Tree-line changes along the Andes: implications of spatial patterns and dynamics

The Andes provide an extensive latitudinal and topographical framework for studying the factors that control the spatial patterns of forests (timberlines) and their species components expressed through the presence of tree growth forms (tree lines). Despite consistent overall similarities in landscape patterns, many processes must be unique, given the dramatic differences in species richness and biophysical constraints along the Andes. In all cases evaluated to date, morphological plasticity is a common trait of plant species that dominate at tree lines. In fact, many changes observed can be related to species-specific traits. Physiological limitations on tree growth form only explain species limits, while disturbances and cyclical climate fluctuations interact to affect many landscape patterns. Over long periods of time, tree lines provide unique habitats and perhaps opportunities for speciation. Understanding the spatial organization of tree-line dynamics is one viable research approach for evaluating the likely past fluxes and possible future changes.     

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.     

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.     

cGMP-dependent protein kinase I interacts with TRIM39R, a novel Rpp21 domain-containing TRIM protein

Nitric oxide modulates vascular smooth muscle cell (SMC) cytoskeletal kinetics and phenotype, in part, by stimulating cGMP-dependent protein kinase I (PKGI). To identify molecular targets of PKGI, an interaction trap screen in yeast was performed using a cDNA encoding the catalytic region of PKGI and a human lung cDNA library. We identified a cDNA that encodes a putative PKGI-interactor that is a novel variant of TRIM39, a member of the really interesting new gene (RING) finger family of proteins. Although this TRIM39 variant encodes the NH(2)-terminal RING finger (RF), B-box, and coiled-coil (RBBC) domains of TRIM39, instead of a complete COOH-terminal B30.2 domain, this TRIM39 isoform contains the COOH-terminal portion of Rpp21, a component of RNase P. RT-PCR demonstrated that the TRIM39 variant, which we refer to as TRIM39R, is transcribed in the human fetal lung and in rat pulmonary artery SMC. Indirect immunofluorescence using an antibody generated against the conserved domains of TRIM39 and TRIM39R revealed the proteins in speckled intranuclear structures in human acute monocytic leukemia (THP-1) and human epidermal carcinoma line (HEp-2) cells. PKGI phosphorylated a typical PKGI/PKA phosphorylation domain in a conserved region of TRIM39 and TRIM39R. Additional studies demonstrated that PKGI interacts with both isoforms of TRIM39 in yeast cells and phosphorylates both isoforms of TRIM39 in human cell lines. Although PKGI has been observed to interact with proteins that regulate cytoskeletal function and gene expression, this investigation shows for the first time that PKGI interacts with tripartite motif (TRIM) proteins, which, through diverse molecular pathways, are often observed to regulate important aspects of cellular homeostasis.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^- / -  and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in Ogg1^- / -  mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^- / -  mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^- / -  mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^- / -  mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States. Isolates belonging to the same VCG had similar sets of single nucleotide polymorphisms regardless of isolate origin. The six VCGs formed four genetically distinct groups. Although recombination between certain pairs of VCGs could not be excluded, none was found for YV36, the VCG that includes the atoxigenic A. flavus isolate currently used to mitigate aflatoxin contamination in cotton in Arizona.     

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Humans have seven alcohol dehydrogenase genes (ADH) falling into five classes. Three out of the seven genes (ADH1A, ADH1B and ADH1C) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: >90%; introns: >70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total>15.0 kb) for five non-human primates--four great apes and one Old World Monkey (OWM)--and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B. We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution (dN/dS) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.     

Evolutionary Genomics of Weedy Rice in the USA

Red rice is an interfertiie, weedy form of cultivated rice (Oryza sativa L.) that competes aggressively with the cropin the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America andthe weed has been proposed to have evolved through multiple mechanisms, including "de-domestication" of UScrop cultivars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedyOryza strains. The phenotype of US red rice ranges from "crop mimics", which share some domestication traitswith the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity haveindicated that many weed strains are closely related to Asian taxa (including indica and aus rice varieties, whichhave never been cultivated in the US, and the Asian crop progenitor O. rufipogon), whereas others show geneticsimilarity to the tropical japonica varieties cultivated in the southern US. Herein, we review what is known aboutthe evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to furthercharacterize the evolutionary genomics of this aggressive weed.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.