Human immunodeficiency virus type 1 protease regulation of tat activity is essential for efficient reverse transcription and replication

The human immunodeficiency virus type 1 (HIV-1) Tat protein enhances reverse transcription, but it is not known whether Tat acts directly on the reverse transcription complex or through indirect mechanisms. Since processing of Tat by HIV protease (PR) might mask its presence and, at least in part, explain this lack of data, we asked whether Tat can be cleaved by PR. We used a rabbit reticulocyte lysate (RRL) system to make Tat and PR. HIV-1 PR is expressed as a Gag-Pol fusion protein, and a PR-inactivated Gag-Pol is also expressed as a control. We showed that Tat is specifically cleaved in the presence of PR, producing a protein of approximately 5 kDa. This result suggested that the cleavage site was located in or near the Tat basic domain (amino acids 49 to 57), which we have previously shown to be important in reverse transcription. We created a panel of alanine-scanning mutations from amino acids 45 to 54 in Tat and evaluated functional parameters, including transactivation, reverse transcription, and cleavage by HIV-1 PR. We showed that amino acids 49 to 52 (RKKR) are absolutely required for Tat function in reverse transcription, that mutation of this domain blocks cleavage by HIV-1 PR, and that other pairwise mutations in this region modulate reverse transcription and proteolysis in strikingly similar degrees. Mutation of Tat Y47G48 to AA also down-regulated Tat-stimulated reverse transcription but had little effect on transactivation or proteolysis by HIV PR, suggesting that Y47 is critical for reverse transcription. We altered the tat gene of the laboratory strain NL4-3 to Y47D and Y47N so that overlapping reading frames were not affected and showed that Y47D greatly diminished virus replication and conveyed a reverse transcription defect. We hypothesize that a novel, cleaved form of Tat is present in the virion and that it requires Y47 for its role in support of efficient reverse transcription.

Sesamin modulation of lipid class and fatty acid profile in early juvenile teleost, Lates calcarifer, fed different dietary oils

Sesamin, a major sesame seed lignan, has diverse biological functions including the modulation of molecular actions in lipid metabolic pathways and reducing cholesterol levels. Vertebrates have different capacities to biosynthesize long-chain PUFA from dietary precursors and sesamin can enhance the biosynthesis of ALA to EPA and DHA in marine teleost. Early juvenile barramundi, Lates calcarifer, were fed for two weeks on diets rich in ALA or SDA derived from linseed or Echium plantagineum, respectively. Both diets contained phytosterols and less cholesterol compared with a standard fish oil-based diet. The growth rates were reduced in the animals receiving sesamin regardless of the dietary oil. However, the relative levels of n-3 LC-PUFA in total lipid, but not the phospholipid, increased in the whole body by up to 25% in animals fed on sesamin with ALA or SDA. Sesamin reduced the relative levels of triacylglycerols and increased polar lipid, and did not affect the relative composition of phospholipid subclasses or sterols. Sesamin is a potent modulator for LC-PUFA biosynthesis in animals, but probably will have more effective impact at advanced ages. By modulating certain lipid metabolic pathways, sesamin has probably disrupted the body growth and development of organs and tissues in early juvenile barramundi.

The growth performance of Jade Tiger cultured abalone fed diets supplemented with fish oil and vegetable oils

Background
The effects of fish oil (FO) supplementation and the dietary replacement of FO with flaxseed oil (FlaxO) and canola oil (CO) on the growth of cultured abalone was investigated. The study involved three growth experiments: (E1) diets containing 0.5, 1.0, 1.5, 2.0 and 2.5% of FO, respectively; (E2) diets in which FO was serially replaced by 25, 50, 75 and 100% FlaxO, respectively; and (E3) diets in which FO was serially replaced by 25, 50, 75 and 100% CO, respectively.

Results
In Experiment 1, abalone fed a diet supplemented with 1.5% FO showed a significantly higher (121.2 ± 1.1 mg day−1) daily growth rate of weight (DGRw) compared to control (70.1 ± 1.71 mg day−1). In Experiment 2, abalone fed 1.5% FO diet and diets containing 25–75% FlaxO showed no significant differences in DGRw. The diet containing 100% FlaxO showed significantly lower (63.3 ± 6.7 mg day−1) DGRw. In Experiment 3, abalone fed diets containing 25% and 50% CO showed similar DGRw as those fed a 1.5% FO diet. The diet containing 75% and 100% CO showed significantly lower (63.7 ± 5.0 to 95.4 ± 5.1 mg day−1) DGRw.

Conclusion
Supplementation with 1.5% of dietary FO can improve growth performance in cultured abalone. It is feasible to replace 75% of dietary FO with FlaxO and 50% of dietary FO with CO, without negative effect on growth performance.

Bone marrow chimeras and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Molecular sex-typing in shorebirds: a review of an essential method for research in evolution, ecology and conservation

Knowing the correct sex of individuals is essential both for research in evolutionary ecology and for practical conservation. Recent molecular advances have produced cheap, quick and reliable methods for sexing birds including chicks, juveniles, immatures and adults. Shorebird researchers have not yet fully utilised these advances. Here we provide an overview of work in this area to date with two objectives: (i) to review the major applications of molecular sexing and findings of shorebird research so far, and (ii) to provide an essential guide on how to carry out molecular sexing using current methods whilst avoiding methodological pitfalls. We encourage shorebird researchers to make better use of molecular sex-typing techniques in studies of conservation, migration, foraging ecology and breeding behaviour.

Abolishing defensive homicide offence essential

The Victorian government's proposal to abolish the offence of defensive homicide is a welcome step forward for a law that has been plagued by controversy since the abolition in 2005 of the partial defence of provocation. The offence of defensive homicide was introduced in November 2005 in response to concerns that the abolition of provocation would disadvantage those who killed in response to prolonged family violence.