Methods of authenticating already canned fish were developed, using polymerase chain reaction (PCR) followed by sequencing and restriction site analysis. The canning process degrades DNA to fewer than 123 base pairs (bp) in length. Therefore, degenerate PCR primers were designed to amplify short (<123 bp) mitochondrial cytochrome b gene sequences known to differ at specific nucleotides among the species of interest. Sequences of canned tuna (Thunnus albacares, Thuunus alalunga, and Katsuwonus pelamis), bonito (Euthynnus affinis), and frigate mackerel (Auxis thazard) were reproducibly identified, and were used to determine which species or whether more than one species was present in individual cans. Restriction site analysis of two amplified regions of the cytochrome b gene demonstrated a faster and less expensive method than sequencing for distinguishing PCR products of different species. Thus, restriction site analysis of PCR products can be used in conjunction with sequencing to authenticate species in canned fish products. Keywords: Polymerase chain reaction; authentication; fish; tuna; bonito; Thunnus; Katsuwonus; Euthynnus; mitochondrial DNA; cytochrome b gene

Cardiac calsequestrin concentrates in junctional sarcoplasmic reticulum in heart and skeletal muscle cells by an undefined mechanism. During transit through the secretory pathway, it undergoes an as yet uncharacterized glycosylation and acquires phosphate on CK2-sensitive sites. In this study, we have shown that active calsequestrin phosphorylation occurred in nonmuscle cells as well as muscle cells, reflecting a widespread cellular process. To characterize this post-translational modification and resolve individual molecular mass species, we subjected purified calsequestrin to mass spectrometry using electrospray ionization. Mass spectra showed that calsequestrin glycan structure in nonmuscle cells was that expected for an endoplasmic reticulum-localized glycoprotein and showed that each glycoform existed as four mass peaks representing molecules that also had 0 -3 phosphorylation sites occupied. In heart, mass peaks indicated carbohydrate modifications characteristic of transit through Golgi compartments. Phosphorylation did not occur on every glycoform present, suggesting a far more complex movement of calsequestrin molecules in heart cells. Significant amounts of calsequestrin contained glycan with only a single mannose residue, indicative of a novel post-endoplasmic reticulum mannosidase activity. In conclusion, glyco-and phosphoforms of calsequestrin chart a complex cellular transport in heart, with calsequestrin following trafficking pathways not present or not accessible to the same molecules in nonmuscle.

We conclude that the point mutation D307H leads to a profoundly altered conformation that no longer responds normally to Ca(2+) and fails to bind normally to triadin and junctin.

Calsequestrin (CSQ) concentrates in junctional sarcoplasmic reticulum (SR) where it functions in regulation of Ca2+ release. When purified from heart tissue, cardiac CSQ contains phosphate on a cluster of C-terminal serine residues, but little is known about the cellular site of kinase action, and the identity of the kinase remains uncertain. To determine basic features of the phosphorylation, we examined the reaction in canine heart preparations. CSQ phosphorylation was observed in [32P]metabolically-labeled heart cells after adenoviral overexpression, and its constitutive phosphorylation was limited to a CK2-sensitive C-terminal serine cluster. The CSQ kinase was oriented intralumenally, as was CSQ, inside membrane vesicles, such that exposure to each required detergent permeabilization. Yet even after detergent permeabilization, CSQ was phosphorylated much less efficiently by protein kinase CK2 in cardiac microsomes than was purified CSQ. Reduced phosphorylation was strongly dependent upon protein concentration, and phosphorylation time courses revealed a phosphatase activity that occurred constitutively as phosphorylated substrate accumulates. Evidence of selective dephosphorylation of CSQ glycoforms in heart homogenates was also seen by mass spectrometry analysis. Molecules with greater mannose content, a feature of early secretory pathway compartments, were more highly phosphorylated, while greater dephosphorylation was apparent in more distal compartments. Taken together, the analyses of CSQ phosphorylation in heart suggest that a constitutive process of phosphate turnover occurs for cardiac CSQ perhaps associated with its intracellular transport.

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