Sex determination in insects: a binary decision based on alternative splicing

The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks.     

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.     

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.     

In vitro and in vivo evaluation of intravesical docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols in an orthotopic model of bladder cancer

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.