Comparative Phylogenomics and Evolution of the Brucellae Reveal a Path to Virulence

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as “stealth pathogens” that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing “atypical” strains and a highly conserved “classical” core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.     

Intact cannabinoid CB1 receptors in the Alzheimer's disease cortex

The cannabinoid CB1 receptor has gained much attention as a potential pharmacotherapeutic target in various neurodegenerative diseases including Alzheimer's disease (AD). However, the relation of CB1 receptors to cognitive function in AD is at present unclear. In this study, postmortem brain tissues from a cohort of prospectively assessed, neuropathologically confirmed AD patients and aged controls were used to measure CB1 receptors by immunoblotting, and a subset of subjects also had [(3)H]SR141716A binding. Correlational analyses were then performed for the neurochemical and cognitive data. We found that CB1 receptor levels in were unchanged AD in the brain regions assessed (frontal cortex, anterior cingulate gyrus, hippocampus, caudate nucleus). Within the AD group, frontal cortical CB1 immunoreactivity correlated with cognitive scores assessed within a year of death. Our study suggests that CB1 receptors are intact in AD and may play a role in preserving cognitive function. Therefore, CB1 receptors should be further assessed as a potential therapeutic target in AD.     

Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes

Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central α-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.Neurofibromatosis type 2 is an inherited autosomal disorder that is characterized by bilateral schwannomas of the eighth cranial nerve. The tumor suppressor gene responsible for this disorder, NF2, was cloned in 1993 (45). Biallelic inactivation of the NF2 gene is also seen in spontaneous schwannoma, meningioma, and malignant mesothelioma (22). In mouse models, deletion of the Nf2 gene is embryonic lethal, indicating an essential role for NF2 in development (24). Heterozygous mice develop a variety of aggressive metastatic tumors that have lost the wild-type allele (23). Targeted deletion of the Nf2 gene in Schwann cells leads to schwannoma formation (7). In vitro, Nf2-null cells grow to significantly higher densities (31), suggesting that contact inhibition of growth is impaired in these cells and that mediation of growth arrest at high cell density may be the basis for the tumor suppressor function of the NF2 gene. In normal fibroblasts, merlin is inactive as a growth suppressor in subconfluent cells, becoming activated as they approach confluence, thereby effecting contact inhibition of growth (26).The NF2 gene encodes a 70-kDa protein called merlin (for moesin, ezrin, radixin-like protein), which shares significant homology with members of the ezrin-radixin-moesin (ERM) branch of the Band 4.1 superfamily (45). The domain structure of merlin, also shared with other ERM proteins, consists of an N-terminal FERM domain, followed by a central α-helical region (CH) and a C-terminal tail domain (CTD). The merlin FERM domain has relatively high sequence similarity with other ERM family members, a 60 to 70% identity over the first 300 amino acids. The CH domain and the CTD show much lower identity (28 to 36%); however, the α-helical character of the CH domain is preserved, as is the heptad repeat pattern typical of α-helices that form coiled coils (46).The critical point of regulation of all the ERM proteins is a high-affinity intramolecular interaction between the C-terminal domain and the FERM domain (4) (Fig. ​(Fig.1).1). The FERM domain folds into a three-lobed cloverleaf structure that acts as a multifaceted docking site for protein binding partners (16, 39). The CTD, consisting of four major and two minor helices and a beta sheet, binds to the FERM domain by extending across the face of the F2 and F3 lobes (32). This intramolecular head-to-tail binding results in a “closed” conformation, with the C-terminal domain covering much of the surface of the FERM domain (32, 44). For ezrin, radixin and moesin, the CTD functions as a mask, blocking access of effector molecules, such as the cell surface receptors CD44 and ICAM2 and adaptor molecules, like EBP50/NHERF, to sites on the surface of the FERM domain (11, 25, 44). The interaction between the CTD and FERM domain is regulated by phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) binding to the FERM domain and by phosphorylation of a critical residue in the CTD (3, 6, 10, 49). This residue, threonine 567 in ezrin, is conserved throughout the ERM family (21). Phosphorylation introduces a negative charge and a bulky side group that effectively reduces the affinity of the interaction, releasing the CTD from the FERM domain and causing a transition to an open conformation. Low-angle rotary shadowing electron microscopy (13) and biochemical studies (12) of purified radixin suggest that in the open conformation it is an extended filamentous structure with globular N and C termini that is greater than 240 Å in length. Signal transduction systems, such as the epidermal growth factor and Rho A pathways, induce phosphorylation of ERM proteins at the conserved C-terminal threonine via a number of kinases, including Rho kinase and protein kinase Cα (21, 28). Thus, conformational regulation of ERM proteins can be a point of integration of ERM activity with signal transduction pathways. The overall concept of ERM regulation, then, is centered upon a transition between an inactive, closed conformation that is mediated by the FERM-CTD interaction and an active, open conformation that is regulated by phosphorylation. In these two states, ERM proteins likely interact with different sets of binding partners, resulting in distinct functional outcomes.Open in a separate windowFIG. 1.ERM tertiary structure as represented by the crystal structure of full-length Sf-moesin (20), but with the merlin amino acid sequence substituted for Sf-moesin. Approximate boundary amino acid residues for all domains appear at the top of the figure. Each domain is assigned a different color. The ERM structure consists of an N-terminal FERM domain folded into three lobes, F1, F2, and F3. This is followed by a central α-helical domain containing three subhelices (αA, αB, and αC) and a CTD with four short helices. An ERM protein is thought to have an open conformation, an extended structure with the FERM domain and the CTD separated by the α-helical domain, that is more than 240 Å long. In the closed conformation, the α-helical domain bends at the αA-αB junction and again at the αB-αC junction, causing the CTD to be positioned over F2 and F3 of the FERM domain. More than half of the surface of the FERM domain is masked by interaction with the CTD, αA, and parts of αB and αC.Like the classical ERMs, merlin is also thought to be regulated by changes in conformation. The FERM domain and the CTD of merlin interact with each other, albeit at a lower level of affinity than the ezrin FERM domain and the CTD (29). There are important differences, however, between merlin and the other ERM proteins. First, phosphorylation of the conserved C-tail threonine T576 has not been reported to occur in mammalian merlin, and nonphosphorylatable and phosphomimetic substitutions at this site have no effect on merlin activity (42). Instead, merlin is phosphorylated at serine 518 in the CTD, a target of the p21-activated kinase PAK and protein kinase A (1, 18, 47). The growth-suppressive function of merlin is activated by dephosphorylation of S518 by the phosphatase PP1δ in a density-dependent manner (14). Second, it was reported in a study using FERM domain and CTD truncates of merlin that only cotransfection of both the N-and C-terminal halves resulted in growth suppression (38). Together, these observations suggested a model of inactive, phosphorylated merlin in an open conformation that, upon cell-to-cell contact, is dephosphorylated and transitions to a closed, growth suppressive conformation.The existing model for conformational regulation described above is inferred from indirect data and assays that generally measure the interaction of isolated FERM and CTD truncates rather than full-length molecules (9, 29, 38). It has been impossible to test directly because tools have not been available to specifically assay for either the open or the closed form of merlin. Therefore, we have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer (FRET), both as purified protein and in live cells. Using these tools, we show that merlin exists predominately as a monomer in a stable, largely closed conformation. Additionally, we find that the closed conformation is largely mediated by the central α-helical domain; the contribution of the FERM-CTD interaction appears to be less significant than previously thought. Finally, we find that phosphorylation and protein interaction cause unexpectedly small changes in merlin conformation. We propose a new and more refined model for merlin regulation, in which merlin function is regulated by specific but subtle conformational changes that modulate the interaction between the FERM domain and the CTD.     

Opposing Roles of Dnmt1 in Early- and Late-Stage Murine Prostate Cancer

Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.Changes in DNA methyltransferase (Dnmt) expression and DNA methylation are observed in human prostate cancer (3, 38, 41). Of particular interest, genes with tumor suppressive function become hypermethylated and silenced, which correlates with the development of specific disease phenotypes (2, 3, 38). Although an association between prostate cancer and alterations in DNA methylation has been established, in vivo models are required to determine whether these changes functionally contribute to the disease. In this context, studies in which pharmacological inhibitors of Dnmts were shown to inhibit prostate cancer in murine models have proven informative (34, 56). However, it remains unknown whether genetic disruption of epigenetic components, such as Dnmts, also impacts prostate cancer development. This is a critical question since the pharmacological inhibitors of Dnmts have pleiotropic effects, including those unrelated to activation of methylation-silenced genes (21, 23, 31). Moreover, no studies to date have examined whether Dnmts or DNA methylation play roles in normal prostate development; this information is vital to fully understanding the effects that inhibiting DNA methylation may have on prostate cancer.Dnmt1 is a maintenance DNA methyltransferase that propagates preexisting DNA methylation patterns in genomic DNA (44). Dnmt1 also is involved in de novo DNA methylation in cancer cells and interacts with other key epigenetic control molecules, including histone-modifying enzymes (11, 19). Murine models have been used to investigate the in vivo functions of Dnmt1. Complete genetic knockout of Dnmt1 is embryonic lethal in mice (29). However, hypomorphic expression of Dnmt1 allows murine development to proceed but causes global DNA hypomethylation and impacts cancer development and progression (7, 14, 28). Specifically, hypomorphic expression of Dnmt1 can lead to the development of lymphoma (14). Furthermore, crossing Dnmt1 hypomorphic mice with murine tumor models alters tumor progression, resulting in either increased or decreased tumor development, depending on the disease stage and tissue site (1, 7, 53). For example, reduced expression of Dnmt1 dramatically decreases intestinal polyp formation in Apc^Min/+ mice, either alone or in combination with 5-aza-2′-deoxycytidine treatment (7, 27). However, it was later noted that reduced expression of Dnmt1 has a dual effect on intestinal cancer in Apc^Min/+ mice, in which the development of early stage intestinal microadenomas is accelerated, whereas the formation of adenomatous polyps is significantly reduced (53). In addition, Apc^Min/+ Dnmt1 hypomorphic mice develop liver cancer associated with the loss of heterozygosity of Apc (53). Similarly, in Dnmt1 hypomorphic mice crossed to Mlh1^−/− mice, a dual effect was noted wherein mice developed fewer intestinal cancers but displayed increased T- and B-cell lymphomas (52). In addition, a recent study demonstrated that hypomorphic Dnmt1 expression is associated with reduced squamous cell carcinoma of the tongue and esophagus, resulting in decreased invasive cancer (1). Taken together, the data suggest that Dnmt1 has diverse effects on cancer development, which are dependent on tissue context and tumor stage.TRAMP is a well-established transgenic prostate cancer model driven by prostate-specific expression of the simian virus 40 (SV40) T/t oncogenes (16). TRAMP mice are characterized by Dnmt mRNA and protein overexpression, altered DNA methylation, and altered gene expression during prostate cancer development (2, 33, 35, 37). Of the three enzymatically active Dnmts, Dnmt1 shows the greatest level of overexpression in TRAMP, and this correlates with Rb inactivation, a key genetic event driving prostate cancer in the model (37). Most critically, global DNA hypomethylation occurs during early and late disease stages, while DNA hypermethylation occurs primarily at late disease stages in TRAMP (35).Here, we utilized Dnmt1 hypomorphic mice and the TRAMP model to assess the role of DNA methylation in both normal prostatic development and prostate cancer. The Dnmt1 hypomorphic mouse model used involves two different hypomorphic alleles (N and R), resulting in four genotypes with progressively reduced DNA methylation (Dnmt1^+/+, Dnmt1^R/+, Dnmt1^N/+, and Dnmt1^N/R) (7, 52). The N allele consists of a PGK-Neo insertion that deletes a portion of exon 4 of Dnmt1, resulting in severely reduced Dnmt1 expression, while the R allele involves a lacO insertion into intron 3 of Dnmt1, which partially reduces Dnmt1 expression (7, 52). Based on our previous work establishing the timing of DNA hypomethylation and DNA hypermethylation in TRAMP, we hypothesized that hypomorphic Dnmt1 expression in TRAMP may have tumor-promoting effects at early disease stages and tumor-inhibitory effects at later stages of prostate cancer progression. Our data are consistent with this hypothesis and, more importantly, reveal a critical and unanticipated role for Dnmt1 in prostate cancer metastasis.     

Eutrophication impacts on a river macrophyte

The postulated relationship between eutrophication, enhanced standing macrophyte crop and flow impedance was assessed in 14 rivers across the UK. We sampled the July standing crop of Ranunculus subgenus Batrachium at 14 rivers across England and southern Scotland, at sites where there is a known relationship between standing crop and flow impedance. We relate standing crop to variation in carbon concentrations and to elevated phosphorus concentrations, using regression analysis. Standing crop increased significantly with P availability as soluble reactive phosphorus (filtered SRP) and total phosphorus (TP). Best subsets multiple regression analysis suggests that, based on this sample of sites, there is evidence that macrophyte biomass increases with SRP concentrations and also increases with the amount of carbon as HCO₃ for a given concentration of SRP in the water. Thus eutrophication is found to increase the standing crop of a submerged aquatic plant in UK rivers. Current targets for P reduction may not be sufficient and managers should now also recognise that eutrophication can exacerbate flood risk by elevating macrophyte standing crop.     

Historical Stem‐Mapped Permanent Plots Increase Precision of Reconstructed Reference Data in Ponderosa Pine Forests of Northern Arizona

Forest structural reference conditions are widely used to understand how ecosystems have been altered and guide restoration and management objectives. We used six stem‐mapped permanent plots established in the early twentieth century to provide precise structural reference conditions for ponderosa pine forests of northern Arizona prior to Euro‐American settlement. Reference conditions for these plots in 1873–1874 included the following historical attributes: tree densities of 45–127 trees/ha, mean tree diameter at breast height (dbh) of 43.8 cm with a corresponding quadratic mean diameter range of 41.5–51.3 cm, and a stand basal area of 9.2–18.0 m²/ha. The reconstructed diameter distributions (for live ponderosa pine trees with dbh ≥9.14 cm) prior to fire exclusion varied in shape but generally displayed an irregular unimodal distribution. We suggest that management objectives for the structural restoration of ponderosa pine forests of northern Arizona emphasize: (1) conservation and retention of all pre‐settlement (>130 years) trees; (2) reduction of tree densities with a restoration objective ranging between 50 and 150 trees/ha having a large‐tree component between 25 and 50% of the total trees per hectare, respectively; (3) manipulation of the diameter distribution to achieve a unimodal or irregular, uneven‐aged shape (possibly targeting a balanced, uneven‐aged shape on cinder soil types) through the use of harvest and thinning practices that mimic gap disturbances (i.e., individual tree selection system); and (4) retention of 3–11 snags and logs per hectare resulting from natural mortality.     

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.     

Dibutyltin activates MAP kinases in human natural killer cells,in vitro

Previous studies have shown that dibutyltin (DBT) interferes with the function of human natural killer (NK) cells, diminishing their capacity to destroy tumor cells, in vitro. DBT is a widespread environmental contaminant and has been found in human blood. As NK cells are our primary immune defense against tumor cells, it is important to understand the mechanism by which DBT interferes with their function. The current study examines the effects of DBT exposures on key enzymes in the signaling pathway that regulates NK responsiveness to tumor cells. These include several protein tyrosine kinases (PTKs), mitogen-activated protein kinases (MAPKs), and mitogen-activated protein kinase kinases (MAP2Ks). The results showed that in vitro exposures of NK cells to DBT had no effect on PTKs. However, exposures to DBT for as little as 10 min were able to increase the phosphorylation (activation) of the MAPKs. The DBT-induced activations of these MAPKs appear to be due to DBT-induced activations of the immediate upstream activators of the MAPKs, MAP2Ks. The results suggest that DBT-interference with the MAPK signaling pathway is a consequence of DBT exposures, which could account for DBT-induced decreases in NK function.