Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis.

Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses.

Results: When challenged with hydrogen peroxide (H₂O₂), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H₂O₂ challenge.

Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.     

Xer recombinase and genome integrity in Helicobacter pylori, a pathogen without topoisomerase IV

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.     

Why complexity and entropy matter: information, posttranslational modifications, and assay fidelity

Computationally aided protein identification of mass spectrometry (MS) data has been a challenge for the proteomic community since the field's inception. As the community attempts to address challenges such as computational site assignment of posttranslational modifications (PTMs), selective reaction monitoring data analysis, and the transition of proteomic assays to the clinic a robust framework with defined metrics is essential. The frameworks used for protein identification are currently score based, not hypothesis driven nor deterministic, resulting in a gradation from poor to good but without the ability to recognize when a given tandem MS spectrum has insufficient information in the context of the proteome to be identified. Means of computing deterministic and pseudodeterministic assays have been proposed by both us and others [Sherman, J., McKay, M.J., Ashman, K., Molloy, M.P., Unique ion signature mass spectrometry, a deterministic method to assign peptide identity. Mol. Cell. Proteomics 2009, 8, 2051-2062; Kiyonami, R., Schoen, A., Prakash, A., Peterman, S., et al., Increased selectivity, analytical precision, and through-put in targeted proteomics. Mol. Cell. Proteomics 2011, 10, M110.002931]. We believe it is crucial to incorporate the complexity of the proteome into the design of the experiment/data analysis upstream and that by doing so proteomic data analysis will become more robust. The seminal paper by Claude Shannon published in 1948 provides the robust mathematical framework now called Information Theory, to achieve this goal. Information Theory defines appropriate metrics for information and complexity as well as means to account for interference, all of which is directly applicable to peptide identification. We attempt to encourage the adoption of Information Theory as a means to ensure that appropriate metrics are used to measure the uncertainty in a peptide identification with or without PTM site assignment.     

Comparison of central versus peripheral delivery of pregabalin in neuropathic pain states

Background

Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaV??₂??-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects.

Results

We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaV??₂??-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaV??₂??-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaV??₂??-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaV??₂??-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation.

Conclusions

Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaV??₂??-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.     

Cytosolic manganese superoxide dismutase genes from the white shrimp Litopenaeus vannamei are differentially expressed in response to lipopolysaccharides, white spot virus and during ontogeny

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme usually located in mitochondria. There are only a few examples of cytosolic MnSOD (cMnSOD). In the shrimp Litopenaeus vannamei, we have previously characterized three cMnSOD cDNAs and their differential tissue-specific expression. To obtain insights about their genomic organization, we characterized the three corresponding cMnSOD genes, named them cMnsod1, cMnsod2, and cMnsod3 and studied their specific expression during ontogeny, response to lipopolysaccharides (LPS) and white spot virus infection (WSSV) in hemocytes from shrimp. The first two genes contain five introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions, while the third one is intron-less. We analyzed 995 nucleotides upstream cMnsod2, but no classical promoter sequences were found. The deduced products of the three cMnSOD genes differ in two amino acids and there are four silent changes. cMnsod3 expression is modulated by WSSV and cMnsod2 by LPS. cMnsod2 is expressed from eggs to post larval stage during ontogeny. This is the first report of crustacean cMnSOD multigenes that are differently induced during the defense response and ontogeny.     

Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity.     

Systematic functional prioritization of protein posttranslational modifications

Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.