bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sat, the Secreted Autotransporter Toxin of Uropathogenic
Escherichia coli
, Is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells
Authors: Guyer Debra M. , Radulovic Suzana , Jones Faye-Ellen , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

The secreted autotransporter toxin (Sat) of uropathogenic
Escherichia coli
exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a
sat
mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type
E. coli
CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type
E. coli
CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic
E. coli
CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.
Helicobacter pylori-Induced Activation of Human Endothelial Cells
Authors: Innocenti M. , Thoreson A.-C. , Ferrero R. L. , Strömberg E. , Bölin I. , Eriksson L. , Svennerholm A.-M. , Quiding-Järbrink M. 
Published: July 2002

ABSTRACTHelicobacter pyloriinfection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa. To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterizedH. pyloristrains as well as purified proteins. Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation withH. pyloribacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-α) and the cytokine IL-6. However, there were large variations in the ability of the differentH. pyloristrains to stimulate endothelial cells. These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens. In addition, one or several unidentified proteins which act via NF-κB activation seem to induce endothelial cell activation. In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certainH. pyloristrains. These effects probably contribute to the continuous recruitment of neutrophils toH. pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.
Fusobacterium necrophorumLeukotoxin Induces Activation and Apoptosis of Bovine Leukocytes
Authors: Narayanan Sanjeevkumar , Stewart George C. , Chengappa M. M. , Willard Lloyd , Shuman Wilma , Wilkerson Melinda , Nagaraja T. G. 
Published: July 2002

ABSTRACTFusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. Its major virulence factor is leukotoxin, a high-molecular-weight secreted protein, primarily toxic to ruminant leukocytes. In this study, bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin and the cytotoxicity was analyzed by flow cytometry and scanning and transmission electron microscopy. At very low toxin concentrations, polymorphonuclear leukocytes (PMNs) showed activation, as indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability ofF. necrophorumleukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.
Expression of Inducible Nitric Oxide Synthase in Skin Lesions of Patients with American Cutaneous Leishmaniasis
Authors: Qadoumi Muna , Becker Inge , Donhauser Norbert , Röllinghoff Martin , Bogdan Christian 
Published: July 2002

ABSTRACT

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of
Leishmania major
or
Leishmania donovani
infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human
Leishmania
infections in vivo.
Role of the High-Affinity Zinc Uptake
znuABC
System in
Salmonella enterica
Serovar Typhimurium Virulence
Authors: Campoy Susana , Jara Mónica , Busquets Núria , Pérez de Rozas Ana M. , Badiola Ignacio , Barbé Jordi 
Published: July 2002

ABSTRACT

The
Salmonella enterica
serovar Typhimurium
znuABC
genes encoding a high-affinity zinc uptake system and its regulatory
zur
gene have been cloned.
Salmonella
serovar Typhimurium
zur
and
znuC
knockout mutants have been constructed by marker exchange. The 50% lethal dose of the
znuC
mutant increased when either orally or intraperitoneally inoculated in BALB/c mice, while virulence of the
zur
mutant decreased only when mice were intraperitoneally challenged.
Inactivation of Cytochrome
o
Ubiquinol Oxidase Relieves Catabolic Repression of the
Pseudomonas putida
GPo1 Alkane Degradation Pathway
Authors: Dinamarca M. Alejandro , Ruiz-Manzano Ana , Rojo Fernando 
Published: July 2002

ABSTRACT

Expression of the alkane degradation pathway encoded by the OCT plasmid of
Pseudomonas putida
GPo1 is regulated by two control systems. One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes. The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes. This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway. To identify the factors involved, a screening method was developed to isolate mutants without this control. Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome
o
ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions. Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source. This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell. Since inactivation of the
crc
gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome
o
terminal oxidase. The two mutations had an additive effect in relieving catabolic repression in rich medium. This suggests that
crc
and
cyo
belong to different regulation pathways, both contributing to catabolic repression.
Family Shuffling of a Targeted
bphA
Region To Engineer Biphenyl Dioxygenase
Authors: Barriault Diane , Plante Marie-Michèle , Sylvestre Michel 
Published: July 2002

ABSTRACT

In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of
bphA
that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of
bphA
genes from
Burkholderia
sp. strain LB400,
Comamonas testosteroni
B-356, and
Rhodococcus globerulus
P6. Unlike all parents, these variants exhibited high activity toward 2,2′-, 3,3′-, and 4,4′-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (
335
TFNNIRI
341
) of LB400 BphA by the corresponding segment (
333
GINTIRT
339
) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.
The RNA Polymerase α Subunit from
Sinorhizobium meliloti
Can Assemble with RNA Polymerase Subunits from
Escherichia coli
and Function in Basal and Activated Transcription both In Vivo and In Vitro
Authors: Peck Melicent C. , Gaal Tamas , Fisher Robert F. , Gourse Richard L. , Long Sharon R. 
Published: July 2002

ABSTRACT

Sinorhizobium meliloti
, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in
S. meliloti
, we cloned and characterized the gene for the α subunit of RNA polymerase (RNAP).
S. meliloti rpoA
encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding
rpoA
identified six open reading frames that are found in the conserved gene order
secY
(SecY)-
adk
(Adk)-
rpsM
(S13)-
rpsK
(S11)-
rpoA
(α)-
rplQ
(L17) found in the α-proteobacteria. In vivo, S
. meliloti rpoA
expressed in
Escherichia coli
complemented a temperature sensitive mutation in
E. coli rpoA
, demonstrating that
S. meliloti
α supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of
E. coli.
In vitro, we reconstituted RNAP holoenzyme from
S. meliloti
α and
E. coli
β, β′, and σ subunits. Similar to
E. coli
RNAP, the hybrid RNAP supported transcription from an
E. coli
core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from
S. meliloti
. Our results demonstrate that
S. meliloti
α functions are conserved in heterologous host
E. coli
even though the two α subunits are only 51% identical. The ability to utilize
E. coli
as a heterologous system in which to study the regulation of
S. meliloti
genes could provide an important tool for our understanding and manipulation of these processes.
Activity of
Sinorhizobium meliloti
NodAB and NodH Enzymes on Thiochitooligosaccharides
Authors: Southwick Audrey M. , Wang Lai-Xi , Long Sharon R. , Lee Yuan C. 
Published: July 2002

ABSTRACT

Rhizobium
bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated β-(1,4)-
N
-acetylglucosamine, are synthesized by the nodulation (
nod
) gene products. We tested the ability of three
Sinorhizobium meliloti nod
gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.
RsmA and the Quorum-Sensing Signal,N-[3-Oxohexanoyl]-l-Homoserine Lactone, Control the Levels ofrsmBRNA inErwinia carotovorasubsp.carotovoraby Affecting Its Stability
Authors: Chatterjee Asita , Cui Yaya , Chatterjee Arun K. 
Published: July 2002

ABSTRACTRsmA (for regulator of secondary metabolism), RsmC, andrsmBRNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity inErwinia carotovorasubsp. carotovora.RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay.rsmBRNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA andrsmBRNA by positively regulatingrsmAand negatively controllingrsmB.The level ofrsmBRNA is substantially higher in RsmA+bacteria than in RsmA−mutants. We show thatrsmBRNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of anrsmB-lacZtranscriptional fusion; in fact, the β-galactosidase level is somewhat higher in RsmA−bacteria than in RsmA+bacteria. We also investigated the basis for increased levels ofrsmAandrsmBRNAs in the absence of the quorum-sensing signal,N-[3-oxohexanoyl]-l-homoserine lactone (OHL). The absence of OHL activates transcription ofrsmAbut not ofrsmB.Instead, increased stability ofrsmBRNA in the presence of RsmA accounts for the elevated levels of thersmBRNA in OHL−bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels ofrsmBRNA, high levels ofrsmBRNA occur in the absence of RsmC or OHL only in RsmA+bacteria, indicating a critical role for RsmA in modulating the levels ofrsmBRNA. The findings reported here firmly establish that the quorum-sensing signal is channeled inE. carotovorasubsp.carotovoravia thersmA-rsmBposttranscriptional regulatory system.
Evidence for Temporal Regulation of the Two
Pseudomonas cellulosa
Xylanases Belonging to Glycoside Hydrolase Family 11
Authors: Emami Kaveh , Nagy Tibor , Fontes Carlos M. G. A. , Ferreira Luis M. A. , Gilbert Harry J. 
Published: July 2002

ABSTRACT

Pseudomonas cellulosa
is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of
xyn10A
,
xyn10B
,
xyn10C
,
xyn10D
, and
xyn11A
, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene,
xyn11B
, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound.
xyn10C
,
xyn10D
,
xyn11A
, and
xyn11B
were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of
xyn11A
and
xyn11B
was apparent on xylan-containing media. Transcription of
xyn11A
occurred earlier than transcription of
xyn11B
, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of
P. cellulosa
in which
xyn11A
and
xyn11B
were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type
P. cellulosa
. Based on the data presented here, we propose a model for xylan degradation by
P. cellulosa
in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.
The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway
Authors: Kim Alexander D. , Baker Angela S. , Dunaway-Mariano Debra , Metcalf W. W. , Wanner B. L. , Martin Brian M. 
Published: July 2002

ABSTRACT

The 2-aminoethylphosphonate transaminase (AEPT; the
phnW
gene product) of the
Salmonella enterica
serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and
l
-alanine (
l
-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log

V
m

and log

V
m

/

K
m

versus pH) revealed a pH optimum of 8.5. At pH 8.5,
K
eq
is equal to 0.5 and the
k
cat
values of the forward and reverse reactions are 7 and 9 s
−1
, respectively. The

K
m

for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for
l
-Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.
Characterization of
Salmonella
Serovars by PCR-Single-Strand Conformation Polymorphism Analysis
Authors: Nair Satheesh , Lin Thong Kwai , Pang Tikki , Altwegg Martin 
Published: July 2002

ABSTRACT

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb
groEL
gene from 41 strains of 10 different
Salmonella
serovars. Three
Hae
III RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the
groEL
genes of various
Salmonella
serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different
Salmonella
serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11
Salmonella enterica
serovar Enteritidis strains, and two SSCP profiles were generated for the 7
S. enterica
serovar Infantis and five
S. enterica
serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of
Salmonella
serovars due to its rapidity, simplicity, and typeability.
Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP
Authors: Kitamoto Noritoshi , Tanaka Tomoyuki , Natori Katsurou , Takeda Naokazu , Nakata Shuji , Jiang Xi , Estes Mary K. 
Published: July 2002

ABSTRACT

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family
Caliciviridae.
The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.
Nosocomial Spread of a
Staphylococcus capitis
Strain with Heteroresistance to Vancomycin in a Neonatal Intensive Care Unit
Authors: Van Der Zwet Wil C. , Debets-Ossenkopp Yvette J. , Reinders Erik , Kapi Maria , Savelkoul Paul H. M. , Van Elburg Ruurd M. , Hiramatsu Keiichi , Vandenbroucke-Grauls Christina M. J. E. 
Published: July 2002

ABSTRACT

A premature infant in a neonatal intensive care unit (NICU) developed a bloodstream infection caused by coagulase-negative staphylococci (CoNS) sensitive to vancomycin. The infection persisted for 3 weeks, despite therapy with vancomycin and replacement of all intravenous catheters. The neonate died due to necrotizing enterocolitis which developed during the ongoing sepsis. We screened this strain and 216 other strains of CoNS from cultures of blood obtained from neonates between 1997 and 2000 for heteroresistance to vancomycin. Forty-eight isolates, including the strain that caused ongoing sepsis, proved heteroresistant. All isolates were identified as
Staphylococcus capitis
and were identical, just as their resistant stable subcolonies were, when they were genetically fingerprinted by amplified-fragment length polymorphism analysis. The heteroresistant phenotype of this endemic strain was confirmed by population analysis. We conclude that heteroresistance to vancomycin occurs in
S. capitis
and might be the cause of therapeutic failures in NICUs. Moreover, heteroresistant strains can become endemic in such units.