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A central event in angiogenesis is proliferation of blood vessels, which plays a major role in the progression of a number of inflammatory and neoplastic diseases. It is responsible for the switch of endothelial cells from an antiangiogenic to an angiogenic phenotype. To identify novel activated/proliferating-related proteins in human endothelial cells, a subtractive immunization approach was used to elicit a host antibody response against human dermal microvascular endothelial cells (HDMECs) stimulated with a potent angiogenic cytokine such as VPF/VEGF165. In this study, a new mAb, LY9, which is highly specific to VPF/VEGF165-activated HDMECs, was isolated. Stimulation of HDMECs by VPF/VEGF165 or basic fibroblast growth factor (bFGF) resulted in a dose-dependent and time-dependent increase in the binding of LY9. On Western-blot analysis, LY9 identified an 85-kDa protein (p85) in the lysates of several endothelial cells derived from microvascular or large vessel sources, the expression of which is dramatically increased by VPF/VEGF165. The mAb also identified p85 in primary cell cultures of human foreskin keratinocytes but failed to recognize human fibroblasts (MRC5) and a number of different human tumor cell lines, including MG63 osteosarcoma and MCF7 breast carcinoma cells. Immunological screening of a human keratinocyte lambdagt11 cDNA expression library with LY9 identified a partial cDNA clone of 750 bp. DNA sequencing of this clone and predicted amino acids showed more than 93% homology to RING3 kinase, a member of a newly described family of bromodomain-containing proteins that transactivates in the nucleus the promoters of a number of the E2F family of transcription factors. This molecule may represent a new signaling target activated by VPF/VEGF165 and bFGF that allows endothelial cells to enter the proliferative phase of the angiogenic process.

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International audience; Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

A 5500 base-pair fragment including the β-globin gene downstream from codon 122 and about 4000 base-pairs of its 5′ flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5′ extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.