Vanin genes are clustered (human 6q22-24 and mouse 10A2B1) and encode isoforms of pantetheinase ectoenzymes.

The mouse Vanin-1 molecule plays a role in thymic reconstitution following damage by irradiation. We recently demonstrated that it is a membrane pantetheinase (EC 3.56.1.-). This molecule is the prototypic member of a larger Vanin family encoded by at least two mouse (Vanin-1 and Vanin-3) and three human (VNN1, VNN2, VNN3) orthologous genes. We now report (1) the structural characterization of the human and mouse Vanin genes and their organization in clusters on the 6q22-24 and 10A2B1 chromosomes, respectively; (2) identification of the human VNN3 gene and the demonstration that the mouse Vanin-3 molecule is secreted by cells, and (3) that the Vanin genes encode different isoforms of the mammalian pantetheinase activity. Thus, the Vanin family represents a novel class of secreted or membrane-associated ectoenzymes. We discuss here their possible role in processes pertaining to tissue repair in the context of oxidative stress.

Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice.

Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy.

P/Q-type voltage-gated Ca(2+) channels (VGCC) regulate neurotransmitter release in the hippocampus and molecular alterations of their alpha(1A) pore-forming subunits are involved in various animal and human CNS diseases. We evaluated, using RT-PCR and in situ hybridization, the spatio-temporal activation of two alpha(1A) subunits splice variants (alpha(1A-a) and alpha(1A-b)) in control and kainic acid (KA)-treated rats. Six hours after KA treatment, alpha(1A-a) and alpha(1A-b) mRNAs increased, decreased or remained unchanged with area specific patterns. These changes were evidenced in the hippocampus and the dentatus gyrus and absent in the cerebellum. The alpha(1A) mRNA upregulation lasted for at least 7 days after KA treatment. Altogether, these results indicate that alpha(1A-a) and alpha(1A-b) mRNAs following seizure onset exhibit a complex and specific spatio-temporal pattern. The long-lasting changes in alpha(1A) subunit mRNA contents suggests that VGCC may be involved in the mechanisms generating chronic focal hyperexcitability and/or cellular damage in temporal lobe epilepsy.

Rat embryonic hippocampal neurons express a new class A calcium channel variant.

The aim of this study was to investigate, using a RT-PCR strategy, rat voltage-gated class A calcium channel (alpha1A) splice variants during rat hippocampus development. Results demonstrate the presence of multiple alpha1A mRNAs with the hippocampus formation and revealed a new variant of the rat alpha1A subunit (alpha1A-EFe) that diverges from alpha1A-a in the EF-hand domain. alpha1A-EFe expression in hippocampal neurons is restricted to the embryonic period. This in vivo developmental program is recapitulated in dissociated cultures of E17 embryonic hippocampal neurons. These data demonstrate that rat hippocampus neurons express a unique alpha1A splice variant during the embryonic period and suggest that alternative RNA splicing may modulate neuronal calcium channel properties during development.

L-type calcium channel activation controls the in vivo transduction of the neuralizing signal in the amphibian embryos.

We have analyzed the transduction pathways involved in the triggering of neural induction, in amphibian embryos, in vivo. Using a plasmid construction, we have targetted the bioluminescent calcium probe aequorin to the plasma membrane of ectoderm cells of the amphibian Pleurodeles waltl before gastrulation. We have demonstrated that the in vivo triggering of neural induction depends on the activation of calcium-dependent pathways and involves L-type calcium channels. Furthermore, on excised ectoderm taken at the gastrula stage, we show that noggin, a protein currently considered as one of the natural inducers, also activates L-type calcium channels. This activation represents the first necessary event to determine cells of the dorsal ectoderm toward the neural pathway.

Prolonged treatment of breast cancer cells with antiestrogens increases the activating protein-1-mediated response: involvement of the estrogen receptor.

At micromolar (pharmacological) concentrations, the action of tamoxifen on the proliferation of estrogen-dependent cells can be mediated not only by the estrogen receptor (ER), but also by other target molecules, such as protein kinase-C (PKC), which are easily inhibited by antiestrogens in cell-free experiments. By developing MTLN and MDT cell lines, in which any modulation of PKC activity is reflected by a variation of the expression of an activating protein-1 (AP-1)-controlled firefly luciferase gene, we investigated whether such antiestrogen inhibitory effects on PKC occurred in intact breast cancer cells. Firstly, in short term (4-h) treatment of both cell lines, antiestrogens only inhibited the 12-O-tetradecanoyl-phorbol-13-acetate-induced luciferase activity at very high concentrations (30 microM). A cytolytic effect was also observed. Secondly, in prolonged (4-day) treatments of MTLN (ER-positive) cells, low antiestrogen concentrations (nanomolar) decreased the basal AP-1 response by about 2 and increased the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated AP-1 response by about 3-4. This stimulation was mediated by ER, because 1) dose-response curves established with tamoxifen and hydroxytamoxifen were in agreement with their affinity for ER; 2) when present with antiestrogens, estradiol abolished this phenomenon; and 3) this effect was not observed in MDT (ER-negative) cells. Such a latent activation of AP-1 pathway could appear in the course of breast cancer antiestrogen treatment, in conditions where natural PKC activators are abnormally produced with unexpected consequences on the results of a long term antiestrogen treatment.

Role of the A/B region of the human mineralocorticoid receptor in aldosterone response selectivity.

In transfection experiments performed with wild-type MR, the maximal transcriptional activation of a mineralocorticoid response induced by glucocorticoid was generally similar to that of aldosterone, the natural mineralocorticoid hormone. However, compared to aldosterone, glucocorticoid activity decreased when the A/B region of MR was absent. We describe in this study the synthesis and biological activities of seven mutated MRs differently truncated in the N-terminal region. Using transient expression conditions in MCF-7 cells, the N-terminal domain of MR has been shown to contain a region (residues 254-390) whose deletion led to an apparent "aldosterone selectivity". These results suggest that this region could help to maintain the most transcriptionally active conformation of MR even in the presence of ligands which are not by themselves able to fully induce such a conformation.

Structure and expression of the human GATA3 gene.

GATA3, a member of the GATA family that is abundantly expressed in the T-lymphocyte lineage, is thought to participate in T-cell receptor gene activation through binding to enhancers. To understand GATA3 gene regulation, we cloned the human gene and the 5' end of the mouse GATA3 gene. We show that the human GATA3 gene contains six exons distributed over 17 kb of DNA. The two human GATA3 zinc fingers are encoded by two separate exons highly conserved with those of GATA1, but no other structural homologies between these two genes can be found. The human and mouse GATA3 transcription units start at a major initiation site. The promoter sequence analysis of these two genes revealed that they are embedded within a CpG island and share structural features often found in the promoters of housekeeping genes. Finally, we show that a DNA fragment containing the human GATA3 transcription unit, 3 kb upstream from the initiation site and 4 kb downstream from the polyadenylation site, displays T-cell specificity.

Stable luciferase transfected cells for studying steroid receptor biological activity.

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.

[Using examples of firefly luciferase gene for the study of biological activities (estrogens, retinoids, phorbol esters)]. / Exemples d'utilisation du gène de la luciférase de Luciole pour l'étude d'activités biologiques diverses (oestrogènes, rétinoïdes, esters de phorbol).

Some possible applications of chimeric cellular models, specifically responding to an effector through firefly luciferase induction are presented with the help of examples in relation with the biological activity of estradiol or retinoic acid, or phorbol ester. A comparison of experiments on either chimeric or natural responses shows that: i) the responses of both type of cellular models are effector concentration-dependent; ii) these concentrations are in the same order of magnitude; partial agonist compounds and antagonist compounds; iv) potencies (EC50) of test-compounds are similarly classified. Moreover we show that a chimeric cellular model allows the observation of interactions between hormone or effector pathways: it allows readily performed kinetic studies and long-term experiments in intact cells that permit to investigate the effect of a given effector, its reversibility and time-dependent action. Therefore, various steps of a cellular signalling pathway involved in the action of an effector may be observed with such a valuable tool.

Identification of a new mutation responsible for hepatoerythropoietic porphyria.

A deficiency in the activity of uroporphyrinogen decarboxylase (URO-D), the fifth enzyme of the haem biosynthetic pathway, is found in two hereditary diseases, familial porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP). Little is known about the genetic relationship between those two diseases and it has been postulated that HEP is the homozygous form of PCT. A URO-D cDNA was cloned from an HEP patient and the comparison between the mutant and the wild-type sequences showed a single base difference within the coding sequence leading to the replacement of a glutamic acid by a lysine at codon 167 of the mutant protein. This replacement produced a protein which is rapidly degraded in the presence of cell lysate. On the basis of hybridization of synthetic oligomers to amplified genomic DNA, we demonstrated that this patient is homozygous for this single base mutation. In order to look for any relationship between HEP and PCT, we tested six unrelated patients with familial PCT and could not detect the codon 167 mutation in any of them. These results indicate an heterogeneity in the mutations responsible for the PCT and HEP phenotypes.

Regulated expression of the overlapping ubiquitous and erythroid transcription units of the human porphobilinogen deaminase (PBG-D) gene introduced into non-erythroid and erythroid cells.

The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.

Structure of the gene for human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase, the fifth enzyme of the heme biosynthetic pathway, is an housekeeping enzyme whose activity is enhanced during erythropoietic differentiation. We have previously shown that this increased activity was in part accounted for by an enhanced transcription of the gene in erythropoietic tissues. To elucidate further the tissue specific regulation of an housekeeping gene we have isolated the human URO-D gene and determined its organization. The cloned gene comprises 10 exons spread over 3 Kb. Two transcriptional start sites were determined and analysis of 900 bp of the 5' flanking region suggests a very simple structural organization for the URO-D gene promoter. We also show that this gene is functional when transfected into mouse fibroblasts, and that its promoter is sensitive to a viral enhancer.

A de novo intragenic deletion of the potential EGF domain of the factor IX gene in a family with severe hemophilia B.

We have studied a family of three patients who were severely afflicted with hemophilia B without inhibitor for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (30% of normal) factor IX antigen. DNA hybridization analysis demonstrated that these patients had a partial intragenic deletion in their factor IX gene. This 2.8-kb deletion included exon d and the surrounding sequences. This exon codes for the amino acid sequence from No. 47 through 84 of the factor IX protein and contains its first potential EGF domain; the de novo occurrence of the mutation in the grandfather's germ cells was established by linkage analysis. This specific gene has been named F IXStrasbourg.

Prenatal diagnosis of sickle-cell anemia in the first trimester of pregnancy.

To investigate the usefulness of chorionic biopsy for prenatal diagnosis of sickle-cell anemia by restriction-endonuclease analysis of fetal DNA, we studied 30 pregnancies before elective abortion. When the reproducibility of the technique for obtaining adequate DNA samples was established, we successfully applied the test to five pregnancies at risk for sickle-cell anemia. In two cases, sickle-cell disease of the fetus led to a decision to terminate the pregnancy. In three other cases, a normal or AS genotype was demonstrated. One normal infant has been born, and one other pregnancy is continuing normally. In one case in which fetal death was observed three weeks after sampling, placental abnormalities found on histologic examination were compatible with a chromosomal aberration. Our study shows that chorionic biopsy is feasible for the prenatal diagnosis of sickle-cell disease before the 10th gestational week. If subsequent experience demonstrates this technique to be safe enough for mother and fetus, the ability to test in early pregnancy may make prenatal diagnosis acceptable to more couples at risk for serious genetic disorders.

Antenatal diagnosis of haemoglobinopathies by Biorex chromatography of haemoglobin.

Biorex chromatograhpy of haemoglobin has been compared to the standard chromatographic separation of radioactive globin chains in 60 fetal blood samples obtained for the antenatal diagnosis of haemoglobinopathies. Biorex chromatography of haemoglobin permitted two measurements, the optical density at 418 nm and the radioactivity incorporated into fetal and adult haemoglobin. The two measurements were highly correlated (r2=0.96) and enabled a distinction between homozygous from heterozygous states of the diseases to be made, particularly in beta thalassaemia. A single column was used for 50 analyses. This fast and very sensitive method is proposed for the antenatal diagnosis of haemoglobinopathies using fetal blood.

New techniques for the prenatal diagnosis of hemoglobinopathies.

Two new techniques have been devised for the prenatal diagnosis of hemoglobinopathies performed on fetal blood samples. Isoelectric focusing (IEF) of hemoglobins was compared to the classical chromatography of labelled globin chains for 51 fetal blood samples, 40 being obtained for prenatal diagnosis of hemoglobinopathies, in Paris. In all cases the two methods provided identical results. Adult hemoglobin was quantitatively evaluated. In addition blood samples obtained in other centers after abortion of 22 fetuses homozygous for beta thalassemia did not exhibited measureable amounts of Hb A by IEF. The fetal blood must be free of maternal contamination. If present maternal red blood cells can be completely eliminated by selective lysis using the 0RSKOV reaction. A chromatography of hemoglobins on Biorex 70 has been devised very recently to overcome the two limitations of IEF: First the contamination of fetal blood samples by maternal cells, and second the impossibility to evaluate Hb A when present in proportion below 1%. The chromatography of hemoglobins on Biorex 70 is performed in 75 minutes with 0.1 mg of hemoglobin present in membrane free hemolysate. The optical density recording allows to evaluate .5% of Hb A and to detect .1% of Hb A. In addition, the radioactivity profile of the chromatography can be determined. It has to be used in case of maternal contamination.

A gamma and G gamma globin chain synthesis in BFU-E colonies from adult, newborn, and fetal subjects and from thalassemic patients.

The G gamma and A gamma content of Hb F produced in cultures of BFU-Es from the blood of normal fetuses, neonates, and adults was determined. The results show that erythroid progenitors produce A gamma and G gamma chains in a ratio characteristic of their ontogenic stage. The analysis of the G gamma/A gamma ratio in culture of BRU-Es from thalassemic patients showed a marked heterogeneity, resembling that observed in freshly drawn cells. These results afford evidence that the type of gamma chain produced is programmed at the level of early erythroid progenitors.