Search

Cocksfoot/orchard grass (Dactylis glomerata) anther cDNA clones encoding the group II allergen Dac g II were previously isolated on the basis of immunoreactivity of human, rabbit, and murine antibodies with a 24-kDa protein expressed as a fusion protein with beta-galactosidase. Nucleotide sequencing reveals an open reading frame predicting expression of a 98-amino-acid (11-kDa) polypeptide exhibiting > 90% homology with the group II allergen of Lolium perenne, Lol p II. In vitro translation of different sized clone fragments generated by polymerase chain amplification confirms eukaryotic expression of a 10-12-kDa polypeptide by SDS-PAGE and the position of a translational stop apparently unrecognized during expression of lambda gt11 in E. coli. The unusual characteristics of the prokaryote-expressed fusion proteins may be exerting conformational alterations in Dac g II, as reflected by previous demonstrations of differences in human IgE immunoreactivity. Northern blot analysis using PCR-generated partial and full-length probes suggests that group II allergens may be encoded by a different family or families of temporally expressed genes from those encoding group I major allergens, although a group I gene may have been the progenitor.

Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH.

Immobilization of oligonucleotides containing 5'-terminal thiol groups on thiol-terminated paramagnetic Biomag beads via disulphide bond formation was investigated. Oligonucleotides are demonstrated to couple at high yields, the linkage is stable at 90 degrees C and is reversible, and the immobilized oligonucleotide is available for complementary, but not non-complementary, hybridization. Specific hybridization capacity per micrograms of immobilized oligonucleotide exceeds that achieved with other forms of immobilization chemistries employing random attachment and/or specific end attachment of the oligonucleotide to the solid support. Adsorption of DNA on the surface of the beads was decreased by incubation in 0.2% SDS; other non-specific blocking agents had no effect. Brief heating of the beads possessing immobilized oligonucleotides at 90 degrees C before hybridization increased the amount of specific hybridization dependent upon the inclusion of poly(dT) spacer sequences 5' to the immobilized oligonucleotide and 3' to the thiol group. Increasing lengths of spacers [up to a poly(dT16) spacer] linearly increased hybridization of complementary sequences.

A method is described for simultaneously determining retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin in 0.25 mL of plasma. Plasma mixed with sodium dodecyl sulfate is deproteinized with ethanol containing tocopherol acetate, then extracted with heptane. The evaporated organic layer is reconstituted with mobile phase (methanol/acetonitrile/chloroform, 47/47/6 by vol) and injected onto a 100 x 4.6 mm 3-micron column of Spherisorb ODS-2 (LKB) at 1.5 mL/min. The alpha- and beta-carotenes are well resolved during the 6.5-min run. Retinol is monitored at 325 nm, the tocopherols at 292 nm, and the carotenoids at 450 nm. Extraction of concentrations as great as 135 mumol/L is complete. Intrabatch CVs were 1.7%, 2.3%, 4.1%, 10.4%, 6.4%, and 3.6% for retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin, respectively. Interbatch CVs for measurements on 30 occasions over 11 weeks were about 10% for all components except alpha-tocopherol (5.3%). Results agree well with those for retinol, alpha-tocopherol, and beta-carotene in quality-control samples.

Urinary protein excretion has been investigated using the iso-dalt technique in pregnant patients with hypertension and proteinuria, without hypertension but with proteinuria and without either hypertension or proteinuria. This high resolution electrophoretic technique has shown a great variation in the pattern of protein excreted by these patients, particularly those with hypertension. These showed an increased number of protein species in the urine, some of unknown identity. Two urines from this group appeared to contain no detectable albumin. Currently this technique is inappropriate for the routine analysis of urine proteins. However, further studies may determine the diagnostic value of these changes in urine excretion and allow the development of more specific assays.