The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 Å by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of 60 Å · 40 Å · 50 Å and an overall b-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the ureadenatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.

Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. beta-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss.

The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid ) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.

Nanotechnology has vast potential for expanded development and novel application in numerous sectors of society. With growing use and applications, substantial production volumes and associated environmental release can be anticipated. Exposure effect of nanoparticles (NP) on biological systems may be intrinsic to their physicochemical properties introducing unknown associated risk. Herein, we expand the knowledge of health and environmental impact of silver nanoparticles (AgNPs), testing the acute toxicity of 14 AgNP preparations on developing zebrafish embryos (Danio rerio). Toxicological end points, including mortality, hatching rate, and heart rate were recorded. Concentration, stabilization agent and physicochemical properties were monitored as contributing outcome factors. Our findings indicate wide ranging LC50 24 h postfertilization values (0.487 ppm (0.315, 0.744 95% CI) to 47.89 ppm (18.45, 203.49 95% CI)), and indicate surface charge and ionic dissolution as key contributory factors in AgNP exposure outcome.

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play antiapoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.