Early expression of antiinsulin autoantibodies of humans and the NOD mouse: Evidence for early determination of subsequent diabetes

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16–18 weeks of age (NOD mice IAA+ at 8 weeks: 83% (5/6) diabetic by 18 weeks versus 11% (1/9) of IAA negative at 8 weeks; P < .01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively “fixed” early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.