Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2(-/-) or C5aR(-/-) mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2(-/-) mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.     

The significance of p16INK4a in cell defenses against transformation

Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.     

Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor

Primary erythroid cells and erythroid cell lines may synthesize and secrete tumor necrosis factor-alpha (TNF-alpha) following stimulation with erythropoietin (EPO). The effect of triggering TNF-alpha synthesis and secretion was investigated in erythroleukemia and myeloid cell lines: HCD57, DA3-EPOR, and BAF3-EPOR. The EPO-induced, membrane-bound form of autocrine TNF-alpha seemed to enhance proliferation of HCD57 and DA3-EPOR cells; however, the concentration of secreted autocrine/paracrine TNF-alpha was never sufficient to have an effect. Autocrine TNF-alpha acts through TNFRII receptors to stimulate proliferation. Modulation of mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK-1/2) activity by the membrane-bound form of autocrine TNF-alpha apparently played a central role in the control of EPO-dependent proliferation of HCD57 and DA3-EPOR cells. Primary erythroid cells and DA3-EPOR cells were found to express similar, high levels of both TNFRI and TNFRII, showing that differential expression of TNF-alpha receptors does not explain why primary cells are inhibited and DA3-EPOR cells are stimulated by autocrine TNF-alpha. BAF3 cells expressing a mutant EPOR with no cytoplasmic tyrosine residues were capable of triggering EPO-dependent TNF-alpha synthesis and secretion, indicating that tyrosine-docking sites in the EPOR were not required for EPO-dependent TNF-alpha secretion.     

ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells

Background  

Unrepaired DNA double-stranded breaks (DSBs) cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ) pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID) and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms.     

Impact of urban fragmentation on the genetic structure of the eastern red-backed salamander

Urban development is a major cause of habitat loss and fragmentation. Few studies, however, have dealt with fragmentation in an urban landscape. In this paper, we examine the genetic structure of isolated populations of the eastern red-backed salamander (Plethodon cinereus) in a metropolitan area. We sampled four populations located on a mountain in the heart of Montréal (Québec, Canada), which presents a mosaic of forested patches isolated by roads, graveyards and buildings. We assessed the genetic structure of these populations using microsatellite loci and compared it to the genetic structure of four populations located in a continuous habitat in southern Québec. Our results indicate that allelic richness and heterozygosity are lower in the urban populations. Exact differentiation tests and pairwise F _ST also show that the populations found in the fragmented habitat are genetically differentiated, whereas populations located in the continuous habitat are genetically homogeneous. These results raise conservation concerns for these populations as well as for rare or threatened species inhabiting urban landscapes.