Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

Background  

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.     

Circulating parasite antigen in Brugia pahangi-infected jirds

The Mongolian jird is used widely in filariasis research for studies of protective immunity, pathogenesis, and therapy. The purpose of this study was to evaluate parasite antigen detection as a means of noninvasively monitoring Brugia pahangi infection in jirds. A parasite antigen with Mr of 105-110 kDa was identified in sera from i.p.- and s.c.-infected jirds by immunoblot with a monoclonal antibody to phosphorylcholine. The same antibody was used in a direct sandwich enzyme immunoassay to measure antigen in jird sera. Parasite antigen was detectable as early as 2 wk after i.p. or s.c. injection of L3. Antigen titers increased between 2 and 12 wk and stabilized between 12 and 36 wk after infection in s.c.-infected animals. A different pattern was seen in i.p.-infected jirds with antigen titers peaking at 16 wk and falling significantly between 16 and 32 wk after infection. Parasite antigen titers correlated significantly with adult worm infection intensities in jirds with mature i.p. and s.c. infections. Antigenemia was also detectable in sera from jirds after i.p. implantation of adult parasites of either sex. However, antigen was not detected in sera from infant offspring of antigenemic infected mothers. We conclude that parasite antigen detection allows B. pahangi development and survival as well as infection intensity to be monitored in living animals with unprecedented sensitivity and accuracy. This technique should facilitate drug and vaccine studies in this important experimental filariasis model.     

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioactivation and Toxicity of Acetaminophen in a Rat Hepatocyte Micropatterned Coculture System

We have recently shown that primary rat hepatocytes organized in micropatterned cocultures with murine embryonic fibroblasts (HepatoPac?) maintain high levels of liver functions for at least 4 weeks. In this study, rat HepatoPac was assessed for its utility to study chemical bioactivation and associated hepatocellular toxicity. Treatment of HepatoPac cultures with acetaminophen (APAP) over a range of concentrations (0–15 mM) was initiated at 1, 2, 3, or 4 weeks followed by the assessment of morphological and functional endpoints. Consistent and reproducible concentration‐dependent effects on hepatocyte structure, viability, and basic functions were observed over the 4‐week period, and were exacerbated by depleting glutathione using buthionine sulfoximine or inducing CYP3A using dexamethasone, presumably due to increased reactive metabolite‐induced stress and adduct formation. In conclusion, the results from this study demonstrate that rat HepatoPac represents a structurally and functionally stable hepatic model system to assess the long‐term effects of bioactivated compounds. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:471‐478, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21512     

Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin

Toxin A, one of several virulence factors secreted by the gram-negative bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precursor with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside within the region defined by the toxin A LP and the first 30 amino acids (aa) of mature toxin A. In the present study, we have used exonuclease Ba131 deletion analysis to examine the specific role of the first 30 as in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue region fused to a toxin A carboxy-terminal region, were identified. In addition, a gene fusion encoding a hybrid protein consisting of the LP of P. aeruginosa elastase and the final 305 residues of toxin A, was generated. The cellular location of the toxA subclone products in P. aeruginosa was determined by immunoblotting analysis. Toxin A CRMs (cross-reacting material) encoded by different subclones were detected in different fractions of P. aeruginosa including the periplasm and the supernatant. Results from these studies suggest that (1) mature toxin A contains two separate secretion signals one within the N-terminal region and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.     

Brugia pahangi: circulating antibodies to adult worm antigens in uninfected progeny of homologously infected female jirds

Serum IgG antibody levels to adult Brugia pahangi antigens were measured in uninfected offspring from uninfected and B. pahangi-infected female jirds. Antibody titers to B. pahangi antigens in sera of offspring from infected females mimicked the maternal titer during the suckling period. Neonate titers peaked at 2 weeks of age at levels as high as 1:4100, then decreased to levels well below maternal titers by 8-12 weeks of age. Concurrent maternal and 2-week-old neonate sera recognized identical B. pahangi antigens in Western blots. Spleen cells from 2-week-old filariae-exposed and unexposed offspring failed to produce measurable antibody to B. pahangi in vitro. Progeny of uninfected mothers nursed by B. pahangi-infected females showed circulating IgG antibody titers to adult worm antigens similar to those of homologously reared offspring. Conversely, offspring born to B. pahangi-infected females and nursed by an uninfected female had no serum antibodies to B. pahangi antigens. Blastogenic responses of spleen cells to the mitogens phytohemagglutinin and pokeweed mitogen, and adult B. pahangi antigens, were not different between offspring groups. Mean areas of pulmonary granulomas induced by the intravenous inoculation of B. pahangi antigen-coated beads also did not differ between 4- and 8-week-old progeny of uninfected or infected females. These results suggest that the circulating IgG antibodies to adult B. pahangi antigens demonstrated in offspring of infected female jirds are maternally derived via the milk and do not alter the cellular responses of uninfected offspring to B. pahangi antigens as measured by antigen-stimulated blastogenesis or pulmonary granulomatous inflammatory response.