Showing papers by "Collège de France published in 1987"

Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells

Abstract: QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.

Anatomical and electrophysiological evidence for a direct projection from ammon's horn to the medial prefrontal cortex in the rat

Abstract: Following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the medial prefrontal cortex (defined as the neocortical area innervated by the thalamic mediodorsal nucleus) labelled cells were observed in the pyramidal layer of the CA1 field of Ammon's horn. Observations made using antidromic stimulation confirmed these results, and revealed the slow conduction velocity of the fibres of the hippocampal cells innervating the prefrontal cortex. Taken together, these data provide evidence for a direct projection of CA1 cells to the medial prefrontal cortex.

A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies

Abstract: The Escherichia coli lacZ gene has been used as an indicator gene for the study of cell lineage in vivo. To adapt this marker for gene expression studies, a sequence encoding a modified beta-galactosidase and including the simian virus 40 large tumor nuclear location signal (nls-beta-Gal) has been introduced into vectors. In differentiated cells, multipotential cells, and embryos, the constructs led to the expression of an enzymatically active protein. Its location was examined by its beta-galactosidase activity or by using antibodies and electron microscopy. The results show that the nls-beta-Gal protein remains mainly located at the nuclear periphery (probably at the nuclear pores) but does not reach the nucleoplasm. It suggests that an interaction with the nuclear membrane is necessary but not sufficient for protein uptake into the nucleus. In multipotential cells, the expression of nuclear location signal LacZ (nls-LacZ) interferes neither with cell growth nor with differentiation. Using various lacZ constructs, the transcriptional activity of embryos was studied. At the two-cell stage, the promoters of the Rous sarcoma virus, simian virus 40, and the beta-actin gene are functional but the Moloney murine leukemia virus long terminal repeat is not. Thus, transcriptional specificity must already be present at the stage of activation of the embryonic genome.

MAP2 expression and neuritic outgrowth and branching are coregulated through region-specific neuro-astroglial interactions

Abstract: Embryonic neurons from the rat striatum and mesencephalon were plated on mesencephalic or striatal astrocytes in 4 possible combinations. It was found that specific traits are expressed by the neurons when they are grown on homotopic astrocytes (neurons and astrocytes from the same region). These traits are the following: 1. The number of cells stained with an antibody raised against the microtubule-associated protein 2 (MAP2) is higher in homotopic than in heterotopic cocultures. This is true for both mesencephalic and striatal neurons. 2. In homotopic conditions, there is an increase in the number of cells having more primary neurites and branching points. This effect is observed for both neuronal populations but is more pronounced in mesencephalic neurons. 3. The intensity of MAP2 staining was correlated with the branching ability of the neurons. First, on comparing MAP2-positive and MAP2-negative cells, it was found that, in any combination (homotopic and heterotopic cocultures), the number of primary neurites and branching points was much higher in MAP2-positive cells. In fact, almost no branching activity was found in MAP2-negative neurons. Second, within the MAP2-positive neuronal population, the higher number of branching points observed under homotopic neuro-astroglial conditions was mostly due to the neuritic compartment, which was strongly and homogeneously stained with the anti-MAP2 antibody. These observations strongly suggest that the astrocytic environment regulates the synthesis and/or intracellular distribution of MAP2, as well as the morphology of the neurons, and that this regulation is region specific.

In vivo effect of brain-derived neurotrophic factor on the survival of developing dorsal root ganglion cells.

Abstract: Implantation of silastic membranes between neural tube and somites at somitic levels 20-24 in 30-somite-stage chick embryos results in separation of early migrated neural crest cells of the dorsal root ganglion (DRG) anlage from the neural tube and their death within a few hours [Kalcheim and Le Douarin, (1986) Dev. Biol., 116, 451-460]. The in vivo effects of brain-derived neutrotrophic factor (BNDF) on survival of HNK-1 immunoreactive DRG cells separated from the tube were examined by implantation of laminin-treated silastic membranes (controls) or BDNF/laminin-treated membranes. In the presence of BDNF/laminin-treated membranes, 20/25 grafted embryos fixed 10 h after implantation, contained many rescued cells on the operated side. In contrast, only a few rescued cells on the operated side. In contrast, only a few rescued cells were observed in sections on the operated in 2/11 embryos implanted with laminin-treated silastic membranes, and no rescued cells at all could be detected in embryos implanted with NGF/laminin-treated (seven embryos) or untreated silastic membranes (12 embryos). The data presented support the hypothesis that early survival and differentiation of neural crest-derived sensory cells depend on central nervous system-derived factor(s). Moreover, this is the first evidence for the in vivo activity of BDNF on survival of developing DRG cells.

How does a droplet spread

Abstract: Because of their practical interest, wetting phenomena have been the subject of many experimental and theoretical investigations in the past. However, no coherent description of this field had been obtained until recently. Some intriguing paradoxes remained unsolved. For example, once spreading is achieved, the rate of spreading of a droplet on a solid surface is usually found to be independent of the nature of the solid surface. New theoretical approaches, emphasizing the role of long-range forces, have led to a general framework in which all the experimental observations on spreading harmoniously find their place. The universal laws, experimentally found at the macroscopic level, result from processes occurring on a microscopic scale, typically between 10 and 1000 A.

Electrophoretic mobility of λ phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

Abstract: The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 102 to 2.36 × 104 base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ0) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ0, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.

Structure and expression of HLA-DQ alpha and -DX alpha genes: interallelic alternate splicing of the HLA-DQ alpha gene and functional splicing of the HLA-DQ alpha gene using a retroviral vector.

Abstract: The nucleotide sequences of the two closely related HLA-DQ α and HLA-DX α , genes have been determined. Exons coding for the signal peptide, α2 and transmembrane domains are 94–99% homologous, whereas the α1 exon and the promoter region have diverged as much as or more than introns and the 3′ untranslated region. The promoter regions of both genes contain two short sequences thought to be important for regulation of transcription by γ-interferon. Transfection studies established that the DQ α and DQ β genes encode the HLA-DQ antigen. Transcripts of varying length are produced from different alleles as the result of the use of alternate splice and polyadenylation signals at the 3′ end of the DQ α gene. Thus typing at the DQ α locus can be achieved by Northern blot analysis. No transcript of DX α was detected in B lymphocytes. The DX α gene was accurately spliced when introduced into a retroviral vector, suggesting that the lack of expression of DX α is not due to aberrant splice signals.

[3H]neurokinin B and 125I-Bolton Hunter eledoisin label identical tachykinin binding sites in the rat brain.

Abstract: Three different tachykinins have been identified in the rat CNS, substance P (SP), neurokinin A (NKA or substance K) (1) and neurokinin B (NKB or neuromedin K) (1,2). Since there is evidence of multiple receptors (3) for the tachykinins particularly in the periphery, much interest has been focused lately to determine whether each tachykinin binds to a corresponding specific binding site in the CNS.

Fate of uncrossed retinal projections following early or late prenatal monocular enucleation in the mouse

Abstract: In mammals binocular vision is made possible by the existence in the temporal retina of ipsilaterally projecting ganglion cells (IGCs) (with axons that do not cross the brain midline and join optic fibers from the opposite eye). To learn whether early interactions between fibers of each eye play a role in generating a mixed ipsi + contralateral projection pattern, we studied with horseradish peroxidase the origin of uncrossed retinal projections in mice that developed after one eye was destroyed at very early embryonic ages. One eye was removed on embryonic day 16 (E16; when optic fibers have grown past the chiasm bilaterally, but very few have grown into the visual centers) or on E13 or E12 (when few or no optic fibers have passed the presumptive chiasm region). Normal adult mice have a mean of 946 IGCs (range: 784–1,073) within the temporal sector of the retina, and less than 25 in the rest of the retina. In adult mice enucleated at E16, an average of 1,354 (1,215–1,484) IGCs are present within a clearly demarcated temporal sector of the remaining retina and 265 (152–312) are present throughout the rest of the retina. In both the temporal and nasal retina the excess IGCs in these mice have, generally, very small somas. In some of these mice the most peripheral part of the temporal sector contains fewer IGCs. In E12 or E13 enucleates, IGCs are also generally located in a narrow (often narrower than normal) region along the temporo-inferior retinal border, but their number is less than in normal or E16-enucleated mice: E13 enucleates have a mean of 639 cells (range: 361–875) in the temporal sector and 109 (8–275) in the rest of the retina. Following enucleation of one mouse at E12, the respective values are 349 and 31 cells. The reduction in numbers of IGCs in these mice is especially pronounced for ganglion cells with small cell bodies. These findings suggest that the development of uncrossed projections in mice depends on selective guidance mechanisms of axons from temporal retina through the chiasm. These may consist of interactions of optic axons with guidance cues distributed in the presumptive chiasm (possibly at early stages) and also of fiber-fiber guidance mechanisms, in particular between fibers from each eye.

Spin manifolds, Killing spinors and universality of the Hijazi inequality

Abstract: In terms of the Dirac operator P, we introduce on any field a first-order operator D and show that the operator (Δ−ρ) on the spinors (ρ=(n/4(n−1))R; dim W=n) is positive. By means of a universal formula, we show that, on a compact spin manifold of dimension ≥3, the Hijazi inequality [8] holds for every spinor field such that (Pψ, Pψ) = λ 2(ψ, ψ) (λ=const.). In the limiting case, the manifold admits a Killing spinor which can be evaluated in terms of ψ. Different properties of spin manifolds admitting Killing spinors are proved. D is nothing but the ‘twistor operator’.

3H-neurokinin A labels a specific tachykinin-binding site in the rat duodenal smooth muscle.

Abstract: 3H-Neurokinin A (3H-NKA) with high specific activity (75 Ci/mmol) was synthesized to study NKA (NK-2)-binding sites on membrane preparations of various tissues in the rat, including brain, spinal cord, duodenum, vas deferens, and ileum. The binding capacity of 3H-NKA (0.9 nM) was very low in membrane preparations of different central nervous system regions and the ileum smooth muscle (0.2-2 fmol/mg of protein). In contrast, relatively high specific binding was found in membrane suspensions of the rat duodenal smooth muscle (18 fmol/mg of protein) and the vas deferens (8 fmol/mg of protein). 3H-NKA-binding sites were further characterized on the rat duodenal smooth muscle. The specific binding of 3H-NKA was shown to be temperature dependent, saturable, reversible, and increased in parallel with the protein concentration. Scatchard analyses and Hill plots of equilibrium binding studies in the concentration range of 0.40-30 nM revealed that 3H-NKA bound to a single class of noninteracting binding sites (Bmax = 270 fmol/mg of protein, KD = 13.3 nM). Displacement of 3H-NKA with different tachykinin-related peptides gave the following rank order of potencies: NKA greater than NKA (4-10) greater than kassinin greater than eledoisin greater than NKB much greater than substance P greater than physalaemin, which suggests that the binding site labeled by 3H-NKA is different from substance P (NK-1)-and NKB (NK-3)-binding sites. The biological activities of tachykinins and related peptides were tested by measuring their contractile effects on the rat duodenum and rabbit pulmonary artery, two tissues known to be sensitive for NKA. Ki values were correlated with the EC50 obtained in biological assays. The results revealed a significant correlation (r = 0.86, p less than 0.01) between Ki and EC50 values obtained in the isolated rabbit pulmonary artery, whereas there was no significant correlation between binding affinities and biological responses on the rat duodenum (r = 0.62, p greater than 0.05).