Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.     

Genetics of postzygotic isolation in Eucalyptus: whole-genome analysis of barriers to introgression in a wide interspecific cross of Eucalyptus grandis and E. globulus

The genetic architecture of hybrid fitness characters can provide valuable insights into the nature and evolution of postzygotic reproductive barriers in diverged species. We determined the genome-wide distribution of barriers to introgression in an F(1) hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and E. globulus (Labill.). Two interspecific backcross families (N = 186) were used to construct comparative, single-tree, genetic linkage maps of an F(1) hybrid individual and two backcross parents. A total of 1354 testcross AFLP marker loci were evaluated in the three parental maps and a substantial proportion (27.7% average) exhibited transmission ratio distortion (alpha = 0.05). The distorted markers were located in distinct regions of the parental maps and marker alleles within each region were all biased toward either of the two parental species. We used a Bayesian approach to estimate the position and effect of transmission ratio distorting loci (TRDLs) in the distorted regions of each parental linkage map. The relative viability of TRDL alleles ranged from 0.20 to 0.72. Contrary to expectation, heterospecific (donor) alleles of TRDLs were favored as often as recurrent alleles in both backcrosses, suggesting that positive and negative heterospecific interactions affect introgression rates in this wide interspecific pedigree.     

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.     

Situational repair of replication forks: roles of RecG and RecA proteins

Replication forks often stall or collapse when they encounter a DNA lesion. Fork regression is part of several major paths to the repair of stalled forks, allowing nonmutagenic bypass of the lesion. We have shown previously that Escherichia coli RecA protein can promote extensive regression of a forked DNA substrate that mimics a possible structure of a replication fork stalled at a leading strand lesion. Using electron microscopy and gel electrophoresis, we demonstrate that another protein, E. coli RecG helicase, promotes extensive fork regression in the same system. The RecG-catalyzed fork regression is very efficient and faster than the RecA-promoted reaction (up to 240 bp s(-1)), despite very limited processivity of the RecG protein. The reaction is dependent upon ATP hydrolysis and is stimulated by single-stranded binding protein. The RecA- and RecG-promoted reactions are not synergistic. In fact, RecG functions poorly under the conditions optimal for the RecA reaction, and vice versa. When both RecA and RecG proteins are incubated with the DNA substrate, high RecG concentrations inhibit the RecA protein-promoted fork regression. The very different reaction profiles may reflect a situational application of these proteins to the rescue of stalled replication forks in vivo.     

Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity

Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).