The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874–2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid

The impact of DNA methylation in Alphaproteobacteria.

Alphaproteobacteria include bacteria with very different modes of life, from free-living to host-associated and pathogenic bacteria. Their genomes vary in size and organization from single circular chromosomes to multipartite genomes and are often methylated by one or more adenine or cytosine methyltransferases (MTases). These include MTases that are part of restriction/modification systems and so-called orphan MTases. The development of novel technologies accelerated the analysis of methylomes and revealed the existence of epigenetic patterns in several Alphaproteobacteria. This review describes the known functions of DNA methylation in Alphaproteobacteria and also discusses its potential drawbacks through the accidental deamination of methylated cytosines. Particular emphasis is given to the strong connection between the cell cycle-regulated orphan MTase CcrM and the complex network that controls gene expression and cell cycle progression in Alphaproteobacteria

Model Assessment of Alternatives for Reducing Seepage from Buried Uranium Mill Talings at the Morton Ranch Site in Central Wyoming

The purpose of this study is to examine potential ground water contamination by seepage from buried tailings under four alternatives of clay liners and tailings placement, which have been proposed for possible use at the Morton Ranch Site. To accomplish this comparison of alternatives, laboratory work and numerous measurements were made on materials typical of the Morton Ranch Site. These measurements provide the soil characteristics necessary for input to the hydrologic flow and transport models

TRUST: A Computer Program for Variably Saturated Flow in Multidimensional, Deformable Media

The computer code, TRUST. provides a versatile tool to solve a wide spectrum of fluid flow problems arising in variably saturated deformable porous media. The governing equations express the conservation of fluid mass in an elemental volume that has a constant volume of solid. Deformation of the skeleton may be nonelastic. Permeability and compressibility coefficients may be nonlinearly related to effective stress. Relationships between permeability and saturation with pore water pressure in the unsaturated zone may include hysteresis. The code developed by T. N. Narasimhan grew out of the original TRUNP code written by A. L. Edwards. The code uses an integrated finite difference algorithm for numerically solving the governing equation. Narching in time is performed by a mixed explicit-implicit numerical procedure in which the time step is internally controlled. The time step control and related feature in the TRUST code provide an effective control of the potential numerical instabilities that can arise in the course of solving this difficult class of nonlinear boundary value problem. This document brings together the equations, theory, and users manual for the code as well as a sample case with input and output

AF17 Competes With AF9 for Binding to DOT1A to up-Regulate Transcription of Epithelial NA\u3csup\u3e+\u3c/sup\u3e Channel α

We previously reported that Dot1a*AF9 complex represses transcription of the epithelial Na+ channel subunit α (α-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na+ currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an α-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na+ currents. AF17 over-expression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a*AF9-mediated repression of α-ENaC and other target genes

Stochastic Dynamics of Lexicon Learning in an Uncertain and Nonuniform World

We study the time taken by a language learner to correctly identify the meaning of all words in a lexicon under conditions where many plausible meanings can be inferred whenever a word is uttered. We show that the most basic form of cross-situational learning - whereby information from multiple episodes is combined to eliminate incorrect meanings - can perform badly when words are learned independently and meanings are drawn from a nonuniform distribution. If learners further assume that no two words share a common meaning, we find a phase transition between a maximally-efficient learning regime, where the learning time is reduced to the shortest it can possibly be, and a partially-efficient regime where incorrect candidate meanings for words persist at late times. We obtain exact results for the word-learning process through an equivalence to a statistical mechanical problem of enumerating loops in the space of word-meaning mappings.Comment: 7 pages, 3 figures. Version 2 contains additional discussion and will appear in Phys. Rev. Let

Matrix isolation and spectroscopic properties of the methylsulfinyl radical CH3(O)S˙

Publikacja w ramach programu Royal Society of Chemistry "Gold for Gold" 2013 finansowanego przez Uniwersytet ŁódzkiThe atmospherically highly relevant methylsulfinyl radical CH3(O)S˙ was generated thermally under flash pyrolysis conditions and isolated in Ar matrices at 10 K; the allyl radical is a byproduct. CH3(O)S˙ and its D3- and 13C-isotopologues were characterized through the excellent agreement between experimental and computed IR and UV/Vis spectra

Activity of 2-Aryl-2-(3-indolyl)acetohydroxamates Against Drug-Resistant Cancer Cells

Many types of tumor, including glioma, melanoma, non-small cell lung, esophageal, head and neck cancer, among others, are intrinsically resistant to apoptosis induction and poorly responsive to current therapies with proapoptotic agents. In addition, tumors often develop multi-drug resistance based on the cellular efflux of chemotherapeutic agents. Thus, novel anticancer agents capable of overcoming these intrinsic or developed tumor resistance mechanisms are urgently needed. We describe a series of 2-aryl-2-(3-indolyl)acetohydroxamic acids, which are active against apoptosis- and multidrug-resistant cancer cells as well as glioblastoma neurosphere stem-like cell cultures derived from patients. Thus, the described compounds serve as a novel chemical scaffold for the development of potentially highly effective clinical cancer drugs

Robot-assisted pelvic floor reconstructive surgery: an international Delphi study of expert users.

Robotic surgery has gained popularity for the reconstruction of pelvic floor defects. Nonetheless, there is no evidence that robot-assisted reconstructive surgery is either appropriate or superior to standard laparoscopy for the performance of pelvic floor reconstructive procedures or that it is sustainable. The aim of this project was to address the proper role of robotic pelvic floor reconstructive procedures using expert opinion. We set up an international, multidisciplinary group of 26 experts to participate in a Delphi process on robotics as applied to pelvic floor reconstructive surgery. The group comprised urogynecologists, urologists, and colorectal surgeons with long-term experience in the performance of pelvic floor reconstructive procedures and with the use of the robot, who were identified primarily based on peer-reviewed publications. Two rounds of the Delphi process were conducted. The first included 63 statements pertaining to surgeons' characteristics, general questions, indications, surgical technique, and future-oriented questions. A second round including 20 statements was used to reassess those statements where borderline agreement was obtained during the first round. The final step consisted of a face-to-face meeting with all participants to present and discuss the results of the analysis. The 26 experts agreed that robotics is a suitable indication for pelvic floor reconstructive surgery because of the significant technical advantages that it confers relative to standard laparoscopy. Experts considered these advantages particularly important for the execution of complex reconstructive procedures, although the benefits can be found also during less challenging cases. The experts considered the robot safe and effective for pelvic floor reconstruction and generally thought that the additional costs are offset by the increased surgical efficacy. Robotics is a suitable choice for pelvic reconstruction, but this Delphi initiative calls for more research to objectively assess the specific settings where robotic surgery would provide the most benefit