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61.
Knowledge of DNA evolution is central to our understanding of biological history, but how fast does DNA change? Previously, pedigree and ancient DNA studies--focusing on evolution in the short term--have yielded molecular rate estimates substantially faster than those based on deeper phylogenies. It has recently been suggested that short-term, elevated molecular rates decay exponentially over 1-2 Myr to long-term, phylogenetic rates, termed "time dependency of molecular rates." This transition has potential to confound molecular inferences of demographic parameters and dating of many important evolutionary events. Here, we employ a novel approach--geologically dated changes in river drainages and isolation of fish populations--to document rates of mitochondrial DNA change over a range of temporal scales. This method utilizes precise spatiotemporal disruptions of linear freshwater systems and hence avoids many of the limitations associated with typical DNA calibration methods involving fossil data or island formation. Studies of freshwater-limited fishes across the South Island of New Zealand have revealed that genetic relationships reflect past, rather than present, drainage connections. Here, we use this link between drainage geology and genetics to calibrate rates of molecular evolution across nine events ranging in age from 0.007 Myr (Holocene) to 5.0 Myr (Pliocene). Molecular rates of change in galaxiid fishes from calibration points younger than 200 kyr were faster than those based on older calibration points. This study provides conclusive evidence of time dependency in molecular rates as it is based on a robust calibration system that was applied to closely related taxa, and analyzed using a consistent and rigorous methodology. The time dependency observed here appears short-lived relative to previous suggestions (1-2 Myr), which has bearing on the accuracy of molecular inferences drawn from processes operating within the Quaternary and mechanisms invoked to explain the decay of rates with time. 相似文献
62.
Background
Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.Methodology/Principal Findings
In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.Conclusions/Significance
Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury. 相似文献63.
Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration 总被引:1,自引:0,他引:1
Sandquist JC Swenson KI Demali KA Burridge K Means AR 《The Journal of biological chemistry》2006,281(47):35873-35883
The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions. 相似文献
64.
65.
Hamel B Monaghan-Benson E Rojas RJ Temple BR Marston DJ Burridge K Sondek J 《The Journal of biological chemistry》2011,286(14):12141-12148
SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors. 相似文献
66.
Ram Krishna Thakur Vinod Kumar Yadav Akinchan Kumar Ankita Singh Krishnendu Pal Luke Hoeppner Dhurjhoti Saha Gunjan Purohit Richa Basundra Anirban Kar Rashi Halder Pankaj Kumar Aradhita Baral MJ Mahesh Kumar Alfonso Baldi Bruno Vincenzi Laura Lorenzon Rajkumar Banerjee Praveen Kumar Viji Shridhar Debabrata Mukhopadhyay Shantanu Chowdhury 《Nucleic acids research》2014,42(18):11589-11600
67.
Marieke?Pingen Ramin?Sarrami-Forooshani Annemarie?MJ?Wensing Petra?van Ham Agata?Drewniak Charles?AB?Boucher Teunis?BH?GeijtenbeekEmail author Monique?NijhuisEmail author 《Retrovirology》2014,11(1):113
Background
Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.Results
In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5+ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5+ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.Conclusions
Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.68.
Anna Brüniche-Olsen Menna E. Jones Jeremy J. Austin Christopher P. Burridge Barbara R. Holland 《Biology letters》2014,10(11)
The Tasmanian devil (Sarcophilus harrisii) was widespread in Australia during the Late Pleistocene but is now endemic to the island of Tasmania. Low genetic diversity combined with the spread of devil facial tumour disease have raised concerns for the species’ long-term survival. Here, we investigate the origin of low genetic diversity by inferring the species'' demographic history using temporal sampling with summary statistics, full-likelihood and approximate Bayesian computation methods. Our results show extensive population declines across Tasmania correlating with environmental changes around the last glacial maximum and following unstable climate related to increased ‘El Niño–Southern Oscillation’ activity. 相似文献
69.
70.
Subarna Bhattacharya Paul W. Burridge Erin M. Kropp Sandra L. Chuppa Wai-Meng Kwok Joseph C. Wu Kenneth R. Boheler Rebekah L. Gundry 《Journal of visualized experiments : JoVE》2014,(91)
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described. 相似文献