The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were whown to comprise three major polypeptides of M _r 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and-negative mammlian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3t3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.     

Vaccine-induced neutralizing antibodies directed in part to the simian immunodeficiency virus (SIV) V2 domain were unable to protect rhesus monkeys from SIV experimental challenge.

The potential of the simian immunodeficiency virus (SIV) variable 2 (V2) domain as an effective region to boost SIV-neutralizing antibodies and to protect against live SIV challenge was tested in rhesus macaques. In this study, two rhesus macaques were primed with vaccinia virus recombinants expressing the surface glycoprotein gp140 of SIVmac and were given booster injections with the SIVmac V2 domain presented by a highly immunogenic carrier, the hepatitis B surface antigen (HBsAg). The two vaccinated macaques exhibited SIV-neutralizing antibodies after primer injections that were enhanced by the V2/HBsAg injections. Part of these SIV-neutralizing antibodies were directed specifically to the V2 region, as shown by neutralization-blocking experiments. However, despite having consistent SIV-neutralizing antibody titers, animals were not protected against homologous challenge with BK28, the molecular clone of SIVmac251. No SIV envelope-specific cellular cytotoxic response was detected throughout the immunization protocol, suggesting that neutralizing antibodies directed to SIV envelope gp140 and especially to the V2 domain were unable on their own to protect against SIV challenge. Furthermore, the vaccinees seemed to have higher viral loads than control animals after challenge, raising the question of whether neutralizing antibodies induced by vaccination and directed to the SIV envelope selected viral escape mutants, as shown previously in SIV-infected macaques. This mechanism is certainly worthy of intensive investigation and raises some concern for SIV envelope-targeted immunization.     

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37° C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37° C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunola-belling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.     

Induction of the murine “W phenotype” in long-term cultures of human cord blood cells by c-kit antisense oligomers

The murine white (W) spotting locus is the site of the c-kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c-kit antisense oligonucleotides to long-term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c-kit gene expression and the preferential suppression of the generation of erythroid burst-forming cells (BFU-E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c-kit gene function in early erythropoiesis. Because the generation of BFU-E was suppressed even after c-kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley-Liss, Inc.     

In Vitro and Molecular Docking Evaluation of Larvicidal Effects of Essential Oils of Five Aromatic Plants on the Fall Armyworm Spodoptera frugiperda JE. Smith (Lepidoptera: Noctuidae) from Ivory Coast

Faced with the serious consequences resulting from the abusive and repeated use of synthetic chemicals, today rethinking crop protection is more than necessary. It is in this context that the essential oils of the Lamiaceae Ocimum gratissimum and Ocimum canum, the Poaceae Cymbopogon citratus and nardus and a Rutaceae Citrus sp. of known chemical compositions were experimented. The evaluation of the larvicidal potential of the essential oils was done by the method of topical application of the test solutions, on the L1−L2 stage larvae from the first generation of S. frugiperda obtained after rearing in an air-conditioned room. Lethal concentrations (LC₁₀, LC₅₀ and LC₉₀) were determined after 48 h. After assessing the larvicidal potential of essential oils, molecular docking was carried out to study protein-ligand interactions and their propensity to bind to insect enzyme sites (AChE). The essential oil of O. gratissimum was the most effective with the lowest lethal concentrations (LC₁₀=0.91 %, LC₅₀=1.91 % and LC₉₀=3.92 %). The least toxic oil to larvae was Citrus sp. (LC₁₀=5.44 %, LC₅₀=20.50 % and LC₉₀=77.41 %). Molecular docking revealed that p-cymene and thymol from O. gratissimum essential oil are structurally similar and bind to the AChE active site via predominantly hydrophobic interactions and a H-bond with Tyr374 in the case of thymol. The essential oil of O. gratissimum constitutes a potential candidate for the development of biological insecticides for the fight against insect pests and for the protection of the environment.     

Antibiofilm Activity of Invasive Plants against Candida albicans: Focus on Baccharis halimifolia Essential Oil and Its Compounds

The extracts of five invasive plants were investigated for antifungal and antibiofilm activities against Candida albicans, C. glabrata, C. krusei, and C. parapsilosis. The antifungal activity was evaluated using the microdilution assay and the antibiofilm effect by measurement of the metabolic activity. Ethanol and ethanol-water extracts of Reynoutria japonica leaves inhibited 50 % of planktonic cells at 250 μg mL⁻¹ and 15.6 μg mL⁻¹, respectively. Ethanol and ethanol-water extracts of Baccharis halimifolia inhibited >75 % of the mature biofilm of C. albicans at 500 μg mL⁻¹. The essential oil (EO) of B. halimifolia leaves was the most active (50 % inhibition (IC₅₀) at 4 and 74 μg mL⁻¹against the maturation phase and 24 h old-biofilms of C. albicans, respectively). Oxygenated sesquiterpenes were the primary contents in this EO (62.02 %), with β-caryophyllene oxide as the major component (37 %). Aromadendrene oxide-(2), β-caryophyllene oxide, and (±)-β-pinene displayed significant activities against the maturation phase (IC₅₀=9–310 μ mol l⁻¹) and preformed 24 h-biofilm (IC₅₀=38–630 μ mol l⁻¹) of C. albicans with very low cytotoxicity for the first two compounds. C. albicans remained the most susceptible species to this EO and its components. This study highlighted for the first time the antibiofilm potential of B. halimifolia, its EO and some of its components.     

NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a non-fluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.     

Effect of exercise intensity on the postexercise sweating threshold.

The hypothesis that the magnitude of the postexercise onset threshold for sweating is increased by the intensity of exercise was tested in eight subjects. Esophageal temperature was monitored as an index of core temperature while sweat rate was measured by using a ventilated capsule placed on the upper back. Subjects remained seated resting for 15 min (no exercise) or performed 15 min of treadmill running at either 55, 70, or 85% of peak oxygen consumption (V(o2 peak)) followed by a 20-min seated recovery. Subjects then donned a liquid-conditioned suit used to regulate mean skin temperature. The suit was first perfused with 20 degrees C water to control and stabilize skin and core temperature before whole body heating. Subsequently, the skin was heated ( approximately 4.0 degrees C/h) until sweating occurred. Exercise resulted in an increase in the onset threshold for sweating of 0.11 +/- 0.02, 0.23 +/- 0.01, and 0.33 +/- 0.02 degrees C above that measured for the no-exercise resting values (P < 0.05) for the 55, 70, and 85% of V(o2 peak) exercise conditions, respectively. We did note that there was a greater postexercise hypotension as a function of exercise intensity as measured at the end of the 20-min exercise recovery. Thus it is plausible that the increase in postexercise threshold may be related to postexercise hypotension. It is concluded that the sweating response during upright recovery is significantly modified by exercise intensity and may likely be influenced by the nonthermal baroreceptor reflex adjustments postexercise.