cDNA in CHO cells. No expression was observed when the same vector was transiently transfected into NIH3T3 or LA4 cells or stably integrated in CHO cells. These results demonstrate the ability to constitute recombinant glutathione reductase expression in eukaryotic cells but suggest that this expression may be toxic.

Absence of correlation between graft-versus-host associated immunosuppression and cytotoxic T cell activity in response to major histocompatibility antigens.

Studies in mice suggest that the T cell subset involved in graft-versus-host-reaction (GvHR) across the major histocompatibility complex (MHC) depends on the class of MHC antigens recognized by the donor cells. However, the correlation between phenotype and function is not absolute. Using a functional approach, we investigated in a parent --> F1 hybrid model differing at the whole MHC, whether graft-versus-host (GvH) associated immunosuppression was correlated with donor cytotoxic T cell activity. The immunodeficiency was tested by the ability of the F1 mice to generate a cytotoxic T cell response against trinitrophenyl-modified syngeic cells (TNF-self) or an alloantigen. F1 specific parental cytotoxic T cells, generated in vitro, induced less immunosuppression than naive parental cells. Specific in vivo priming increased the cytotoxicity of parental spleen cells, but decreased their capacity to induce GvH-associated immunosuppression. In contrast, nonspecific priming resulted in the usual immunodeficiency. In conclusion, there was no correlation between GvH-associated immunosuppression and cytotoxic T cell activity of the parental cells.

[Immune deficiency associated with experimental graft-versus-host reaction. Lack of correlation with T-cell cytotoxic activity]. / Déficit immunitaire associé à la réaction expérimentale du greffon contre l'hôte. Absence de corrélation avec l'activité cytotoxique des lymphocytes T.

Graft-versus-host reactions (GvHR) are initiated by T lymphocytes. In mice, the T cell subset involved depends on the incompatibility for minor or major histocompatibility antigens between donor and host. However, the correlation between phenotype and function is not absolute, and anti-host cytotoxic T cells can be detected in recipients without GvHR. We have presently investigated in a P----F1 model differing at the MHC, whether GvH associated immunosuppression was correlated with donor cytotoxic T cell activity. The immunodeficiency was tested by the ability of the F1 mice to generate a cytotoxic T cell response against TNP self or an alloantigen. F1 specific parental cytotoxic T cells generated in vitro induced less immunosuppression than naive parental spleen cells. Specific in vivo priming increased the cytotoxicity of parental spleen cells, but decreased their capacity to induce GvH associated immunosuppression. In contrast, non specific priming resulted the usual immunodeficiency. Spleens of the F1 mice injected with specific cytotoxic T cells were very enlarged, suggesting that these cells remained capable of inducing a GvHR without generating immunosuppression.

Mucosal immunity in adult primary glomerulonephritis. I. Evaluation of salivary IgA subclasses and components.

Salivary components (proteins, albumin, IgA1, IgA2, IgG, IgM, beta 2-microglobulin, neopterin and peroxidase) were investigated in 3 adult types of primary glomerulonephritis (PGN): IgA mesangial glomerulonephritis (IgAGN; n = 14); idiopathic membranous glomerulonephritis (n = 8); idiopathic nephrotic syndrome (INS; n = 14), and a control group (n = 11). Salivary IgA1 levels were significantly increased in all these PGN whereas salivary IgA2 levels were only higher than controls in INS. Albumin and proteins did not differ between PGN and controls, while the IgA1 + IgA2/protein ratio was significantly increased in these 3 PGN. Salivary neopterin levels were enhanced in the 3 types of PGN, whereas beta 2-microglobulin levels were not. The other salivary components did not differ from controls. These results demonstrate the nonspecificity of the IgA increase at mucosal sites previously found in IgAGN and raise the hypothesis of an activation of mucosal immunity of PGN or of a disturbed isotypic network or lymphokine secretion in these diseases.

Biologic activity of a C2-derived peptide. Demonstration of a specific interaction with guinea pig lung tissues.

A synthetic peptide derived from the carboxy terminus of C2b has been investigated for its ability to induce the contraction of guinea pig lung parenchymal strips. This peptide is known to enhance vascular permeability in guinea pig and human skin, and to induce contraction of estrous rat uterus. This C2 peptide (C2 207-223) is active from 5 x 10(-5) M to 5 x 10(-4) M and is not tachyphylactic to itself. No cross-activity between C2 207-223 and C5a or C3a could be demonstrated. C2 207-223 is not inhibited by antihistamines or cyclooxygenase inhibitors. These data indicate that the peptide exerts its action via a mechanism distinct from those of the C3a and C5a anaphylatoxins.

Angioedema induced by a peptide derived from complement component C2.

Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.

A method facilitating the demonstration of synergism in the tissue-responses to mixed peptide stimuli.

A methodology for the pharmacodynamical assay of mixtures of 2 spasmogenic peptides is described, illustrated and discussed, with reference to a method intended for the chemists (P. Job, 1927). This allows a quick appreciation of the existence of synergism, of its own order of magnitude and of that of the doses for which it is significant. Also given is a fast way of curve-fitting the dose response to individual peptides, according to Clark's approximation.