A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

Cleavage of dT8 and dT8 phosphorothioyl analogues by Escherichia coli DNA topoisomerase I: product and rate analysis

Escherichia coli DNA topoisomerase I catalyzes the cleavage of short, single-stranded oligodeoxynucleotides with dT8 as the shortest cleavable oligo(thymidylic acid). The 5'-32P-labeled products formed from the cleavage of [5'-32P]dT8 are dT5, dT4, and dT3 with over 70% of the substrate cleaved to dT4. Mg(II) ions affect this product distribution by increasing the percentage of dT4 formed. The substitution of a sulfur atom for a nonbridging oxygen atom in a phosphodiester linkage yields oligodeoxynucleotide phosphorothioyl (PS) analogues. The epimers of the analogues were separated, and the position and stereochemistry of the phosphorothiodiester bond were determined. Topoisomerase I is stereospecific in its reactivity toward these analogues. With the oligodeoxynucleotide PS analogue substrates, the rate of cleavage, the stereospecificity, and the product distribution depend upon the position and the stereochemistry of the phosphorothiodiester linkage.     

A statistical method for correlating tRNA sequence with amino acid specificity.

A statistical method for finding the nucleotide positions in tRNA sequences that correlate with amino acid specificity has been developed. The procedure involves finding the subset of nucleotide positions and groups of positions where the marginal density of one amino acid tRNA class does not overlap that of any other amino acid class. The procedure is an application of a statistical method known as the Expectation Maximization algorithm.     

Developmental Patterns of Catechol-O-Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies

Abstract: Catechol- O -methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50–60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011