125,208 research outputs found
Studies on CheA(B): Phosphorylation, methylation and behavioral control during chemotactic signal transduction in Bacillus subtilis
No full textChemotaxis in Bacillus subtilis is a prototypical behavior and signal transduction process, and CheA\sb{\rm B} plays an important role in it. The cheA\sb{\rm B} gene was isolated, sequenced, expressed and mutated. It encoded a large negatively charged protein with a molecular weight of approximately 74,000. The predicted protein sequence has 33% homology with the Escherichia coli and CheA\sb{\rm E}. Such proteins have been found to autophosphorylate and are members of the same histidine kinase signal modulating family. CheA\sb{\rm B} has several conserved regions (including the histidine that is phosphorylated in CheA\sb{\rm E}) that coincide with the other autophosphorylated signal transducers. In vitro experiments on partially purified CheA\sb{\rm B} indicates that it also appears to autophosphorylate. CheA\sb{\rm E} mediates the effect of binding of attractants or repellents at the MCPs (receptors) by altering its rate of autophosphorylation and subsequent phosphorylation of CheY, to bring about behavioral change, and CheB, to bring about adaptation. CheA\sb{\rm B} appears to play a similar central controlling role, for a null mutant and all point mutants tested were defective in attractant-induced methanol production and showed no behavioral response upon addition of chemoeffectors. Furthermore, methylation of the MCPs, in which methyl groups are transferred away to an acceptor and replaced by methyl groups from S-adenosylmethionine, is regulated by CheB\sb{\rm B}, whose activity is controlled by CheA\sb{\rm B}.Although the chemotactic mechanism in B. subtilis thus appears to involve phosphoryl transfer, it is significantly different from that in E. coli. First, the cheA\sb{\rm B} null mutant mostly tumbles, whereas cheA\sb{\rm E} mutants swim smoothly. Since the null mutant in cheY\sb{\rm B} also tumbles whereas that in cheY\sb{\rm E} swims smoothly, it is likely that CheY\sb{\rm B}-P causes smooth swimming, whereas CheY\sb{\rm E}-P is known to cause tumbling. Second, only in B. subtilis does excitation lead to methyl transfer and methanol formation, probably to help bring about adaptation, and CheA\sb{\rm B} controls this. It also is required for some, but not all, changes in methylation of MCPs following addition of the attractant aspartate. All of these experiments suggest a unique mechanism for chemotaxis in B. subtilis.Made available in DSpace on 2011-05-07T14:20:44Z (GMT). No. of bitstreams: 2
license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5)
9305529.pdf: 2932358 bytes, checksum: 45078ce1bb5a19f8d68f88ae5bd84044 (MD5)
Previous issue date: 1992Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding ([email protected]) on 2011-05-07T15:05:41Z
Item is restricted indefinitely.Restriction data tranferred 2014-07-01T11:31:29-05:00
Original Data
Group with Access UIUC Users [automated]
Release Date: none
Reason: ETDs are only available to UIUC Users without author permissionETDs are only available to UIUC Users without author permissionU of I Onl
CheW and CheA counts per TD show different trends.
No full text<p>(<b>A</b>) For complexes with rest groups, the number of CheW molecules per TD is independent of complex size. For species with no rest groups, this ratio increases with the number of TDs in the species. Raising the expression level of CheW results in the formation of larger complex sizes. (<b>B</b>) The number of CheA dimers per TD shows two opposing trends with respect to complex size. As the ratio is highest for single TDs with CheA<sub>2</sub>-including rest groups, raising the expression level of CheA results in smaller complex sizes but an increased number of CheA molecules contributing to the signal amplitude.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Biochemistry and regulation of CheA in Bacillus subtilis chemotaxis
No full textBacterial chemotaxis is one of the most well understood signal transduction processes in biology. By controlling the direction of flagella rotation, this process allows the organism to sense changes in its surroundings and migrate toward more favorable environments. The process of chemotaxis is controlled by the activity of an autophosphorylating histidine kinase, CheA. The work described in this thesis shows that Bacillus subtilis CheA differs from other members of this family of bacterial histidine kinases in two respects: it is able to achieve its maximum phosphorylation potential at very low ATP concentrations, and the phosphorylated form of the enzyme is very stable in the presence of ADP and other potential phosphoryl group acceptors. We have long hypothesized that attractant-bound methyl-accepting chemotaxis proteins (MCPs) in B. subtilis increase the activity of CheA. Asparagine-bound McpB has been shown here to increase CheA activity in vitro, the first example of activation of a two-component signal transducing kinase by its proposed environmental ligand to date. Studies on the enteric chemotaxis system revealed that taxis toward certain sugars is mediated solely through a separate phosphoenolpyruvate-dependent phosphotransferase system (PTS), involved in sugar transport. In B. subtilis, we have determined that taxis toward PTS carbohydrates is mediated both by MCPs and the PTS. Finally, a restriction map of the distal end of the 26kb fla/che operon has been deduced, which will be of value in attempts to clone additional chemotaxis genes in B. subtilis.Made available in DSpace on 2011-05-07T12:51:56Z (GMT). No. of bitstreams: 2
license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5)
9712277.pdf: 4229278 bytes, checksum: b99e8e9e353cdb76ac7932cf8f3cd2de (MD5)
Previous issue date: 1996Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding ([email protected]) on 2011-05-07T14:46:55Z
Item is restricted indefinitely.Restriction data tranferred 2014-07-01T11:21:07-05:00
Original Data
Group with Access UIUC Users [automated]
Release Date: none
Reason: ETDs are only available to UIUC Users without author permissionETDs are only available to UIUC Users without author permissionU of I Onl
Growth-stage-dependent interaction of HtpG with FliN and CheA.
No full text<p>(<b>A</b>) Efficiency of FRET between HtpG-YFP or HtpG(E34A)-YFP and FliN-CFP or CFP-CheA as a function of growth stage (indicated by OD<sub>600</sub> value), measured by acceptor photobleaching in wild-type cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003631#s4" target="_blank">Methods</a>). Error bars indicate standard errors from three replicates. For these assays, a truncated form of CheA lacking the first 97 amino acids (CheA<sub>s</sub>) was used because this fusion was more stable against spontaneous proteolysis than the fusion to full-length CheA, but showed similar interaction with HtpG (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003631#pgen.1003631.s011" target="_blank">Table S4</a>) (<b>B</b>) Growth-stage dependence of motility in cultures used for FRET measurements in (A), assayed as a percentage of motile cells The onset of cell motility is substantially delayed in cells expressing HtpG(E34A). Error bars indicate standard errors from three replicates.</p
Identification of intramolecular phosphoryl transfer within CheA<sub>3</sub> and CheS<sub>3</sub>.
No full text<p>(A) CheA<sub>3</sub>∼P is acid- and alkaline-labile, whereas the REC mutant CheA<sub>3</sub>:D663A∼P is acid-labile and base-resistant. (B) Both CheS<sub>3</sub>∼P and its REC mutant CheS<sub>3</sub>:D54A are acid-labile and alkaline-stable. (C) CheA<sub>3</sub>:D663A∼P phosphorylates CheA<sub>3</sub>-REC truncation protein in Buffer 15 containing K<sup>+</sup> and 18 mM Mg<sup>2+</sup>. (D) Phosphoryl transfer from CheS<sub>3</sub>:D54A∼P to CheS<sub>3</sub>-REC1 truncation protein was not observed in Buffer 15.</p
Pharmacokinetic profiles of the two major active metabolites of metamizole (dipyrone) in cats following three different routes of administration
No full textThis study was performed to determine pharmacokinetic profiles of the two active metabolites of the analgesic drug metamizole (dipyrone, MET), 4-methylaminoantipyrine (MAA), and 4-aminoantipyrine (AA), after intravenous (i.v., intramuscular (i.m.), and oral (p.o.) administration in cats. Six healthy mixed-breed cats were administered MET (25 mg/kg) by i.v., i.m., or p.o. routes in a crossover design. Adverse clinical signs, namely salivation and vomiting, were detected in all groups (i.v. 67%, i.m. 34%, and p.o. 15%). The mean maximal plasma concentration of MAA for i.v., i.m., and p.o. administrations was 148.63 ± 106.64, 18.74 ± 4.97, and 20.59 ± 15.29 Î1⁄4g/ml, respectively, with about 7 hr of half-life in all routes. Among the administration routes, the area under the plasma concentration curve (AUC) value was the lowest after i.m. administration and the AUCEV/i.v. ratio was higher in p.o. than the i.m. administration without statistical significance. The plasma concentration of AA was detectable up to 24 hr, and the mean plasma concentrations were smaller than MAA. The present results suggest that MET is converted into the active metabolites in cats as in humans. Further pharmacodynamics and safety studies should be performed before any clinical use. 2017 John Wiley & Sons Ltd
Sequence comparison of phoR, gyrB, groEL, and cheA genes as phylogenetic markers for distinguishing Bacillus amyloliquefaciens and B. subtilis and for identifying Bacillus strain B29
No full textGiven the close genetic relationship between Bacillus amyloliquefaciens and B. subtilis, distinguishing the two solely based on their physiological and biochemical characteristics and 16S rRNA sequences is difficult. Molecular identification was used to discover suitable genes for distinguishing the two bacteria, and to identify the bio-controlling strain B29, due to molecular identification has been paid more and more attention. The similarity of four genes, cheA, gyrB, groEL and phoR, of the two species was compared by the software BLASTN and MAGA, and phylogenetic tree was constructed. The B29 strain was re-identified by using the screened genes. The similarities of the four genes, gyrB, groEL, cheA and phoR, of the two species were 93-95%, 82-84%, 76-78% and 76-77%, respectively. The homologies of the four genes of the strain B29 and the strains of B. amyloliquefaciens strains were more than 95%. We determined how well the phoR and cheA genes could be used to differentiate B. amyloliquefacien and B. subtilis. The previously isolated biological control strain B29, initially classified as B. subtilis, was re-classified as B. amyloliquefaciens. Our data indicate that other than the phoR gene, the cheA gene might be a useful phylogenetic marker for differentiating B. subtilis and B. amyloliquefaciens.</jats:p
In vitro reconstitution of the TlpD, CheW, and CheA chemotaxis signaling complex.
No full text(A) Kinetics of HpCheA with varying concentrations of ATP are shown. Experiments were conducted in triplicate with 4 μM CheA and varying amounts of ATP in 50 mM Tris (pH 7.5), 100 mM NaCl, and 10 mM MgCl2. The average kobs for three replicate time courses are shown at various concentrations of ATP (black dots), error bars are the sample standard deviation, and these measurements are fit to the Michaelis-Menten curve (black line). (B) Representative analytical ultracentrifugation data (black axes) are shown for TlpD at 1 μM (black dotted black line) in PBS buffer (pH 7) with 1 mM TCEP. At this concentration, peaks corresponding to the TlpD monomer and dimer occur near 3.5 [S] and 5.2 [S], respectively. The average dimer KD for the recombinant TlpD construct calculated across various protein concentrations was found to be 188 nM (S1 Table). Shown in red on secondary axes are fluorescence anisotropy data for a titration of TlpD under identical conditions with experiments run in triplicate. See also S1 Fig. for a comparison of the TlpD dimer KD with the N-terminal His tag present or cleaved off and simulated data showing the expected monomer and dimer populations expected based on measured KD values. (C) Shown on top are representative raw data from radio-ATP labeling experiments of 15-minute reactions with CheA alone (“A”) and additions of CheW (“AW”) and TlpD (“AWD”). Below are reactions of 1 mM ATP and 4 μM CheA (black circles), +8 μM CheW (blue triangles), and +8 μM CheW; 24 μM TlpD (red squares) run in triplicate and fit to a pseudo–first-order reaction curve (solid lines). (D) CheW was titrated against 4 μM CheA and resulting kobs measurements were fit to a binding isotherm to estimate a kinetically-defined KD of 14.6 μM for the CheA↔CheW interaction. (E) TlpD was titrated against 4 μM CheA and 40 μM CheW and fit to a binding isotherm as in panel C to approximate the thermodynamics of the CheA, CheW↔TlpD interaction to have a KD of 15.2 μM. (F) A titration of TlpD against 4 μM CheA shows no activation (gray squares). For CheA in the presence of saturating [CheW], a 2.7-fold activation occurs (blue line), and with saturating [CheW] and [TlpD], this is increased to a 14.6-fold activation (red line) over CheA alone (black line). See S1 Table for a summary of reaction parameters and statistics. CheA, chemotaxis protein A; CheA-Pi, phosphorylated CheA; CheW, chemotaxis protein W; HpCheA, Helicobacter pylori CheA; S, Sedverg; TCEP, tris(2-carboxyethyl)phosphine; TlpD, transducer-like protein D.</p
- …
