Detecting outbreaks of E coli O157 infection in nurseries.
Explore the source record for details and available documents.
Child, Preschool↗
Biomedical subjects
R J Roberts
Publications and source records attributed to R J Roberts.
Theta bursts, closed head injury, and partial seizure-like symptoms: a retrospective study.
The purpose of this retrospective, chart-review study was to provide additional information on the clinical correlates ofparoxysmal theta activity on EEG and to further examine the previously proposed relation (Varney, Hines, Bailey, & Roberts, 1992) between episodic symptoms in the context of prior closed-head injury and theta bursts. Allparticipants were veterans receiving their health care at a single, tertiary-care, Veterans Affairs medical center. Compared with patients with normal EEGs (comparison group, n = 30) and nonparoxysmal theta-delta slowing (comparison group, n = 30), consecutive patients with theta bursts (n = 38) were found to have significantly more episodic (ie., partial seizure-like) symptoms documented in their medical records. History of closed-head trauma was also associated with increased reporting of episodic symptoms across all three clinical groups. These findings suggest that, in the presence of theta bursts on EEG, clinicians may wish to interview systematicallyfor episodic, partial seizure-like phenomena, especially when paroxysmal theta activity occurs in the context of prior closed head injury.
Adult↗
Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome.
Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently. About two dozen potential restriction-modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes. Here we describe a functional analysis of the 16 putative Type II R-M systems in the H. pylori J99 genome. To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements. To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine. We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities. Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial 'immune' system, by alternatively turning on/off a subset of numerous R-M systems.
Adenine↗
Identification of type II restriction and modification systems in Helicobacter pylori reveals their substantial diversity among strains.
A total of 22 type II restriction endonucleases with 18 distinct specificities have been identified in six Helicobacter pylori strains. Among these 18 specificities are three completely new endonucleases, Hpy178III, Hpy99I, and Hpy188I, that specifically cleave DNA at TCNNGA, CGWCG, and TCNGA sites, respectively. The set of endonucleases identified in each strain varies, but all have four- or five-base recognition sequences. Among 16 H. pylori strains, examination of the DNA modification status at the recognition sites of 15 restriction endonucleases reveals that each strain has a substantially different complement of type II modification systems. We conclude that the type II restriction-modification systems in H. pylori are highly diverse between strains, a unique characteristic of H. pylori. The diverse methylation status of H. pylori chromosomal DNA may serve as a new typing system to discriminate H. pylori isolates for epidemiological and clinical purposes. This study also demonstrates that H. pylori is a rich source of type II restriction endonucleases.
Adult↗
Comparison of wet combing with malathion for treatment of head lice in the UK: a pragmatic randomised controlled trial.
BACKGROUND: Concern about the effectiveness and toxicity of insecticide lotions has led to promotion of mechanical methods to remove head lice. We compared the effectiveness of "bug-busting" (wet combing with a fine-toothed comb) and malathion lotion. METHODS: We screened 4037 schoolchildren in two counties in Wales, UK (intermediate resistance to malathion). Of 167 found to have head lice, 81 (aged 3-14 years) were eligible to participate in a randomised controlled trial that compared mechanical removal of lice by a commercial kit every 3-4 days for 2 weeks with two applications of 0.5% malathion lotion 7 days apart; parents carried out both treatments. The outcome measure was the presence of live lice 7 days after the end of treatment. Analyses were by intention to treat. FINDINGS: 74 children completed the study and 72 were included in the analysis. The cure rate was 38% (12 of 32) for bug-busting and 78% (31 of 40) for malathion. Children assigned bug-busting were 2.8 (95% CI 1.5-5.2) times more likely than those assigned malathion to have lice at the end of treatment (p=0.0006). INTERPRETATION: Malathion lotion was twice as effective as bug-busting, even in an area with intermediate resistance. Policies advocating bug-busting as first-line treatment for head lice in the general population are inappropriate. Assessment of the outcome of treatment 1-2 weeks after completion is essential for successful management. Only about 50% of participants complied fully with treatment, so future trials should be pragmatic in design, avoid false incentives, and study representative samples of children.
Adolescent↗
Purification of the novel endonuclease, Hpy188I, and cloning of its restriction-modification genes reveal evidence of its horizontal transfer to the Helicobacter pylori genome.
We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.
Amino Acid Sequence↗
Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain.
The mouse (cytosine-5) DNA methyltransferase (Dnmt1) consists of a regulatory N-terminal and a catalytic C-terminal domain, which are fused by a stretch of Gly-Lys dipeptide repeats. The C-terminal region contains all of the conserved motifs found in other cytosine-5 DNA methyltransferases including the relative position of the catalytic Pro-Cys dipeptide. In prokaryotes, the methyltransferases are simpler and lack the regulatory N-terminal domain. We constructed three hybrid methyltransferases, containing the intact N-terminus of the murine Dnmt1 and most of the coding sequences from M.HhaI (GCGC), M.HpaII (CCGG) or M.SssI (CG). These hybrids are biologically active when expressed in a baculovirus system and show the specificity of the parental C-terminal domain. Expression of these recombinant constructs leads to de novo methylation of both host and viral genomes in a sequence-specific manner. Steady-state kinetic analyses were performed on the murine Dnmt1-HhaI hybrid using poly(dG-dC).poly (dG-dC), unmethylated and hemimethylated oligonucleotides as substrates. The enzyme has a slow catalytic turnover number of 4.38 h(-1) for poly(dG-dC). poly(dG-dC), and exhibits 3-fold higher catalytic efficiency for hemimethylated substrates.
Amino Acid Motifs↗
Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure.
Dnmt1 is the predominant DNA methyltransferase (MTase) in mammals. The C-terminal domain of Dnmt1 clearly shares sequence similarity with many prokaryotic 5mC methyltransferases, and had been proposed to be sufficient for catalytic activity. We show here by deletion analysis that the C-terminal domain alone is not sufficient for methylating activity, but that a large part of the N-terminal domain is required in addition. Since this complex structure of Dnmt1 raises issues about its evolutionary origin, we have compared several eukaryotic MTases and have determined the genomic organization of the mouse Dnmt1 gene. The 5' most part of the N-terminal domain is dispensible for enzyme activity, includes the major nuclear import signal and comprises tissue-specific exons. Interestingly, the functional subdivision of Dnmt1 correlates well with the structure of the Dnmt1 gene in terms of intron/exon size distribution as well as sequence conservation. Our results, based on functional, structural and sequence comparison data, suggest that the gene has evolved from the fusion of at least three genes.
Amino Acid Motifs↗
REBASE - restriction enzymes and methylases.
REBASE is a comprehensive database of information about restriction enzymes and related proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, are also listed. The data is distributed via Email, ftp (ftp.neb.com), and the Web (http://rebase.neb.com).
Amino Acid Sequence↗
Mechanical property predictions for polymorphs of sulphathiazole and carbamazepine.
The mechanical properties of polymorphs of sulphathiazole and carbamazepine have been determined experimentally by three-point beam bending. It is shown that the mechanical properties of sulphathiazole and carbamazepine polymorphs can be predicted using the atom-atom potential model applied to lattice dynamics, as long as account is taken of crystal morphology when considering the experimental results.
Carbamazepine↗
The early days of bioinformatics publishing.
A brief history of the early days of publishing in the bioinformatics field is presented.
Computational Biology↗
Opportunistic immunisation in schools may be more effective than immunisation in hospital.
Explore the source record for details and available documents.
Child↗
Brittle-ductile transitions in sucrose and the influence of lateral stresses during compaction.
Sucrose, in a range of particle sizes, has been compacted to investigate both the effect of brittle-ductile transition and the effect of lateral stresses on the deformation stress as measured using Heckel plots. All particles with a diameter greater than 30 microm exhibited cracking in line with both theoretical predictions and literature data from hammer and ball milling. In addition, crack lengths in compressed particles examined microscopically were very similar to those predicted from the deformation stress, confirming the applicability of the model.
Chemistry, Pharmaceutical↗
Is infection control in residential homes neglected?
We investigated the potential for cross infection during the cleaning of commodes in residential homes in one health authority in order to investigate why residential institutions are the commonest setting for outbreaks of infectious intestinal disease. Most homes used commodes, but half lacked a designated sluice area for cleaning them after use and in half soiled commodes were cleaned in bathroom or toilet washbasins, baths, showers, or toilet pans, creating a risk of cross infection.
Cross Infection↗
Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.
A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.
Animals↗
Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.
Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at </=20 microM AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V(max(app)) resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. We propose that this activation plays a role in vivo in the regulation of maintenance methylation.
Allosteric Regulation↗
Structure of a binary complex of HhaI methyltransferase with S-adenosyl-L-methionine formed in the presence of a short non-specific DNA oligonucleotide.
We have determined a structure for a complex formed between HhaI methyltransferase (M.HhaI) and S-adenosyl-L-methionine (AdoMet) in the presence of a non-specific short oligonucleotide. M.HhaI binds to the non-specific short oligonucleotides in solution. Although no DNA is incorporated in the crystal, AdoMet binds in a primed orientation, identical with that observed in the ternary complex of the enzyme, cognate DNA, and AdoMet or S-adenosyl-L-homocysteine (AdoHcy). This orientation differs from the previously observed unprimed orientation in the M.HhaI-AdoMet binary complex, where the S+-CH3 unit of AdoMet is protected by a favorable cation-pi interaction with Trp41. The structure suggests that the presence of DNA can guide AdoMet into the primed orientation. These results shed new light on the proposed ordered mechanism of binding and explains the stable association between AdoMet and M.HhaI.
Crystallization↗
Examples of successful crystal structure prediction: polymorphs of primidone and progesterone.
The field of crystal structure prediction and its potential value to the pharmaceutical industry is described. The process of structure prediction employed here is summarized and the results of its application to primidone and progesterone are reported. It is shown that the process successfully generates the known polymorphs of these molecules, starting from the molecular structure alone. Observations related to the application of the structure prediction process are reported.
Crystallization↗