bluuebunny/crossref_metadata_2025_split huggingface dataset:
Is it important to correct apparent drug tissue concentrations for blood contamination in the dog?
Authors: Giudicelli Christine , Dricot Eric , Moati Frédérique , Strolin‐Benedetti Margherita , Giudicelli Jean‐François 
Published: May 2004

AbstractThe goal of this study was to quantify in the dog the error that is made in assessing drug tissue concentrations when no correction for blood contamination is performed and hence to determine in which organs such a correction should be made. The organs investigated were the heart, the brain, the liver and the skeletal muscle, and the test drug used was the H1‐antihistamine, cetirizine (0.1 or 0.6 mg/kg/day for 3 days, orally, n = 6 dogs). Radiolabelled serum albumin was used to quantitate blood trapped in the tissues. Blood and tissue samplings were performed 2 h after the last drug administration. Mean (±SEM) volumes of blood trapped in the liver, heart, muscle and brain were 263 ± 12, 91 ± 3, 27 ± 1 and 20 ± 2 μL/g, respectively. Apparent tissue/blood concentration ratios of cetirizine were 2.39 ± 0.33, 1.11 ± 0.09, 0.77 ± 0.07 and 0.37 ± 0.05 in the four organs. When correction for residual blood is not performed, cetirizine concentrations are underestimated (−13.6 ± 3.2%) in the liver, slightly overestimated (+4.7 ± 1.5 to +6.3 ± 2.8%) in the brain, and neither over nor underestimated in the heart and muscle. Simulation data over a wide range of theoretical drug tissue/blood concentration ratios indicate that in the dog: (a) for the liver, correction of apparent tissue concentration for residual blood should be performed when the drug tissue/blood concentration ratio achieved is <0.8 or >4, (b) for the heart, correction should be made when this ratio is ≤0.4 and (c) for the brain and muscle, no correction is necessary unless the ratio is ≤0.1.
Atomic Force Microscopy of Cell Growth and Division in
Staphylococcus aureus
Authors: Touhami Ahmed , Jericho Manfred H. , Beveridge Terry J. 
Published: May 2004

ABSTRACT

The growth and division of
Staphylococcus aureus
was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol.
141:
315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.
Secretion of Virulence Proteins from
Campylobacter jejuni
Is Dependent on a Functional Flagellar Export Apparatus
Authors: Konkel Michael E. , Klena John D. , Rivera-Amill Vanessa , Monteville Marshall R. , Biswas Debabrata , Raphael Brian , Mickelson Joey 
Published: May 2004

ABSTRACT

Campylobacter jejuni
, a gram-negative motile bacterium, secretes a set of proteins termed the
Campylobacter
invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (
flgB
and
flgC
), hook (
flgE2
), and filament (
flaA
and
flaB
) genes, as well as in genes whose products are essential for flagellar protein export (
flhB
and
fliI
). While mutations that affected filament assembly were found to be nonmotile (Mot
−
) and did not secrete Cia proteins (S
−
), a
flaA
(

flaB
+

) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot
−
, S
+
). Complementation of a
flaA flaB
double mutant with a shuttle plasmid harboring either the
flaA
or
flaB
gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the
C. jejuni
mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the
C. jejuni
Cia proteins are secreted from the flagellar export apparatus.
Isolation and Characterization of
rpoS
from a Pathogenic Bacterium,
Vibrio vulnificus
: Role of σ
S
in Survival of Exponential-Phase Cells under Oxidative Stress
Authors: Park Kyung-Je , Kang Min-Jin , Kim Songhee H. , Lee Hyun-Jung , Lim Jae-Kyu , Choi Sang Ho , Park Soon-Jung , Lee Kyu-Ho 
Published: May 2004

ABSTRACT

A gene homologous to
rpoS
was cloned from a fatal human pathogen,
Vibrio vulnificus
. The functional role of
rpoS
in
V. vulnificus
was accessed by using an
rpoS
knockout mutant strain. This mutant was impaired in terms of the ability to survive under oxidative stress, nutrient starvation, UV irradiation, or acidic conditions. The increased susceptibility of the
V. vulnificus
mutant in the exponential phase to H
2
O
2
was attributed to the reduced activity of hydroperoxidase I (HPI). Although σ
S
synthesis was induced and HPI activity reached the maximal level in the stationary phase, the mutant in the stationary phase showed the same susceptibility to H
2
O
2
as the wild-type strain in the stationary phase. In addition, HPII activity, which is known to be controlled by σ
S
in
Escherichia coli
, was not detectable in
V. vulnificus
strains under the conditions tested. The mutant in the exponential phase complemented with multiple copies of either the
rpoS
or
katG
gene of
V. vulnificus
recovered both resistance to H
2
O
2
and HPI activity compared with the control strain. Expression of the
katG
gene encoding HPI in
V. vulnificus
was monitored by using a
katG
::
luxAB
transcriptional fusion. The expression of this gene was significantly reduced by deletion of σ
S
in both the early exponential and late stationary phases. Thus, σ
S
is necessary for increased synthesis and activity of HPI, and σ
S
is required for exponentially growing
V. vulnificus
to develop the ability to survive in the presence of H
2
O
2
.
An Accessory Protein Is Required for Relaxosome Formation by Small Staphylococcal Plasmids
Authors: Smith Matthew C. A. , Thomas Christopher D. 
Published: May 2004

ABSTRACT

Mobilization of the staphylococcal plasmid pC221 requires at least one plasmid-encoded protein, MobA, in order to form a relaxosome. pC221 and closely related plasmids also possess an overlapping reading frame encoding a protein of 15 kDa, termed MobC. By completing the nucleotide sequence of plasmid pC223, we have found a further example of this small protein, and gene knockouts have shown that MobC is essential for relaxosome formation and plasmid mobilization in both pC221 and pC223. Primer extension analysis has been used to identify the
nic
site in both of these plasmids, located upstream of the
mobC
gene in the sense strand. Although the sequence surrounding the
nic
site is highly conserved between pC221 and pC223, exchange of the
oriT
sequence between plasmids significantly reduces the extent of relaxation complex formation, suggesting that the Mob proteins are selective for their cognate plasmids in vivo.
CcpA-Dependent Regulation of
Bacillus subtilis
Glutamate Dehydrogenase Gene Expression
Authors: Belitsky Boris R. , Kim Hyun-Jin , Sonenshein Abraham L. 
Published: May 2004

ABSTRACT

The
Bacillus subtilis rocG
gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway,
rocG
was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of
rocG
readthrough transcription proved to contribute to the slow growth rate of
B. subtilis
cells in glucose-glutamate medium. Increased readthrough expression of
rocG
was shown to be partially responsible for the growth defect of
ccpA
strains in glucose-ammonium medium.
The
davDT
Operon of
Pseudomonas putida
, Involved in Lysine Catabolism, Is Induced in Response to the Pathway Intermediate δ-Aminovaleric Acid
Authors: Revelles Olga , Espinosa-Urgel Manuel , Molin Soeren , Ramos Juan L. 
Published: May 2004

ABSTRACT

Pseudomonas putida
KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and
P. putida
KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to δ-aminovaleric acid and then further degraded to glutaric acid via the action of the
davDT
gene products. We show that the
davDT
genes form an operon transcribed from a single σ
70
-dependent promoter. The relatively high level of basal expression from the
davD
promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be δ-aminovaleric acid because in a mutant unable to metabolize lysine to δ-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the
P. putida
P

davD

promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the
davD
promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.
nsd, a Locus That Affects theMyxococcus xanthusCellular Response to Nutrient Concentration
Authors: Brenner Margaret , Garza Anthony G. , Singer Mitchell 
Published: May 2004

ABSTRACTExpression of the previously reported Tn5lacΩ4469 insertion inMyxococcus xanthuscells is regulated by the starvation response. Interested in learning more about the starvation response, we cloned and sequenced the region containing the insertion. Our analysis shows that the gene fusion is located in an open reading frame that we have designatednsd(nutrient sensing/utilizing defective) and that its expression is driven by a σ70-like promoter. Sequence analysis of thensdgene product provides no information on the potential structure or function of the encoded protein. In a further effort to learn about the role ofnsdin the starvation response, we closely examined the phenotype of cells carrying thensd::Tn5lacΩ4469 mutation. Our analysis showed that these cells initiate development on medium that contains nutrients sufficient to sustain vegetative growth of wild-type cells. Furthermore, in liquid media these same nutrient concentrations elicit a severe impairment of growth ofnsdcells. The data suggest that thensdcells launch a starvation response when there are enough nutrients to prevent one. In support of this hypothesis, we found that, when grown in these nutrient concentrations,nsdcells accumulate guanosine tetraphosphate, the cellular starvation signal. Therefore, we propose thatnsdis used by cells to respond to available nutrient levels.
Bacteriophage Tuc2009 Encodes a Tail-Associated Cell Wall-Degrading Activity
Authors: Kenny John G. , McGrath Stephen , Fitzgerald Gerald F. , van Sinderen Douwe 
Published: May 2004

ABSTRACT

Tuc2009 is a P335-type member of the tailed-phage supergroup
Siphoviridae
and was originally identified as a resident prophage of the gram-positive bacterium
Lactococcus lactis
UC509. A Tuc2009 gene designated

tal
2009

which is located within the morphogenic module was shown to specify a lytic activity within the 3′ portion of its coding region. Comparative sequence analysis indicated that the cell wall-degrading part of Tal
2009
is a member of the M37 protein family and that Tal
2009
lacks a cell-binding domain, a finding supported by binding studies. Tal
2009
appears to undergo self-mediated posttranslational processing in both
L. lactis
and
Escherichia coli
. Antibodies directed against a purified C-terminal portion of Tal
2009
were used for immunoelectron microscopy, which showed that Tal
2009
is located at the tail tip of Tuc2009. Antibody neutralization studies demonstrated that Tal
2009
-directed antibodies inhibited the ability of phage to mediate host lysis by more than 100-fold. These data indicate that

tal
2009

encodes a tail-associated lysin involved in localized cell wall degradation, thus allowing the Tuc2009 DNA injection machinery access to the membrane of its bacterial host.
Transcription by an Archaeal RNA Polymerase Is Slowed but Not Blocked by an Archaeal Nucleosome
Authors: Xie Yunwei , Reeve John N. 
Published: May 2004

ABSTRACT

Archaeal RNA polymerases (RNAPs) are closely related to eukaryotic RNAPs, and in
Euryarchaea
, genomic DNA is wrapped and compacted by histones into archaeal nucleosomes. In eukaryotes, transcription of DNA bound into nucleosomes is facilitated by histone tail modifications and chromatin remodeling complexes, but archaeal histones do not have histone tails and archaeal genome sequences provide no evidence for archaeal homologs of eukaryotic chromatin remodeling complexes. We have therefore investigated the ability of an archaeal RNAP, purified from
Methanothermobacter thermautotrophicus
, to transcribe DNA bound into an archaeal nucleosome by HMtA2, an archaeal histone from
M. thermautotrophicus
. To do so, we constructed a template that allows transcript elongation to be separated from transcription initiation, on which archaeal nucleosome assembly is positioned downstream from the site of transcription initiation. At 58°C, in the absence of an archaeal nucleosome,
M. thermautotrophicus
RNAP transcribed this template DNA at a rate of ∼20 nucleotides per second. With an archaeal nucleosome present, transcript elongation was slowed but not blocked, with transcription pausing at sites before and within the archaeal nucleosome. With additional HMtA2 binding, complexes were obtained that also incorporated the upstream regulatory region. This inhibited transcription presumably by preventing archaeal TATA-box binding protein, general transcription factor TFB, and RNAP access and thus inhibiting transcription initiation.
Purification and Characterization of the AAA+ Domain ofSinorhizobium melilotiDctD, a σ54-Dependent Transcriptional Activator
Authors: Xu Hao , Gu Baohua , Nixon B. Tracy , Hoover Timothy R. 
Published: May 2004

ABSTRACTActivators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain ofSinorhizobium melilotiC4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end ofdctDidentified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD(Δ1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD(Δ1-142)when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD(Δ1-142)displayed multiple species. The DctD AAA+ domain, but not DctD(Δ1-142), formed a stable complex with σ54in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54in addition to its role in recognition of upstream activation sequences.
Evidence for an Arginine Exporter Encoded by
yggA
(
argO
) That Is Regulated by the LysR-Type Transcriptional Regulator ArgP in
Escherichia coli
Authors: Nandineni Madhusudan R. , Gowrishankar J. 
Published: May 2004

ABSTRACT

The anonymous open reading frame
yggA
of
Escherichia coli
was identified in this study as a gene that is under the transcriptional control of
argP
(previously called
iciA
), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either
yggA
or
argP
were supersensitive to the arginine analog canavanine, and
yggA-lac
expression in vivo exhibited

argP
+

-dependent induction by arginine. Lysine supplementation phenocopied the
argP
null mutation in that it virtually abolished
yggA
expression, even in the

argP
+

strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off
yggA
transcription. Dominant missense mutations in
argP
(

argP
d

) that conferred canavanine resistance and rendered
yggA-lac
expression constitutive were obtained. The protein deduced to be encoded by
yggA
shares similarity with a basic amino acid exporter (LysE) of
Corynebacterium glutamicum
, and we obtained evidence for increased arginine efflux from
E. coli
strains with either the

argP
d

mutation or multicopy

yggA
+

. The null
yggA
mutation abolished the increased arginine efflux from the

argP
d

strain. Our results suggest that
yggA
encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it
argO
(for “arginine outward transport”). We propose that the physiological function of
argO
may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.
The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from
Escherichia coli
, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition
Authors: Ren Chuan-Peng , Chaudhuri Roy R. , Fivian Amanda , Bailey Christopher M. , Antonio Martin , Barnes Wayne M. , Pallen Mark J. 
Published: May 2004

ABSTRACT

ETT2 is a second cryptic type III secretion system in
Escherichia coli
which was first discovered through the analysis of genome sequences of enterohemorrhagic
E. coli
O157:H7. Comparative analyses of
Escherichia
and
Shigella
genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3
Salmonella
pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized
E. coli
reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of
E. coli
strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic
E. coli
O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some
E. coli
strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative
E. coli
strain O42.
Novel Genes That Influence Development inStreptomyces coelicolor
Authors: Gehring Amy M. , Wang Stephanie T. , Kearns Daniel B. , Storer Narie Yoo , Losick Richard 
Published: May 2004

ABSTRACTFilamentous soil bacteria of the genusStreptomycescarry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants ofStreptomyces coelicolorthat exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938andSCO2525) and we reidentified two previously described developmental genes (bldAandbldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.
Recognition of Ferric Catecholates by FepA
Authors: Annamalai Rajasekaran , Jin Bo , Cao Zhenghua , Newton Salete M. C. , Klebba Phillip E. 
Published: May 2004

ABSTRACTEscherichia coliFepA transports certain catecholate ferric siderophores, but not others, nor any noncatecholate compounds. Direct binding and competition experiments demonstrated that this selectivity originates during the adsorption stage. The synthetic tricatecholate Fe-TRENCAM bound to FepA with 50- to 100-fold-lower affinity than Fe-enterobactin (FeEnt), despite an identical metal center, and Fe-corynebactin only bound at much higher concentrations. Neither Fe-agrobactin nor ferrichrome bound at all, even at concentrations 106-fold above theKd. Thus, FepA only adsorbs catecholate iron complexes, and it selects FeEnt among even its close homologs. We used alanine scanning mutagenesis to study the contributions of surface aromatic residues to FeEnt recognition. Although not apparent from crystallography, aromatic residues in L3, L5, L7, L8, and L10 affected FepA's interaction with FeEnt. Among 10 substitutions that eliminated aromatic residues,Kdincreased as much as 20-fold (Y481A and Y638A) andKmincreased as much as 400-fold (Y478), showing the importance of aromaticity around the pore entrance. Although many mutations equally reduced binding and transport, others caused greater deficiencies in the latter. Y638A and Y478A increasedKm10- and 200-fold more, respectively, thanKd. N-domain loop deletions created the same phenotype: Δ60-67 (in NL1) and Δ98-105 (in NL2) increasedKd10- to 20-fold but raisedKm500- to 700-fold. W101A (in NL2) had little effect onKdbut increasedKm1,000-fold. These data suggested that the primary role of the N terminus is in ligand uptake. Fluorescence and radioisotopic experiments showed biphasic release of FeEnt from FepA. In spectroscopic determinations,koff1was 0.03/s andkoff2was 0.003/s. However, FepAY272AF329A did not manifest the rapid dissociation phase, corroborating the role of aromatic residues in the initial binding of FeEnt. Thus, the β-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport.