The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.     

In vivo investigations on luteotropic activity of prostaglandins during early diestrus in nonpregnant bitches

The aim of this study was to test for the postulated luteotropic effect of prostaglandin E2 during early diestrus in the dog in an in vivo study. This study was performed on 30 bitches which were randomly assigned to a treatment group (TG) and a control group. Starting on the day of ovulation (Day 0), dogs of the TG were treated for 5, 10, 20, or 30 days with 10 mg firocoxib/kg body weight per day (Previcox, a selective PTGS2 inhibitor) and ovariohysterectomized for collection of corpora lutea on the last day of treatment. Similarly, dogs of the control group were ovariohysterectomized on Days 0, 5, 10, 20, and 30. Blood samples for progesterone measurement were collected every second day; additionally, the area of luteal cell nuclei and the expression of 3β-hydroxysteroid-dehydrogenase at the mRNA and the protein levels were assessed. Mean P4 concentrations were lower in TGs; however, a significant difference was only observed on Day 10. This observation is in line with the finding that treatment with firocoxib reduced expression of 3β-hydroxysteroid-dehydrogenase mRNA and protein (P < 0.05) and the area of luteal cell nuclei (P < 0.05). The results of this study further point to the postulated luteotropic function of prostaglandin E2.     

Phylogeography, phylogeny and hybridization in trichechid sirenians: implications for manatee conservation

Abstract The three living species of manatees, West Indian (Trichechus manatus), Amazonian (Trichechus inunguis) and West African (Trichechus senegalensis), are distributed across the shallow tropical and subtropical waters of America and the western coast of Africa. We have sequenced the mitochondrial DNA control region in 330 Trichechus to compare their phylogeographic patterns. In T. manatus we observed a marked population structure with the identification of three haplotype clusters showing a distinct spatial distribution. A geographic barrier represented by the continuity of the Lesser Antilles to Trinidad Island, near the mouth of the Orinoco River in Venezuela, appears to have restricted the gene flow historically in T. manatus. However, for T. inunguis we observed a single expanding population cluster, with a high diversity of very closely related haplotypes. A marked geographic population structure is likely present in T. senegalensis with at least two distinct clusters. Phylogenetic analyses with the mtDNA cytochrome b gene suggest a clade of the marine Trichechus species, with T. inunguis as the most basal trichechid. This is in agreement with previous morphological analyses. Mitochondrial DNA, autosomal microsatellites and cytogenetic analyses revealed the presence of hybrids between the T. manatus and T. inunguis species at the mouth of the Amazon River in Brazil, extending to the Guyanas and probably as far as the mouth of the Orinoco River. Future conservation strategies should consider the distinct population structure of manatee species, as well as the historical barriers to gene flow and the likely occurrence of interspecific hybridization.     

The P2X7 receptor–pannexin connection to dye uptake and IL-1β release

The P2X₇ receptor (P2X₇R) is uniquely associated with two distinct cellular responses: activation of a dye-permeable pathway allowing passage of molecules up to 900 Da and rapid release of the pro-inflammatory cytokine, interleukin-1β (IL-1β), from activated macrophage. How this dye uptake path forms and whether it is involved in IL-1β release has not been known. Pannexin-1 is a recently identified protein found to physically associate with the P2X₇R. Inhibition of pannexin-1 does not alter P2X₇R ion channel activation or associated calcium flux but blocks one component of P2X₇R-induced dye uptake and unmasks a slower, previously undetected, dye uptake pathway. Inhibition of pannexin-1 blocks P2X₇R-mediated IL-1β release from macrophage as well as release mediated by other stimuli which couple to activation of capase-1 and additionally inhibits the release of interleukin-1α, a member of the IL-1 family whose processing does not require caspase-1 activation. Thus, pannexin-1 is linked to both dye uptake and IL-1β release but via distinct mechanisms.     

Biosynthesis of multiple forms of β-endorphin in the reptile intermediate pituitary

Fractionation of the β-endorphin-sized material from freshly dissected reptile intermediate pituitaries by ion exchange chromatography on sulfopropyl Sephadex (SP) revealed at least three distinct forms of immunoreactive β-endorphin. These forms eluted at 0.25 M NaCl, 0.28 M NaCl, and 0.32 M NaCl and represent respectively, 6%, 65% and 29% of the total immunoreactivity. Only the 0.28 M NaCl peak and the 0.32 M NaCl peak exhibited naloxone reversible opiate bioactivity when tested in the isolated guinea pig ileum bioassay system; taking into account the molar amount of immunoreactive peptides the 0.32 M NaCl peak was 6 fold more potent than the 0.28 M NaCl peak. Intermediate pituitaries in culture were incubated with either [³H]tyrosine, [³H]arginine, or [³⁵S]methionine for periods up to 24 hours and β-endorphin-sized peptides were prepared by immunoprecipitation and gel filtration. Fractionation of the labeled β-endorphin-sized peptides by ion exchange chromatography yielded profiles nearly identical to the immunoassay analyses of freshly dissected tissue. Further analysis of the major labeled forms of reptile β-endorphin by chromatography on Sephadex G-50 equilibrated in 6 M guanidine HCl indicated that the 0.32 M NaCl peak had an apparent molecular weight of 3500±100 and the 0.28 M NaCl peak had an apparent molecular weight of 3200±100. Furthermore, pulse/chase experiments showed that the 0.32 M NaCl peak was the precursor for the 0.28 M NaCl peak. These results coupled with the relative opiate bioactivities of the major forms argue that the principal post-translational modification of reptile β-endorphin is COOH-terminal proteolytic cleavage.