Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lystate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4- and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.     

Diversification in insular plants: inferring the phylogenetic relationship in Aeonium (Crassulaceae) using ITS sequences of nuclear ribosomal DNA

The ITS regions of nuclear ribosomal DNA were sequenced in 37 species of the genus Aeonium . A phylogeny obtained through the use of parsimony agrees to some extent with the sectional division of the genus and confirms the position of two newly described species. It also suggests the potential importance of reticulate evolution in the genus. Based on the geographic distribution of this particular island group and the growth forms of its species, dispersal across similar ecological zones of different islands followed by adaptive radiation and isolation are suggested to be the prominent routes in speciation. As is typical of island genera, large polytomies at the distal nodes in the phylogeny indicate a recent rapid radiation.     

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.     

Reconstructing paternal genotypes to infer patterns of sperm storage and sexual selection in the hawksbill turtle

Postcopulatory sperm storage can serve a range of functions, including ensuring fertility, allowing delayed fertilization and facilitating sexual selection. Sperm storage is likely to be particularly important in wide‐ranging animals with low population densities, but its prevalence and importance in such taxa, and its role in promoting sexual selection, are poorly known. Here, we use a powerful microsatellite array and paternal genotype reconstruction to assess the prevalence of sperm storage and test sexual selection hypotheses of genetic biases to paternity in one such species, the critically endangered hawksbill turtle, Eretmochelys imbricata. In the majority of females (90.7%, N = 43), all offspring were sired by a single male. In the few cases of multiple paternity (9.3%), two males fertilized each female. Importantly, the identity and proportional fertilization success of males were consistent across all sequential nests laid by individual females over the breeding season (up to five nests over 75 days). No males were identified as having fertilized more than one female, suggesting that a large number of males are available to females. No evidence for biases to paternity based on heterozygosity or relatedness was found. These results indicate that female hawksbill turtles are predominantly monogamous within a season, store sperm for the duration of the nesting season and do not re‐mate between nests. Furthermore, females do not appear to be using sperm storage to facilitate sexual selection. Consequently, the primary value of storing sperm in marine turtles may be to uncouple mating and fertilization in time and avoid costly re‐mating.     

New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins

Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases.     

An assessment of ADAMs in bone cells: absence of TACE activity prevents osteoclast recruitment and the formation of the marrow cavity in developing long bones

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.