Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.     

Preparation and functional properties of protein coprecipitate from sheep milk

The optimum conditions for the production of coprecipitate from sheep milk were studied. The best percentage of calcium chloride added to milk was 0.2%, which resulted in a recovery of 97.5% of milk proteins. At low pH (4.5–5), the recovery of protein was low, but it increased at higher pH values (5.5–6.5); recovery was greatest at pH 6.5. The optimum heating temperature to obtain coprecipitate from sheep milk was 85–95°C. The functional properties of the sheep milk coprecipitate were studied. At pH values higher than 6, there were no differences between the solubility of sheep milk coprecipitate and sheep milk sodium caseinate, but the solubility of coprecipitate at pH values lower than 5 was relatively higher than those of the caseinate. At pH ≥6, the emulsion activity index (EAI) for emulsions of sheep milk coprecipitate and caseinate increased as pH increased; at all pH values, the EAI of the coprecipitate was higher than that of the caseinate. Sheep milk coprecipitate showed higher foaming ability and stability than sheep milk sodium caseinate.     

The material basis for reduced mechanical properties in oim mice bones

Osteogenesis imperfecta (OI), a heritable disease caused by molecular defects in type I collagen, is characterized by skeletal deformities and brittle bones. The heterozygous and homozygous oim mice (oim/+ and oim/oim) exhibit mild and severe OI phenotypes, respectively, serving as controlled animal models of this disease. In the current study, bone geometry, mechanics, and material properties of 1-year-old mice were evaluated to determine factors that influence the severity of phenotype in OI. The oim/oim mice exhibited significantly smaller body size, femur length, and moment of area compared with oim/+ and wild-type (+/+) controls. The oim/oim femur mechanical properties of failure torque and stiffness were 40% and 30%, respectively, of the +/+ values, and 53% and 36% of the oim/+ values. Collagen content was reduced by 20% in the oim/oim compared with +/+ bone and tended to be intermediate to these values for the oim/+. Mineral content was not significantly different between the oim/oim and +/+ bones. However, the oim/oim ash content was significantly reduced compared with that of the oim/+. Mineral carbonate content was reduced by 23% in the oim/oim bone compared with controls. Mineral crystallinity was reduced in the oim/oim and oim/+ bone compared with controls. Overall, for the majority of parameters examined (geometrical, mechanical, and material), the oim/+ values were intermediate to those of the oim/oim and +/+, a finding that parallels the phenotypes of the mice. This provides evidence that specific material properties, such as mineral crystallinity and collagen content, are indicative and possibly predictive of bone fragility in this mouse model, and by analogy in human OI.