Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Immunodominant tuberculosis CD8 antigens preferentially restricted by HLA-B

CD8(+) T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-gamma ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8(+) T cells recognizing known immunodominant CD4(+) T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8(+) T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8(+) T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics.     

Putting beta-diversity on the map: broad-scale congruence and coincidence in the extremes

Beta-diversity, the change in species composition between places, is a critical but poorly understood component of biological diversity. Patterns of beta-diversity provide information central to many ecological and evolutionary questions, as well as to conservation planning. Yet beta-diversity is rarely studied across large extents, and the degree of similarity of patterns among taxa at such scales remains untested. To our knowledge, this is the first broad-scale analysis of cross-taxon congruence in beta-diversity, and introduces a new method to map beta-diversity continuously across regions. Congruence between amphibian, bird, and mammal beta-diversity in the Western Hemisphere varies with both geographic location and spatial extent. We demonstrate that areas of high beta-diversity for the three taxa largely coincide, but areas of low beta-diversity exhibit little overlap. These findings suggest that similar processes lead to high levels of differentiation in amphibian, bird, and mammal assemblages, while the ecological and biogeographic factors influencing homogeneity in vertebrate assemblages vary. Knowledge of beta-diversity congruence can help formulate hypotheses about the mechanisms governing regional diversity patterns and should inform conservation, especially as threat from global climate change increases.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5

Flagellin is the major structural protein of the flagella of Gram-negative bacteria. Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants. In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5). The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown. In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation. In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity. In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses. These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.