Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.     

Qualitative study of chromosomal lesions induced by neutrons and neon ions in human lymphocytes at G0 phase

Chromosomal lesions, mitotic index and cell cycle progression delay induced by neutron (protons 34 MeV on beryllium) and neon (250 MeV/i) particles are studied in human lymphocytes. The cell cycle progression is slightly decreased at a dose of 1 Gy. Mitotic indexes are significantly decreased after irradiation by the different particles, except neon in 52-h cultures. By comparison to chromosome damages caused by gamma-rays, previously published, it is found that the lesions observed here are frequently more complex: the number of breaks is higher per abnormal mitosis and higher per rearrangement on the average. This complexity is higher for neon ions than for neutron beams.     

Implications phylogenetiques et biogeographiques des dernieres decouvertes de Muridae en Afghanistan,au Pakistan et en Ethiopie

Based on recent discoveries of Murid species in Pakistan and Afghanistan, two Asiatic autochtonous lineagesare proposed. Two forms from the African Pliocene seem to be related to these lineages. On this set of data, a new faunal province can be defined at the Mio-Pliocene boundary.     

Bioluminescence: an improvement in the enzymatic assay of dehydroepiandrosterone sulfate in biological fluids

Dehydroepiandrosterone sulfate (DHEAS) determination in biological fluids was carried out by enzymatic hydrolysis and conversion into estrogens [estrone (E1) and estradiol (E2)] by the multienzyme system of human placental microsomes. The enzymatic complex consists of sulfatase, 3 beta-hydroxysteroid oxido reductase and 5en----4en isomerase which converts DHEAS into androstenedione (A); the latter component is further converted into estrogens by the aromatase. The resulting estrogens were determined from the NADH formed by the transhydrogenation reactions of human placental dehydrogenase. NADH was measured by bioluminescence. As little as 4 pg was assayable by this rapid enzymatic method, with a coefficient of variation of 8%. The results are in good agreement with radioimmunoassay and the method is suitable for routine use.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Periods of organogenesis in mono- and bicyclic annual shoots of Juglans regia L. (Juglandaceae)

The organogenetic cycle of shoots on main branches of 4-year-old Juglans regia trees was studied. Mono- and bicyclic floriferous and vegetative annual shoots were analysed. Five parent annual shoot types were sampled between October 1992 and August 1993. Organogenesis of summer growth units was monitored between 16 Jun. and 3 Aug. 1993. Variations over time in the number of nodes, cataphylls and embryonic green leaves of terminal buds were studied. The number of nodes of parent shoot buds was compared with the number of nodes of shoots derived from parent shoot buds. The spring growth units of mono- and bicyclic shoots consist exclusively of preformed leaves which were differentiated, respectively, during the spring flush of growth (mid-April until mid-May) or the summer flush of growth (mid-June until early August) in the previous growing season. Thus, winter buds may consist of flower and leaf primordia differentiated in two different periods during annual shoot extension. The summer growth units of bicyclic shoots consist of preformed leaves that were differentiated in spring buds during the spring flush of growth in the current growing season. Bud morphology is compared between spring and summer shoots.     

Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.     

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l -peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1 , Abcd2 , Abcd3 , and Acox1 ) involved in peroxisomal transport or β-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l -peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.     

Zinc status and bone mineralisation in adolescent girls.

The aim of the project was to assess the relationship between zinc status and bone mineralisation in pre-menarcheal adolescent girls. One hundred and thirty-nine healthy pre-menarcheal girls (Tanner pubic hair stage < or = 4), aged 12.4 +/- 1.0 years, had two visits at an interval of 2 years. Serum and urine zinc concentrations (Zn S; Zn U; Zn U/ creatinine), insulin-like growth factor 1 (IGF-I), and markers of bone turn-over, i.e. osteocalcin and parathormone (PTH), concentrations were measured at the first visit. Lumbar (L2-L4) bone mineral content and density (BMC, BMD) were measured at both visits. BMC and BMD and their increase at the follow-up after 2 years were compared with biochemical data by multiple regression. The stage of puberty was added as a covariable in the analysis. At the first visit, a significant correlation was found between sexual maturity and initial BMC, BMD, height, weight, and IGF-I. Zn S was negatively correlated with osteocalcin. Zn U showed a positive correlation with BMC, BMD, IGF-I, height, weight, and PTH. At the second visit, sexual maturity showed a positive correlation with BMD and weight increments and a negative one with BMC and height gains. Zn S was significantly related with BMD increase. These correlations suggest that zinc plays a role in normal growth and bone mineralisation during puberty onset.