Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

The lobatamides, novel cytotoxic macrolides from southwestern Pacific tunicates

Novel macrolides, lobatamides A-F (1-6), have been isolated from shallow water Australian collections of Aplidium lobatum, from a deep water collection of Aplidium sp., and from an unidentified Philippine ascidian. Full details of the isolation and structure elucidation of 1-6 are provided herein, along with results and analyses of the testing of lobatamides A-D (1-4) in the NCI human tumor 60 cell-line screen. The lobatamides share a common core structure with the recently described salicylihalamides, which were isolated from a Haliclona sp. sponge. COMPARE analyses of the mean-graph differential cytotoxicity profiles of the lobatamides and the salicylihalamides showed high correlations with each other but not with members of the NCI's standard agents database. These compounds, therefore, appear to comprise a new mechanistic class, meriting further antitumor investigations.

Sustained gene expression in transplanted skin fibroblasts in rats

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

Friedmanniella spumicola sp nov and Friedmanniella capsulata sp nov from activated sludge foam: Gram-positive cocci that grow in aggregates of repeating groups of cocci

Two Gram-positive, non-motile, non-spore-forming, strictly aerobic, pigmented cocci, strains Ben 107(T) and Ben 108(T), growing in aggregates were isolated from activated sludge samples by micromanipulation. Both possessed the rare type A3 gamma' peptidoglycan. Major menaquinones of strain Ben 107(T) were MK-9(H-4) and MK-7(H-2), and the main cellular fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). In strain Ben 108(T), MK-9(H-4), MK-9(H-2) and MK-7(H-4) were the menaquinones and again the main fatty acid was 12-methyltetradecanoic acid (ai-C-15:0). Polar lipids in both strains consisted of phosphatidyl inositol, phosphatidyl glycerol and diphosphatidyl glycerol with two other unidentified glycolipids and phospholipids also present in both. These data, together with the 16S rDNA sequence data, suggest that strain Ben 107(T) belongs to the genus Friedmanniella which presently includes a single recently described species, Friedmanniella antarctica. Although the taxonomic status of strain Ben 108(T) is far less certain, on the basis of its 16S rRNA sequence it is also adjudged to be best placed in the genus Friedmanniella, The chemotaxonomic characteristics and DNA-DNA hybridization data support the view that Ben 107(T) and Ben 108(T) are novel species of the genus Friedmanniella. Hence, it is proposed that strain Ben 107(T) (=ACM 5121(T)) is named as Friedmanniella spumicola sp. nov. and strain Ben 108(T) (=ACM 5120(T)) as Friedmanniella capsulata sp. nov.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.