Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.     

In vivo resistance of secondary antitumor immune response to cyclophosphamide: effects on T cell subsets

Summary We have analyzed the effects of high doses of cyclophosphamide (Cy) on primary and secondary antitumor immune response against immunogenic (tum^–) variants of Lewis lung carcinoma (3LL) treated in vitro with UV light. Normal mice and mice previously immunized with tum^– clones were inoculated i.p. with Cy (200 mg/kg body weight) and 24 h later challenged intrafootpad with tum^– or parental 3LL cells. Cy treatment suppressed the primary immune response of normal animals and allowed the growth of tum^– cells. In contrast, Cy-treated immune mice rejected the tumor challenge. The in vivo treatment with Cy decreased the total number of lymphoid cells in the spleens, as well as the proportion of B lymphocytes; however, it increased the percentage of both Lyt2⁺ and L3T4⁺ lymphocytes. Thus, the immunosuppressive effects of Cy on the primary antitumor response could not be attributed to elimination of major T lymphocyte subpopulations. Although the treatment of immune mice with Cy did not significantly impair their antitumor resistance, nor the proportion of Lyt2⁺ and L3T4⁺ lymphocytes in their spleens, the in vitro generation of cytotoxic T lymphocytes (CTL) was markedly reduced.After Cy treatment, the proliferative ability of spleen cells in response to interleukin-2 (IL-2) was substantially impaired. Using monoclonal antibodies to the IL-2 receptor, we found that Cy-treated T lymphocytes failed to fully express the IL-2 receptor following in vitro stimulation with irradiated tumor cells. In line with these findings, the in vitro generation of CTL was not restored by addition of recombinant IL-2 to the cultures. In vivo experiments using purified functional subsets of immune T cells showed that Lyt1⁺, but not Lyt2⁺ lymphocytes were able to transfer antitumor immunity in normal irradiated recipients.Therefore, since Ly1⁺ T lymphocytes were responsible for the antitumor resistance in vivo, the Cy-induced impairment of CTL generation did not affect the ability of immune mice to reject a secondary tumor challenge.This project has been funded at least in part with Federal funds from the Department of Health and Human Services, under contract number NO1-CO-23910 with Resources, Inc. The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Variation in eggshell characteristics and in intrauterine egg retention between two oviparous clades of the lizard Lacerta vivipara: insight into the oviparity-viviparity continuum in squamates

The concept of the oviparity-viviparity continuum refers to the wide range in the length of intrauterine egg retention and, hence, in the stage of embryonic development at oviposition existing in squamates. The evolutionary process underlying this continuum may involve not only a lengthening of egg retention in utero, but also a marked reduction in the thickness of the eggshell. The idea that there may exist a negative correlation between the developmental stage reached by the embryo at oviposition and the eggshell thickness within squamates, although supported by the comparison of oviparous vs. viviparous species, has seldom been evaluated by comparing eggshell thickness of oviparous forms with different lengths of intrauterine egg retention. Eggs of two distinct oviparous clades of the lizard Lacerta vivipara were compared. The eggs laid by females from Slovenian and Italian populations have thicker eggshells, contain embryos on average less developed at the time of oviposition, and require a longer incubation period before hatching than the eggs laid by females from French oviparous populations. Our data and several other examples available from the literature support the idea that the lengthening of intrauterine retention of eggs and the shortening of the subsequent external incubation of eggs are associated with reduction in the thickness of the eggshell, at least in some lineages of oviparous squamates. The current hypotheses that may account for this correlation are presented and a few restrictions and refinements to those hypotheses are discussed. In particular, other changes, such as increased vascularization of the oviduct and of the extraembryonic membranes, may play the same role as the decrease of eggshell thickness in facilitating prolonged intrauterine egg retention in squamates. Future studies should also consider the hypothesis that the length of intrauterine retention might directly depend on the extent of maternal-fetal chemical communication through the eggshell barrier.     

Festuca valesiaca Schleich. ex Gaudin newly discovered in the Central Apennines (Italy): a further example of steppe relict in the Abruzzo “dry valleys”

Abstract

Festuca valesiaca Schleich. ex Gaudin subsp. valesiaca is acknowledged as one of the most characteristic grasses of the continental grasslands and steppes of Eurasia. In Italy, the species was known almost only from the continental valleys of the Alpine chain (with the exception of two small populations at the northernmost edge of the Apennines). Here, we report on the first confirmed finding of F. valesiaca subsp. valesiaca in the surroundings of the Fucino Basin, Central Apennines. This finding expands by c. 400?km southwards the species’ range in Italy. The identity of the species was first ascertained by means of leaf cross-sections and stomata measurements, and then confirmed by chromosome count. Its habitat preferences within the area were found to match well with those described in the literature for C-Europe. After discussing some identification issues and the population’s ecological niche, we present the main biogeographic implications of our finding. By recalling and discussing similar examples, we conclude that during the Holocene forest spread some Apennine “dry valleys” performed as refugia for a number of Pleistocene steppic taxa. The grasslands of these areas may thus be ancient and valuable ecosystems maintained by climate and disturbance throughout the Holocene.     

The protein "mycoarray": a novel serological assay for the laboratory diagnosis of primitive endemic mycoses

A protein microarray containing fungal antigens (the "mycoarray") has been set up to provide rapid and appropriate serodiagnosis of primitive endemic mycoses, an important cause of morbidity and mortality in an increasingly high number of patients. The mycoarray consists of three antigen extracts (histoplasmin, coccidioidin and Coccidioides "TP") and antibody dilution curves were spotted on microarray slides. The arrays were processed with coccidioidomycosis and histoplasmosis patients? sera or with control sera and the occurring immunocomplexes were detected by indirect immunofluorescence. In agreement with clinical and microbiological diagnosis, the results distinguished between histoplasmosis and coccidioidomycosis patients. In addition, the assay could clearly discriminate between IgM and IgG antibody reactivity. No reactivity was ever observed in the arrays processed with negative control sera. Therefore, this pilot study demonstrates that the "mycoarray" is sensitive and specific enough to discriminate between healthy individuals and patients with histoplasmosis or coccidioidomycosis. Because of miniaturization and multiparametricity, the new assay cuts costs and processing time. Thus, once clinically validated and implemented as a large-scale array, the "mycoarray" will be ready to be applied to the daily clinical practice.     

Isolation and structure elucidation of cytotoxic polyacetylenes and polyenes from Echinacea pallida

Bioassay-guided fractionation of n-hexane extracts of Echinacea pallida (Asteraceae) roots led to the isolation and structure elucidation of two polyacetylenes (1, 3) and three polyenes (2, 4, 5). Two are known hydroxylated compounds, namely 8-hydroxy-pentadeca-(9E)-ene-11,13-diyn-2-one (1) and 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (2). Two dicarbonylic constituents, namely pentadeca-(9E)-ene-11,13-diyne-2,8-dione (3) and pentadeca-(9E,13Z)-dien-11-yne-2,8-dione (4), were isolated and characterized for the first time. Furthermore, the structure elucidation of pentadeca-(8Z,13Z)-dien-11-yn-2-one (5) is described. The structure of the compounds isolated was determined on the basis of UV, IR, NMR (including 1D and 2D NMR experiments, such as 1H-1H gCOSY, gHSQC-DEPT, gHMBC, gNOESY) and MS spectroscopic data. The cytotoxic activity of the isolated constituents against MIA PaCa-2 human pancreatic adenocarcinoma cells was evaluated in the concentration range 1-100 microg/ml. Results show that the hydroxylated compounds (1, 2) have low cytotoxicity, while the more hydrophobic polyacetylenes (3) and polyenes (4, 5) displayed moderate activity.     

Metal complexes of naphthoquinone based ligand: synthesis,characterization, protein binding,DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (10⁵ M^?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (K_app 10⁵ M^?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.     

TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level

Tributyltin (TBT) exposure at different concentrations (5, 60, and 100 ng TBT as Sn/l) induces a concentration- and time-dependent imposex (=pseudohermaphroditism) development in femaleNucella lapillus andHinia reticulata. In both species the average imposex stage, termed as vas deferens sequence (VDS) index, and the average female penis length increases with increasing TBT concentration and duration of TBT exposure. Testosterone added at a concentration of 500 ng/l induces a faster and more intensive imposex development compared to that induced by the TBT concentrations used in the present experiments. Radioimmunological determination of endogenous steroid content reveals increasing testosterone titres in female gastropods exposed to TBT which correlate with the TBT concentration used and the duration of the experiment. The most marked and highest increase of the endogenous testosterone level is exhibited by females, of both species exposed to testosterone. Simulataneous exposure to TBT and to the antiandrogen cyproterone acetate which suppresses imposex development completely inN. lapillus and reduces imposex development strongly inH. reticulata proves that the imposex-inducing effects of TBT are mediated by an increasing androgen level and are not caused directly by the organotin compound itself. Further-more, TBT-induced imposex development can be suppressed in both snails by adding estrogens to the aqueous medium. These observations suggest that TBT causes an inhibition of the cytochrome P-450 dependent aromatase system which catalyses the aromatization of androgens to estrogens. The increase of the androgen content or the shift of the androgen-estrogen balance in favour of androgens induces the development of pseudohermaphroditism in marine prosobranchs. Artificial inhibition of the cytochrome P-450 dependent aromatase system using SH 489 (1-methyl-1,4-androstadiene-3,17-dione) as a steroidal aromatase inhibitor and flavone as a nonsteroidal aromatase inhibitor induces imposex development inN. lapillus as well as inH. reticulata.