Expressed sequence tags from normalized cDNA libraries prepared from gill and hypodermal tissues of the blue crab, Callinectes sapidus.

Expressed sequence tags (ESTs) were produced from two normalized cDNA libraries from the blue crab, Callinectes sapidus. The gill library represented pooled RNA from respiratory and transporting gills after acclimation to either high or low salinity. The hypodermis library was from arthrodial and dorsal tissue from both pre- and post-molt crabs. Random clones were single-pass sequenced from the 5'-ends, resulting in 11,761 high quality ESTs averaging 652 bases. All the ESTs were assembled using Paracel Transcript Assembler software, producing 2176 potential transcripts-883 contigs and 1293 singlets. Of these, 1235 (56.7%) were sequenced only from the gill library, while 578 (26.6%) were exclusively hypodermal. There were 363 contigs containing ESTs from both tissues (16.7% of the putative transcripts). All contigs and singlets were compared to the public protein database using BLASTx, and descriptions of the three most similar proteins for each were recorded. Additional annotations included an Interpro analysis of protein domains and a listing of Gene Ontology (GO) categories inferred from similar proteins in GO-annotated databases. All sequences are available on a web page (http://firedev.bear.uncw.edu:8080/shaferlab/). The annotations can be searched, and BLAST alignment of user-inputted sequences against the putative transcripts is possible. In addition, the ESTs have been submitted to GenBank.

Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization.

A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.

Evidence that ganglion cells react to retinal detachment.

Growth associated protein 43 (GAP 43) is involved in synapse formation and it is expressed in the retina in a very specific pattern. Although GAP 43 is downregulated at the time of synapse formation, it can be re-expressed following injury such as axotomy or ischemia. Because of this we sought to characterize the expression of GAP 43 after retinal detachment (RD). Immunoblot, immunocytochemical and quantitative polymerase chain reaction (QPCR) techniques were used to assess the level of GAP 43 expression after experimental RD. GAP 43 was localized to three sublaminae of the inner plexiform layer of the normal retina. GAP 43 became upregulated in a subset of retinal ganglion cells following at least 7 days of RD. By immunoblot GAP 43 could be detected by 3 days. QPCR shows the upregulation of GAP 43 message by 6hr of detachment. To further characterize changes in ganglion cells, we used an antibody to neurofilament 70 and 200kDa (NF) proteins. Anti-NF labels horizontal cells, ganglion cell dendrites in the inner plexiform layer, and ganglion cell axons (fasicles) in the normal retina. Following detachment it is upregulated in horizontal cells and ganglion cells. When detached retina was double labelled with anti-GAP 43 and anti-NF, some cells were labelled with both markers, while others labelled with only one. We have previously shown that second order neurons respond to detachment; here we show that third order neurons are responding as well. Cellular remodelling of this type in response to detachment may explain the slow recovery of vision that often occurs after reattachment, or those changes that are often assumed to be permanent.