Skeletal development of the chondrocranium in the tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae).

This study provides a comprehensive description of chondrocranial development before, during and after larval metamorphosis in the tongue sole Cynoglossus semilaevis, a commercially valuable flatfish in China. Samples were collected at regular intervals ranging from 1 to 23 days post hatching (dph). Based on observations of cleared and double-stained specimens and images from sections stained with safranin O-fast green, major morphological events during early development were described. No cartilaginous structure was visible at hatching. From 2 dph onwards, cartilaginous structures such as the trabecular bar and some elements of the mandibular, hyoid and branchial arches appeared. At this time also, cartilaginous structures of the neurocranium started to form. Hypertrophic chondrocytes could be observed in many splanchnocranium elements at 5 dph. The start of ossification was indicated by alizarin red stain visible at 14 dph. At 17 dph, most of the cartilaginous skeleton was ossified. Soon after, the right eye started to migrate and pass through a slit beneath the dorsal-fin base and above the skull. Metamorphosis was complete at 20 dph, at which time the dorsal-fin base cartilage extended onto the anterior region of the head. Meanwhile, extremities of the hyoid and branchial arch elements remained cartilaginous. At 23 dph, endochondral ossification of the splanchnocranium was nearly complete. Unlike previous observations of other Pleuronectiformes, our study indicates that endochondral ossification of C. semilaevis skull cartilage occurs before metamorphosis.

Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae.

PURPOSE: This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. METHODS: The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. RESULTS: Secretion of OX26 scFv was optimal when expression was induced at 20 degrees C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 +/- 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 +/- 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. CONCLUSIONS: The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood-brain barrier (BBB).