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The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V ([V.sub.H]) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

A pair of related classes of full-length coding cDNAs, termed perilipin A and B, from a rat adipocyte cDNA expression library was isolated using the anti-perilipin serum. Perilipin, a protein observed only at the surface of the lipid storage droplets, is the major cAMP-dependent protein kinase substrate in adipocytes.

Nurse shark Ginglymostoma cirratum's immunoglobulin light chain cDNA clone encoding a protein similitude of mammalian kappa light chains, was isolated and characterized. The results implicate the predating of divergent isotypes of L chain prototypes to much before the evolution of elasmobranchs. The homology between gene distributions in these two isotypes typifies the gene infrastructure corresponding to the prototype of L chain.

An experiment was conducted to study the mechanism of hormone stimulated lipolysis (HSL) in adipocytes. Polyclonal antiserum raised against HSL was examined using Western blotting of adipocyte homogenates and the results showed an enzyme shift from the supernatant of the control cells to the floating 'fat cake' of lipolytically stimulated cells. These findings indicate that hormone-stimulated lipolysis was caused by the shift of lipase to the surface of the lipid storage droplet rather than an increase in catalytic activity.