Evaluation of attractant flavours for use in oral vaccine baits for badgers (<Emphasis Type="Italic">Meles meles</Emphasis>)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis infection (tuberculosis) in Ireland and the UK and are implicated in the transmission of infection to livestock. Vaccination of badgers with the human BCG vaccine (Bacille Calmette Guerin) is considered as an important strategy to reduce the burden of disease in this species, and a pragmatic approach is likely to involve oral vaccination. In this study, we evaluated nine different flavours for use as attractants in a prototype oral vaccine bait for European badgers (M. meles): aniseed, apple, cocoa powder, carob powder, curry, fish, garlic, peanut and strawberry. The bait matrix was composed of a natural lipid formulation, developed as a vehicle for oral vaccination against tuberculosis in wildlife. A ‘food for work’ paradigm was employed during the trials to ensure the animals were actively seeking the baits. The trials showed carob and cocoa powders were equally attractive and more attractive than any of the other candidates. Carob and cocoa show potential as bait attractants for badgers and might form part of a novel vaccine delivery system.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.     

Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.     

1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes

Photobiomodulation (PBM) is a non‐plant‐cell manipulation through a transfer of energy by means of light sources at the non‐ablative or thermal intensity. Authors showed that cytochrome‐c‐oxidase (complex IV) is the specific chromophore's target of PBM at the red (600‐700 nm) and NIR (760‐900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3‐hydroxyacyl‐CoA dehydrogenase, an enzyme involved in the β‐oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm² to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm². Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe‐S proteins and Cu‐centers of respiratory complexes and with the water molecules could be supposed.      

Guards and thieves: antagonistic interactions between two ant species coexisting on the same ant-plant

Abstract.  1. The simultaneous occupation of a rare understorey ant-acacia Acacia mayana by its guarding ant Pseudomyrmex ferrugineus , and an apparent opportunist parasite of the mutualism, the generalist ant Camponotus planatus is described. The two ant species occur together in 30.7% of the 26 mature A. mayana plants [23.5% of all trees ( n  = 34)] surveyed, but C. planatus is absent from saplings below 1 m in height ( n  = 8).
2. While P. ferrugineus shows behaviour compatible with effective host-tree defence, C. planatus does not attack phytophagous insects and appears ineffective as an ant-guard. Camponotus planatus does, however, occupy swollen thorns (pseudogalls) on the host tree, and harvests nectar from extrafloral leaf nectaries. It is proposed that C. planatus is a parasite of the Acacia–Pseudomyrmex mutualism.
3. Camponotus planatus does not harvest the second trophic reward produced by the tree for its Pseudomyrmex ant-guards, protein-rich food (Beltian) bodies. Camponotus planatus lack the specialised larval adaptations needed to use Beltian bodies as brood food, suggesting that this resource is potentially more resistant to exploitation by generalists than extrafloral nectar.
4. In competition for access to nectaries, C. planatus effectively displaced P. ferrugineus in 99.8% of encounters. These results suggest not only that C. planatus is a parasite of this mutualism, but also that it is able to effectively counteract the aggression shown to other insects by the resident ant-guards.     

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and wobbler mice

Summary 1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Givenin vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [³H]dexamethasone binding when addedin vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.     

Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hyg^r) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hyg^r–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hyg^r stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.     

The renal urate transporter SLC17A1 locus: confirmation of association with gout

Introduction

Two major gout-causing genes have been identified, the urate transport genes SLC2A9 and ABCG2. Variation within the SLC17A1 locus, which encodes sodium-dependent phosphate transporter 1, a renal transporter of uric acid, has also been associated with serum urate concentration. However, evidence for association with gout is equivocal. We investigated the association of the SLC17A1 locus with gout in New Zealand sample sets.

Methods

Five variants (rs1165196, rs1183201, rs9358890, rs3799344, rs12664474) were genotyped across a New Zealand sample set totaling 971 cases and 1,742 controls. Cases were ascertained according to American Rheumatism Association criteria. Two population groups were studied: Caucasian and Polynesian.

Results

At rs1183201 (SLC17A1), evidence for association with gout was observed in both the Caucasian (odds ratio (OR) = 0.67, P = 3.0 × 10^-6) and Polynesian (OR = 0.74, P = 3.0 × 10^-3) groups. Meta-analysis confirmed association of rs1183201 with gout at a genome-wide level of significance (OR = 0.70, P = 3.0 × 10^-8). Haplotype analysis suggested the presence of a common protective haplotype.

Conclusion

We confirm the SLC17A1 locus as the third associated with gout at a genome-wide level of significance.