Epithelio--mesenchymal interactions are critical for Quox 7 expression and membrane bone differentiation in the neural crest derived mandibular mesenchyme.

In higher vertebrates, branchial arch mesenchyme (ectomesenchyme) is derived from the cephalic neural crest. The ectomesenchyme of the mandibular arch yields the Meckel's cartilage and several membrane bones. We previously reported the isolation of a quail homeobox gene, Quox 7. In common with its mouse counterpart Hox 7, Quox 7 is highly expressed in the medioventral part of the mandibular arch and later in the precursor cells of the membrane bones. Since bone differentiation from ectomesenchyme is strictly dependent upon a signal provided by the mandibular epithelium, we decided to see whether the regulation of Quox 7 gene activity might be correlated with epithelio--mesenchymal interactions. Quox 7 expression was studied in E3 mandibular ectomesenchyme cultured in vitro or grafted on the chick chorioallantoic membrane either alone or recombined with the homotopic and heterotopic epithelia. We found that Quox 7 mRNA was undetectable after 48 h in cultures of mesenchyme alone while it remained abundant in non-cartilaginous tissue of the mandibular arch ectomesenchyme recombined with its own epithelium. The signal provided by the mandibular epithelium for Quox 7 expression can also arise from various heterotopic epithelia, e.g. of dorsal or ventral body wall and of limb bud. Thus the effect of the epithelium on Quox 7 expression in mesenchymal cells strictly parallels that on bone formation. These results strongly suggest that the epithelio-mesenchymal interactions have an essential role on the regulation of Quox 7 gene, the product of which seems to be, in turn, necessary for the execution of the skeletal developmental program in the facial area.     

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.     

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Synthesis and physicochemical properties of oligonucleotides built with either alpha-L or beta-L nucleotides units and covalently linked to an acridine derivative.

Modified deoxynucleosides 2'-deoxy-beta-L-uridine, beta-L-thymidine, alpha-L-thymidine, 2'-deoxy-beta-L-adenosine and 2'-deoxy-alpha-L-adenosine were synthesized and assembled as homooligomers, respectively: octa-beta-L-deoxyuridylates, octa beta-L and alpha-L-thymidylates and tetra beta-L and alpha-L-deoxyadenylates. These unnatural oligomers were then substituted with an acridine derivative. The binding studies of these modified oligonucleotides with D-ribo- and D-deoxyribopolynucleotides were carried out by absorption spectroscopy. While beta-L-d(Up)8m5Acr, beta-L-(Tp)8m5Acr, alpha-L-(Tp)8m5Acr did not interact with poly(rA) and poly(dA), beta-L-d(Ap)4m5Acr and alpha-L-d(Ap)4m5Acr did form double and triple helices with poly(rU) and poly(dT), respectively. Their stability towards nuclease digestion was studied through comparison with that of octa-beta-D-thymidylate and tetra beta-D-deoxyadenylate covalently linked to an acridine derivative. One endonuclease (nuclease P1 from Penicillium citrinum) and two exonucleases (a 3'-exonuclease from Crotalus durissus venom and a 5'-exonuclease extracted from calf thymus) were employed. beta-L- and alpha-L-oligomers demonstrate a high resistance toward nuclease digestion.     

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.     

Use of a simple method for the Epstein-Barr virus transformation of lymphocytes from members of large families of Réunion Island

A simple method for the preparation of lymphoblastoid cell lines from small amounts (100 microliter) of frozen whole blood is described. A success score greater than 90% was obtained for EBV transformations using blood samples which had been collected several months before the infection. Due to the simplicity of the technique, up to 80 samples could be processed per day. This technique was used to prepared 242 permanent cell lines from 13 large families from Réunion Island showing blood group H deficiency. These cell lines are now available for genetic studies.     

Organogenesis at the shoot apex: An attempt at modelization

A geometrical model of the emergence of a primordium at the shoot apex in dicotyledons is proposed. It is based on recent fundamental results on plant morphogenesis, i.e.:

1. the emergence is preceded by the reorganization of the microtubules of the cortical cytoskeleton, leading to a new orientation of the synthesis of the cell wall microfibrils;
2. the resulting global stress is related to the general orientation of the cell growth.

So the model sums up the continuous interactions linking the microtubules, the microfibrils and the cell growth axis. This paper tries to answer three questions which are essential from a botanical point of view:

1. Why does the principal stem shift its growth direction after each lateral emergence?
2. Why do the three axes involved in any ramification (namely the old and the new principal stems and the lateral emergence) exhibit a plane configuration whereas this is an essentially three dimensional phenomenon?
3. Does phyllotaxis exclusively depend upon the local emergence of a primordium?

A come and go between the botanical knowledge and the mathematical model leads to an integrated view of the compatibility mechanisms linking the different microtubules and microfibrils networks, without forgetting the apical dome restoration. A geometrical formalism allows a modern redefinition of both the “generating centre” and the “organizing centre” and their field effects.     

CFC syndrome: a syndrome distinct from Noonan syndrome

We report two children with a common pattern of birth defects. Both have very sparse, curly hair, nystagmus and mental retardation. The first one has Noonan syndrome habitus associated with keratosis plantaris and nystagmus; the second one has a slightly Noonan-like face, macrocephaly, keratosis pilaris, and hypertrophic cardiomyopathy. They represent the extreme of a spectrum of congenital defects recently reported independently as CFC syndrome by Reynolds and as "Noonan-like short stature syndrome with sparse hair" by Baraitser and Patton. The clinical features are reviewed and the autonomy of the syndrome with regards to Noonan syndrome, is disputed, since every sign seems to occur independently in Noonan syndrome. The father of the second case probably has a minor syndrome expression, pointing to probable autosomal dominant inheritance.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.