Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier

Adenovirus vectors with E1 deleted of the human serotype 5 (AdHu5) and the chimpanzee serotype 68 (AdC68) expressing the glycoprotein of the Evelyn Rokiniki Abelseth strain of rabies virus were tested upon oral application for induction of systemic and mucosal transgene product-specific antibody responses in mice. Both vectors induced systemic and mucosal antibodies to rabies virus, including virus-neutralizing antibodies and protection against a severe intracerebral challenge with a mouse-adapted strain of rabies virus. Pre-existing immunity of AdHu5 virus, which dampens induction of transgene product-specific immunity elicited by AdHu5 vectors given systemically did not impair the response induced by oral vaccination. Oral priming-boosting regimens with either heterologous or homologous adenoviral vectors used sequentially increased both mucosal and systemic antibody titers to rabies virus [corrected]     

Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120

IgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.     

The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions

Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.     

Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.     

Detection of open and closed conformations of tryptophan synthase by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance of bound 1-15N-L-tryptophan

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.     

A multiple in silico program approach for the prediction of mutagenicity from chemical structure

We have conducted an evaluation of three of the most widely used commercial toxicity prediction programs, Toxicity Prediction by Komputer Assisted Technology (TOPKAT), Deductive Estimation of Risk from Existing Knowledge (DEREK) for Windows (DfW) and CASETOX. The three programs were evaluated for their ability to predict Ames test mutagenicity using 520 proprietary drug candidate (Test set 1) and 94 commercial (Test set 2) compounds. The study demonstrates that these three commercially available programs are useful, with limitations in their ability to predict mutagenicity over a wide range of chemical space, i.e. global predictivity. Individually, each of the programs performed at an acceptable level for overall accuracy, i.e. the ability to predict the correct outcome. However, analysis of the predictions indicates that the overall accuracy figure is heavily weighted by the ability of the programs to correctly predict non-mutagens, whereas none of the programs individually performed well in the prediction of novel mutagenic structures, i.e. Ames positive compounds. The performance of these programs' in predicting Ames positive mutagens appeared to be independent of the chemical utility of the compound, i.e. industrial, agricultural or pharmaceutical. The combination of program predictions provided some improvement in overall accuracy, sensitivity and specificity.     

Extensive female-mediated gene flow from sub-Saharan Africa into near eastern Arab populations

We have analyzed and compared mitochondrial DNA variation of populations from the Near East and Africa and found a very high frequency of African lineages present in the Yemen Hadramawt: more than a third were of clear sub-Saharan origin. Other Arab populations carried approximately 10% lineages of sub-Saharan origin, whereas non-Arab Near Eastern populations, by contrast, carried few or no such lineages, suggesting that gene flow has been preferentially into Arab populations. Several lines of evidence suggest that most of this gene flow probably occurred within the past approximately 2,500 years. In contrast, there is little evidence for male-mediated gene flow from sub-Saharan Africa in Y-chromosome haplotypes in Arab populations, including the Hadramawt. Taken together, these results are consistent with substantial migration from eastern Africa into Arabia, at least in part as a result of the Arab slave trade, and mainly female assimilation into the Arabian population as a result of miscegenation and manumission.