bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis
Authors: Bottrel R. L. A. , Dutra W. O. , Martins F. A. , Gontijo B. , Carvalho E. , Barral-Netto M. , Barral A. , Almeida R. P. , Mayrink W. , Locksley R. , Gollob K. J. 
Published: July 2002

ABSTRACTLeishmaniasis, caused by infection with the protozoan parasiteLeishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigenLeishmaniahomolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time withLeishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-γ) production by several different sources, listed in order of contribution: CD4+T lymphocytes, CD4−, CD8−lymphocytes, and CD8+T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-γ and tumor necrosis factor alpha (TNF-α) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4+T lymphocytes (IFN-γ+and IL-10−/IL-4−), Tc1 CD8+T cells (IFN-γ+, and IL-10−/IL-4−), and a high frequency of TNF-α-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.
Homogeneity of Antibody Responses in Tuberculosis Patients
Authors: Samanich K. , Belisle J. T. , Laal S. 
Published: July 2002

ABSTRACTThe goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grownMycobacterium tuberculosisthat are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins ofM. tuberculosiswere fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens ofM. tuberculosiswhich suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936–3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.
l-Tryptophan-l-Kynurenine Pathway Metabolism Accelerated byToxoplasmagondiiInfection Is Abolished in Gamma Interferon-Gene-Deficient Mice: Cross-Regulation between Inducible Nitric Oxide Synthase and Indoleamine-2,3-Dioxygenase
Authors: Fujigaki Suwako , Saito Kuniaki , Takemura Masao , Maekawa Naoya , Yamada Yasuhiro , Wada Hisayasu , Seishima Mitsuru 
Published: July 2002

ABSTRACTl-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-γ)-induced antimicrobial effects. In the present study, the effects ofToxoplasmagondiiinfection on IDO were investigated by using wild-type and IFN-γ-gene-deficient (knockout) (IFN-γ KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lungl-tryptophan concentrations were dramatically decreased followingT.gondiiinfection. In contrast, these metabolic changes did not occur inT.gondii-infected IFN-γ KO mice or in uninfected IFN-γ KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-γ KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression ofT.gondiisurface antigen gene 2 (SAG2) induced in lungs and brain byT.gondiiinfection was significantly enhanced in IFN-γ KO mice compared to wild-type mice on day 7 postinfection. Treatment withN-nitro-l-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasmal-kynurenine afterT. gondiiinfection. This in vivo study provides evidence thatl-tryptophan depletion caused byT. gondiiis directly mediated by IFN-γ in the lungs, where iNOS is not induced by IFN-γ. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.
Structural Decomposition and Heterogeneity of Commercial Lipoteichoic Acid Preparations
Authors: Morath Siegfried , Geyer Armin , Spreitzer Ingo , Hermann Corinna , Hartung Thomas 
Published: July 2002

ABSTRACTFractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% forStreptococcus pyogenes, 16% forBacillus subtilis, and 75% forStaphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine andN-acetylglucosamine substituents. All preparations contained—to varying degrees—non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA fromBacillus subtilisandS. pyogenesbut not LTA fromS. aureusinduced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1β), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.
Role of Flagella in Host Cell Invasion by
Burkholderia cepacia
Authors: Tomich Mladen , Herfst Christine A. , Golden Joseph W. , Mohr Christian D. 
Published: July 2002

ABSTRACT

Burkholderia cepacia
is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by
B. cepacia
can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of
B. cepacia
, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of
B. cepacia
, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of
B. cepacia
with reduced invasiveness due to disruption of
fliG
, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in
fliI
, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of
fliG
and
fliI
impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of
B. cepacia
. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by
B. cepacia
, contributing to establishment of infection and systemic spread of the organism.
Regulation of the
Escherichia coli
K5 Capsule Gene Cluster: Evidence for the Roles of H-NS, BipA, and Integration Host Factor in Regulation of Group 2 Capsule Gene Clusters in Pathogenic
E. coli
Authors: Rowe Sonya , Hodson Nigel , Griffiths Gary , Roberts Ian S. 
Published: July 2002

ABSTRACT

The expression of
Escherichia coli
group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20°C. Thermoregulation is at the level of transcription, with no detectable transcription at 20°C. Using the
E. coli
K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20°C. At 37°C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20°C H-NS functions to repress transcription. The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic
E. coli
, was shown to play a similar role to H-NS in regulating transcription at 37 and 20°C. The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37°C and IHF binding demonstrated by bandshift assays. The IHF binding site was 3′ to the site of transcription initiation, suggesting that sequences in the 5′ end of the first gene (
kpsF
) in region 1 may play a role in regulating transcription from this promoter at 37°C. Two additional
cis
-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters. These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in
E. coli
.
Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance
Authors: Lee Angela , Mao Weimin , Warren Mark S. , Mistry Anita , Hoshino Kazuki , Okumura Ryo , Ishida Hiroko , Lomovskaya Olga 
Published: July 2002

ABSTRACT

The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in
Escherichia coli
and
Pseudomonas aeruginosa
. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.
Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within
oriT
in Termination of R64 Transfer
Authors: Furuya Nobuhisa , Komano Teruya 
Published: July 2002

ABSTRACT

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer,
oriT
. We have investigated the
oriT
structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64
oriT
sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated
oriT
s. The
oriT
subregion required for initiation was found to be identical to the 44-bp
oriT
core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal
oriT
sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated
oriT
s was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient
oriT
and initiation-proficient but termination-deficient
oriT
, suggesting that the 3′ terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.
Positive Correlation between Virulence of
Pseudomonas aeruginosa
Mutants in Mice and Insects
Authors: Jander Georg , Rahme Laurence G. , Ausubel Frederick M. 
Published: July 2002

ABSTRACT

Strain PA14, a human clinical isolate of
Pseudomonas aeruginosa
, is pathogenic in mice and insects (
Galleria mellonella
). Analysis of 32 different PA14 mutants in these two hosts showed a novel positive correlation in the virulence patterns. Thus,
G. mellonella
is a good model system for identifying mammalian virulence factors of
P. aeruginosa
.
Molecular Identification of Oligoalginate Lyase of
Sphingomonas
sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate
Authors: Hashimoto Wataru , Miyake Osamu , Momma Keiko , Kawai Shigeyuki , Murata Kousaku 
Published: July 2002

ABSTRACT

A bacterium,
Sphingomonas
sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in
Sphingomonas
sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (
aly
) and the ABC transporter (
algS
,
algM1
, and
algM2
). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.
A Homologue of an Operon Required for DNA Transfer in
Agrobacterium
Is Required in
Brucella abortus
for Virulence and Intracellular Multiplication
Authors: Sieira Rodrigo , Comerci Diego J. , Sánchez Daniel O. , Ugalde Rodolfo A. 
Published: July 2002

ABSTRACT

As part of a
Brucella abortus
2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (
virB
) highly homologous to bacterial type IV secretion systems. The
B. abortus virB
locus is a collinear arrangement of 13 open reading frames (ORFs). Between
virB1
and
virB2
and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of
Brucella
intergenic regions were found. Gene reporter studies demonstrated that the
B. abortus virB
locus constitutes an operon transcribed from
virB1
which is turned on during the stationary phase of growth. A
B. abortus
polar
virB1
mutant failed to replicate in HeLa cells, indicating that the
virB
operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in
virB10
showed different behaviors in mice and in the HeLa cell infection assay, suggesting that
virB10
per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the
virB
operon constitutes a major determinant of
B. abortus
virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of
B. abortus
to an endoplasmic reticulum-related replication compartment.
The
Pseudomonas aeruginosa
Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS
Authors: Winzer Klaus , Falconer Colin , Garber Nachman C. , Diggle Stephen P. , Camara Miguel , Williams Paul 
Published: July 2002

ABSTRACT

In
Pseudomonas aeruginosa
, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators,
N
-(3-oxododecanoyl)-
l
-homoserine lactone (3O-C12-HSL) and
N
-butanoyl-
l
-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the
rhl
locus while, in a
lasR
mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene,
lecA
, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the
lecA
translational start codon. A
lux
box-type element together with RpoS (ς
S
) consensus sequences was identified upstream of the putative promoter region. In
Escherichia coli
, expression of a
lecA
::
lux
reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in
P. aeruginosa
PAO1, the expression of a chromosomal
lecA
::
lux
fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of
rpoS
abolished lectin synthesis in
P. aeruginosa
, demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the
lecA
::
lux
reporter in
E. coli
could be inhibited by the presence of 3O-C12-HSL, this did not occur in
P. aeruginosa
. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of
lecA
expression.
Structural Characterization of the Released Polysaccharide of Desiccation-Tolerant
Nostoc commune
DRH-1
Authors: Helm Richard F. , Huang Zebo , Edwards Devin , Leeson Heidi , Peery William , Potts Malcolm 
Published: July 2002

ABSTRACT

The structure of the viscous extracellular polysaccharide (glycan) of desiccation-tolerant
Nostoc commune
DRH-1 was determined through chromatographic and spectroscopic methods. The polysaccharide is novel in that it possesses a 1-4-linked xylogalactoglucan backbone with
d
-ribofuranose and 3-
O
-[(
R
)-1-carboxyethyl]-
d
-glucuronic acid (nosturonic acid) pendant groups. The presence of
d
-ribose and nosturonic acid as peripheral groups is unusual, and their potential roles in modulating the rheological properties of the glycan are discussed. Nosturonic acid was present in the glycans of
N. commune
from diverse geographic locations, suggesting that this uronic acid is an integral component of this cosmopolitan anhydrophile.
Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme
Authors: Parales Rebecca E. , Lee Kyoung , Resnick Sol M. , Jiang Haiyan , Lessner Daniel J. , Gibson David T. 
Published: July 2002

ABSTRACT

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by
Pseudomonas
sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes
cis
-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of
cis
-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1
R
,2
S
], compared to the enantiomerically pure [>99% (+)-1
R
,2
S
] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.