Techniques for targetting glycoproteins integral to the luminal membrane of the intestinal cells of Haemonchus contortus were used to isolate fractions of whole parasites with protective antigen potential. Sheep immunization trials with various candidate fractions revealed one which selectively bound to lectins with specificity for N-acetylgalactosamine and which reduced mean challenge worm burdens by up to 72% and mean faecal egg counts by up to 93%. The luminal surface of the intestines of the Haemonchus recovered from sheep immunized with this antigen were coated with host immunoglobulin, suggesting that the protective effect was due to antibodies interfering with the function of the gut. Further biochemical characterization of this fraction, which has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP complex), showed that it could be distinguished from previously described protective antigens of this parasite and that it was only detectable in detergent extracts of the worms.

Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function. Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes. Analysis of ⌬nrf1 and ⌬scd1 mutants revealed defects in endocytosis. In addition, overexpression of constitutively active Cdc42 G12V p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in ⌬nrf1 and ⌬scd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (P r -nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age-and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.

The generation of TCR proteins is the result of V(D)J recombinase-mediated genomic rearrangements at recombination signal sequences (RSS) in human lymphocytes. V(D)J recombinase can also mediate rearrangements at nonimmune or “cryptic” RSS in normal and leukemic human peripheral T cells. We previously demonstrated age- and gender-specific developmental differences in V(D)J coding joint processing at cryptic RSS within the HPRT locus in peripheral T cells from healthy children (Murray et al. 2006. J. Immunol. 177: 5393–5404). In this study, we investigated developmentally specific V(D)J recombinase TCRβ immune gene rearrangements and coding joint processing at RSS in peripheral T cells in the same pediatric population. This approach provided a unique opportunity to investigate site-specific V(D)J recombinase rearrangements and coding joint processing at immune and nonimmune genes from the same individual T cell population. We determined the genomic sequence of 244 TCRβ coding junctions from 112 (63 male, 49 female) subjects from the late stages of fetal development through 9 y of age. We observed both age- and gender-specific V(D)J recombinase-mediated TCRβ gene usage and coding joint processing at immune RSS. To the best of our knowledge, these data represent the first description of age- and gender-specific developmental differences in TCR gene usage and coding joint processing that could directly influence TCR diversity and immune specificity. It will be important for future studies to ascertain the mechanistic etiology of these developmental and gender differences in TCR diversity and specificity, as well as their importance with respect to the age and gender risks for infectious and autoimmune diseases in humans.

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.