Effect of caffeine on genes expressions of developing retinas in the chick model.

It has been reported that overconsumption of caffeine during pregnancy leads to a deleterious effect within the nervous tissues during embryonic development. In this study, we further extrapolated the effect of caffeine in the developing retinas, which is known to be one of the most sensitive tissues in chick embryos. Morphological changes of retinal thickness and organization of neuroretinal epithelium were monitored using three gene markers, Atoh7, FoxN4, and Lim1. Upon treating with a single dose of caffeine (15 µmol at embryonic day 1 [E1]), relative thicknesses of developing retinas (particularly of E7 and E9) were significantly altered. Among the three genes studied, the expression pattern of Atoh7 was notably altered while those of FoxN4, and Lim1 mRNA showed only a slight change in these developing retinas. Quantitative polymerase chain reaction results supported the most notable changes of Atoh7 but not FoxN4, and Lim1 gene in the developing retinas, particularly at E7. The effect of caffeine towards other organs during development should be extrapolated and the awareness of its intensive consumption should be raised.

Bone Morphogenetic Protein 2/4 in Mollusk, Haliotis diversicolor: Its Expression and Osteoinductive Function In Vitro.

Bone morphogenetic proteins (BMPs), which are members of the superfamily of transforming growth factor-ß (TGF-ß), are known both in vitro and in vivo for their osteoinduction properties on the osteoblastic cells. Its role in the mollusk shell formation has also been gradually established. Using Haliotis diversicolor as a model, we characterized the HdBMP2/4 gene in the mantle tissue and showed its expression in the outer fold epithelium (particularly at the periostracal groove) the epithelial site which is involved in shell formation, both prismatic and nacreous layers. Shell notching experiments following gene analysis by qPCR revealed the upregulation of the HdBMP2/4 gene up to 3.2-fold than that of the control animals. In vitro treatments of the preosteoblastic cells, MC3T3-E1 with HdBMP2/4 synthetic peptide demonstrated the enhanced effect of many osteogenic genes that are known to regulate bone and shell biomineralization including ALP, Runx2, and OCN with 2-4 fold-change throughout 14 days of culture. In addition, the increased deposition of calcium-based mineral (as assessed by Alizarin red staining) of the treated cells was comparable to the ascorbic acid (Vit C) + glycerophosphate positive control which revealed the enhanced effect of HdBMP2/4 peptide on matrix biomineralization of the preosteoblastic cells. In conclusion, these results indicated the presence of the HdBMP2/4 gene in the mantle tissue at the site involved in shell formation and the effect of the HdBMP2/4 knuckle epitope peptide in osteoinduction in vitro.

Characterization of Thrombospondin Type 1 Repeat in Haliotis diversicolor and Its Possible Role in Osteoinduction.

Thrombospondin repeats (TSR) are important peptide domains present in the sequences of many extracellular and transmembrane proteins with which a variety of ligands interact. In this study, we characterized HdTSR domains in the ADAMTS3 protein of Thai abalone, Haliotis diversicolor, based on the transcriptomic analysis of its mantle tissues. PCR amplification and localization studies demonstrated the existence of HdTSR transcript and protein in H. diversicolor tissues, particularly in both the inner and outer mantle epithelial folds. We, therefore, generated a short recombinant protein, termed HdTSR1/2, based on the existence of the WxxWxxW or WxxxxW motif (which binds to TGF-ß, a known signaling in bone formation/repair) in HdTSR1 and HdTSR2 sequences and used it to test the osteoinduction function in the pre-osteoblastic cell line, MC3T3-E1. This recombinant protein demonstrated the ability to induce the differentiation of MC3T3-E1 cells by the concentration- and time-dependent upregulation of many known osteogenic markers, including RUNX2, COL1A1, OCN, and OPN. We also demonstrated the upregulation of the SMAD2 gene after cell treatment with HdTSR1/2 proteinindicating its possible interaction through TGF-ß, which thus activates its downstream signaling cascade and triggers the biomineralization process in the differentiated osteoblastic cells. Together, HdTSR domains existed in an extracellular ADAMTS3 protein in the mantle epithelium of H. diversicolor and played a role in osteoinduction as similar to the other nacreous proteins, opening up its possibility to be developed as an inducing agent of bone repair.