bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal Structure of the Nucleotide Exchange Factor GrpE Bound to the ATPase Domain of the Molecular Chaperone DnaK
Authors: Harrison Celia J. , Hayer-Hartl Manajit , Liberto Maurizio Di , Hartl F.-Ulrich , Kuriyan John
Published: July 2002

The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase) domain of
Escherichia coli
DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This conformational change is not consistent with tight nucleotide binding. Two long α helices extend away from the GrpE dimer and suggest a role for GrpE in peptide release from DnaK.
Regulation of Abscisic Acid-Induced Stomatal Closure and Anion Channels by Guard Cell AAPK Kinase
Authors: Li Jiaxu , Wang Xi-Qing , Watson Mark B. , Assmann Sarah M.
Published: July 2002

Abscisic acid (ABA) stimulates stomatal closure and thus supports water conservation by plants during drought. Mass spectrometry–generated peptide sequence information was used to clone a
Vicia faba
complementary DNA,
AAPK
, encoding a guard cell–specific ABA-activated serine-threonine protein kinase (AAPK). Expression in transformed guard cells of AAPK altered by one amino acid (lysine 43 to alanine 43) renders stomata insensitive to ABA-induced closure by eliminating ABA activation of plasma membrane anion channels. This information should allow cell-specific, targeted biotechnological manipulation of crop water status.
Selfish DNA in Protein-Coding Genes of
Rickettsia
Authors: Ogata Hiroyuki , Audic Stéphane , Barbe Valérie , Artiguenave François , Fournier Pierre-Edouard , Raoult Didier , Claverie Jean-Michel
Published: July 2002

Rickettsia conorii
, the aetiological agent of Mediterranean spotted fever, is an intracellular bacterium transmitted by ticks. Preliminary analyses of the nearly complete genome sequence of
R. conorii
have revealed 44 occurrences of a previously undescribed palindromic repeat (150 base pairs long) throughout the genome. Unexpectedly, this repeat was found inserted in-frame within 19 different
R. conorii
open reading frames likely to encode functional proteins. We found the same repeat in proteins of other
Rickettsia
species. The finding of a mobile element inserted in many unrelated genes suggests the potential role of selfish DNA in the creation of new protein sequences.
Magnetic Clusters on Single-Walled Carbon Nanotubes: The Kondo Effect in a One-Dimensional Host
Authors: Odom Teri W. , Huang Jin-Lin , Cheung Chin Li , Lieber Charles M.
Published: July 2002

Single-walled carbon nanotubes are ideal systems for investigating fundamental properties and applications of one-dimensional electronic systems. The interaction of magnetic impurities with electrons confined in one dimension has been studied by spatially resolving the local electronic density of states of small cobalt clusters on metallic single-walled nanotubes with a low-temperature scanning tunneling microscope. Spectroscopic measurements performed on and near these clusters exhibit a narrow peak near the Fermi level that has been identified as a Kondo resonance. Using the scanning tunneling microscope to fabricate ultrasmall magnetic nanostructures consisting of small cobalt clusters on short nanotube pieces, spectroscopic studies of this quantum box structure exhibited features characteristic of the bulk Kondo resonance, but also new features due to finite size.
Efficacy of Voriconazole in a Guinea Pig Model of Disseminated Invasive Aspergillosis
Authors: Kirkpatrick William R. , McAtee Robert K. , Fothergill Annette W. , Rinaldi Michael G. , Patterson Thomas F.
Published: July 2002

Voriconazole (VRC) was evaluated in an immunosuppressed-guinea pig model of invasive aspergillosis. VRC was more effective than amphotericin B or similar doses of itraconazole in the clearance of
Aspergillus
from tissues. VRC treatment regimens improved survival and significantly reduced tissue colony counts compared with those of controls.
Fluoroquinolone Resistance Associated with Target Mutations and Active Efflux in Oropharyngeal Colonizing Isolates of Viridans Group Streptococci
Authors: Guerin François , Varon Emmanuelle , Hoï Annie Buu , Gutmann Laurent , Podglajen Isabelle
Published: July 2002

Oropharyngeal samples from 60 hospitalized patients (30 fluoroquinolone [FQ]-treated and 30 non-FQ-treated patients) and 30 untreated nonhospitalized healthy control subjects yielded 20 isolates of viridans group streptococci with reduced susceptibility to FQ, mostly from the hospitalized patients. An efflux phenotype was commonly encountered, expressed either alone or with topoisomerase mutations. Interspecies transfer of the efflux phenotype was demonstrated via transformation of
Streptococcus pneumoniae
R6 with DNA from
S. mitis
and
S. oralis
.
Clinical Isolate of Vancomycin-Heterointermediate
Staphylococcus aureus
Susceptible to Methicillin and In Vitro Selection of a Vancomycin-Resistant Derivative
Authors: Bobin-Dubreux Sophie , Reverdy Marie-Elisabeth , Nervi Chantal , Rougier Martine , Bolmström Anne , Vandenesch François , Etienne Jerome
Published: July 2002

A
Staphylococcus aureus
strain with low-level heteroresistance to vancomycin (designated MER) but susceptible to methicillin was isolated from an outpatient with conjunctivitis who did not receive any glycopeptide antibiotics. Incubation of the parent strain, MER, with increasing concentrations of vancomycin led to rapid selection of a stable progeny homogeneously resistant to vancomycin. Electron micrographs of strain MER showed enhanced cell wall thickness and abnormal septations typically seen with methicillin-resistant
S. aureus
having intermediate susceptibility to vancomycin.
Systematic Analysis of a Conserved Region of the Aminoglycoside 6′-
N
-Acetyltransferase Type Ib
Authors: Shmara Ali , Weinsetel Natalia , Dery Ken J. , Chavideh Ramona , Tolmasky Marcelo E.
Published: July 2002

Alanine-scanning mutagenesis was applied to the aminoglycoside 6′-
N
-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42°C (D. Panaite and M. Tolmasky, Plasmid 39:123–133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.
Outbreak of Cefozopran (Penicillin, Oral Cephems, and Aztreonam)-Resistant
Neisseria gonorrhoeae
in Japan
Authors: Muratani Tetsuro , Akasaka Soichiro , Kobayashi Tomoko , Yamada Yoji , Inatomi Hisato , Takahashi Koichi , Matsumoto Tetsuro
Published: July 2002

We have previously reported that the
Neisseria gonorrhoeae
isolates from clinical failure cases treated with cefdinir and aztreonam, β-lactams exhibited high MICs. These resistant isolates were clearly separated from the isolates exhibiting a low level of resistance to β-lactams as shown by the MIC distribution of cefozopran. Restriction fragment length polymorphism DNA typing revealed that the outbreak of cefozopran-resistant isolates in Kitakyushu, Japan, occurred as a result of clonal spread.
In Vitro Antibacterial Activities of AF 3013, the Active Metabolite of Prulifloxacin, against Nosocomial and Community Italian Isolates
Authors: Montanari Maria Pia , Mingoia Marina , Varaldo Pietro Emanuele
Published: July 2002

AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin.
Pharmacokinetics and Safety of a 7-Day Administration of Intravenous Itraconazole followed by a 14-Day Administration of Itraconazole Oral Solution in Patients with Hematologic Malignancy
Authors: Boogaerts Marc A. , Maertens Johan , Van Der Geest Ronald , Bosly Andre , Michaux Jean-Louis , Van Hoof Achiel , Cleeren Myriam , Wostenborghs Robert , De Beule Karel
Published: July 2002

The pharmacokinetics and safety of an intravenous hydroxypropyl-β-cyclodextrin solution of itraconazole administered for 7 days followed by itraconazole oral solution administered at 200 mg once or twice daily for 14 days were assessed in 17 patients with hematologic malignancies. Steady-state plasma itraconazole concentrations were reached by 48 h after the start of intravenous treatment. The mean trough plasma itraconazole concentration at the end of the intravenous treatment was 0.54 ± 0.20 μg/ml. This concentration was not maintained during once-daily oral treatment but increased further in the twice-daily treatment group, with a trough itraconazole concentration of 1.12 ± 0.73 μg/ml at the end of oral treatment. As expected in the patient population studied, all patients experienced some adverse events (mainly gastrointestinal). Biochemical and hematologic abnormalities were frequent, but no consistent changes occurred. In conclusion, 7 days of intravenous treatment followed by 14 days of twice-daily oral treatment with itraconazole solution enables plasma itraconazole concentrations of at least 0.5 μg/ml to be reached rapidly and to be maintained. The regimen is well tolerated and has a good safety profile.
Domestic Cat Model for Predicting Human Nucleoside Analogue Pharmacokinetics in Blood and Seminal Plasma
Authors: Jordan Holly L. , Pereira Arlene S. , Cohen Myron S. , Kashuba Angela D. M.
Published: July 2002

To establish whether a feline model can predict nucleoside analogue behavior in human semen, zidovudine (ZDV) and lamivudine (3TC) pharmacokinetic parameters (PKs) were determined in the blood and seminal plasma of healthy cats. Our results show considerable similarity in ZDV and 3TC PKs between cats and humans. As in humans, ZDV and 3TC tend to accumulate in feline seminal plasma. Area under the blood plasma concentration-time curve was predictive of seminal plasma excretion. The felid model offers a unique in vivo experimental alternative for investigating the pharmacokinetics of nucleoside analogues in the male genital tract.
Pathovars of
Pseudomonas syringae
Causing Bacterial Brown Spot and Halo Blight in
Phaseolus vulgaris
L. Are Distinguishable by Ribotyping
Authors: González Ana J. , Landeras Elena , Mendoza M. Carmen
Published: July 2002

Ribotyping was evaluated as a method to differentiate between
Pseudomonas syringae
pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in
Phaseolus vulgaris
L. Ribotyping, with restriction enzymes
Bgl
I and
Sal
I and using the
Escherichia coli rrnB
operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported.
Development of a Nonantibiotic Dominant Marker for Positively Selecting Expression Plasmids in Multivalent
Salmonella
Vaccines
Authors: McNeill Hesta V. , Sinha Katharine A. , Hormaeche Carlos E. , Lee Jeong Jin , Khan C. M. Anjam
Published: July 2002

We report the novel application of a herbicide-resistance-based dominant marker for the positive selection of expression plasmids in
Salmonella
serovar vaccines. The β-lactamase gene of the plasmid pTETnir15, which expresses fragment C of tetanus toxin (TetC), has been replaced with the
bar
gene marker. The new plasmid pBAT1 can be positively selected in vitro within
Salmonella
serovars in the presence of the herbicide
dl
-phosphinothricin. The expression of TetC remains unaltered, and the
Salmonella enterica
serovar Typhimurium vaccine strain is stable and immunogenic in vivo.
Development of a Fluorogenic Probe-Based PCR Assay for Detection of
Bacillus cereus
in Nonfat Dry Milk
Authors: Kim Young-Rok , Czajka John , Batt Carl A.
Published: July 2002

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting
Bacillus cereus
in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two
B. cereus
isolates and two
Bacillus thuringiensis
isolates were cloned and sequenced. Three single-base differences in two
B. cereus
strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of
Taq
polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in
B. cereus
. A total of 51 out of 72
B. cereus
strains tested positive with the cerTAQ-1 probe, while only 1 out of 5
B. thuringiensis
strains tested positive. Sequence analysis of the negative
B. cereus
strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative
B. cereus
strains. A total of 35 out of 39
B. cereus
strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58
B. cereus
CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of
B. cereus
with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with
B. cereus
at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were
B. cereus
negative and 19 were contaminated with
B. cereus
at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect
B. cereus
in nonfat dry milk.
Lipopolysaccharides of
Rhizobium etli
Strain G12 Act in Potato Roots as an Inducing Agent of Systemic Resistance to Infection by the Cyst Nematode
Globodera pallida
Authors: Reitz M. , Rudolph K. , Schröder I. , Hoffmann-Hergarten S. , Hallmann J. , Sikora R. A.
Published: July 2002

Recent studies have shown that living and heat-killed cells of the rhizobacterium
Rhizobium etli
strain G12 induce in potato roots systemic resistance to infection by the potato cyst nematode
Globodera pallida
. To better understand the mechanisms of induced resistance, we focused on identifying the inducing agent. Since heat-stable bacterial surface carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential for recognition in the symbiotic interaction between
Rhizobium
and legumes, their role in the
R. etli
-potato interaction was studied. EPS and LPS were extracted from bacterial cultures, applied to potato roots, and tested for activity as an inducer of plant resistance to the plant-parasitic nematode. Whereas EPS did not affect
G. pallida
infection, LPS reduced nematode infection significantly in concentrations as low as 1 and 0.1 mg ml
−1
. Split-root experiments, guaranteeing a spatial separation of inducing agent and challenging pathogen, showed that soil treatments of one half of the root system with LPS resulted in a highly significant (up to 37%) systemic induced reduction of
G. pallida
infection of potato roots in the other half. The results clearly showed that LPS of
R. etli
G12 act as the inducing agent of systemic resistance in potato roots.
Expression of a Tetraheme Protein,
Desulfovibrio vulgaris
Miyazaki F Cytochrome
c
3
, in
Shewanella oneidensis
MR-1
Authors: Ozawa Kiyoshi , Tsapin Alexandre I. , Nealson Kenneth H. , Cusanovich Michael A. , Akutsu Hideo
Published: July 2002

Cytochrome
c
3
from
Desulfovibrio vulgaris
Miyazaki F was successfully expressed in the facultative aerobe
Shewanella oneidensis
MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome
c
3
gene from
D. vulgaris
Miyazaki F was used for transformation of
S. oneidensis
MR-1, resulting in the production of protein product that was indistinguishable from that produced by
D. vulgaris
Miyazaki F, except for the presence of one extra alanine residue at the N terminus.
Monitoring of
Ralstonia eutropha
KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR
Authors: Tani Katsuji , Muneta Masahiro , Nakamura Kanji , Shibuya Katsutoshi , Nasu Masao
Published: July 2002

Ralstonia eutropha
KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10
5
cells/ml and the amount of
Ralstonia
and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10
7
cells/ml 1 h after injection, and the concentration of
Ralstonia
detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.
A Regulatory Role for Interleukin 4 in Differential Inflammatory Responses in the Lung following Infection of Mice Primed with Th1- or Th2-Inducing Pertussis Vaccines
Authors: McGuirk Peter , Mills Kingston H. G.
Published: July 2002

Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge withBordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course ofB. pertussisinfection of naı̈ve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1β (IL-1β) in the lungs. In contrast, neutrophil infiltration and IL-1β production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs followingB. pertussisinfection of IL-4-defective (IL-4−/−) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1β, IL-6, and IL-1ra in Pa-immunized IL-4−/−mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge withB. pertussisand implicate IL-4 as an important regulator of inflammatory-cell recruitment.
Cell Specificity of
Helicobacter pylori
Cytotoxin Is Determined by a Short Region in the Polymorphic Midregion
Authors: Ji Xuhuai , Fernandez Thomas , Burroni Daniela , Pagliaccia Cristina , Atherton John C. , Reyrat Jean-Marc , Rappuoli Rino , Telford John L.
Published: July 2002

There are two alleles of the vacuolating cytotoxin gene from
Helicobacter pylori
, which code for toxins with different cell specificities. By analyzing the phenotypes of natural and artificial chimeras between the two forms of the protein, we have delimited a short stretch of amino acids which determine the cell specificity.
A Functional
cra
Gene Is Required for
Salmonella enterica
Serovar Typhimurium Virulence in BALB/c Mice
Authors: Allen James H. , Utley Maryjane , van den Bosch Han , Nuijten Piet , Witvliet Maarten , McCormick Beth A. , Krogfelt Karen A. , Licht Tine Rask , Brown Derek , Mauel Michael , Leatham Mary P. , Laux David C. , Cohen Paul S.
Published: July 2002

A minitransposon mutant of
Salmonella enterica
serovar Typhimurium SR-11, SR-11 Fad
−
, is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129–134, 1998). Here, evidence is presented that the mutation in SR-11 Fad
−
that renders the strain avirulent is in the
cra
gene, which encodes the Cra protein, a regulator of central carbon metabolism.
Role of Pulmonary Alveolar Macrophages in Defense of the Lung againstPseudomonas aeruginosa
Authors: Cheung Dorothy O. Y. , Halsey Keith , Speert David P.
Published: July 2002

Alveolar macrophages (AM) provide one of the first lines of defense against microbial invasion in the lower airways. The role of AM in the clearance ofPseudomonas aeruginosain mice after intrapulmonary challenge was evaluated. AM were depleted by intranasal administration of liposome-encapsulated dichloromethylene diphosphonate. At 24 h following the instillation of liposomes, a sublethal dose ofP. aeruginosawas inoculated intranasally. Spleen, liver, and lung tissue was then evaluated for viable bacteria and for histopathology. AM depletion of 78 to 88% did not affect the survival rate of infected mice or clearance ofP. aeruginosafrom the spleen, liver, or lung, compared to the control group, but the mice's susceptibility toKlebsiella pneumoniaewas greatly enhanced. The recruitment of neutrophils to the lung was also not affected. Freshly explanted AM were not competent to phagocytose unopsonizedP. aeruginosabut were able to phagocytose zymosan particles. Further studies were conducted to assess the in situ phagocytic activities of AM. Three hours after the intranasal instillation ofP. aeruginosaor other particles, bronchoalveolar lavage was performed. AM phagocytosis of zymosan particles and latex beads exceeded that ofP. aeruginosa. Neutrophils were recruited to the lung in response to a high-dose bacterial challenge. These results suggest that AM do not play an important role in defense of the lung againstP. aeruginosa.
Coincubation of Human Spermatozoa with
Chlamydia trachomatis
In Vitro Causes Increased Tyrosine Phosphorylation of Sperm Proteins
Authors: Hosseinzadeh S. , Brewis I. A. , Pacey A. A. , Moore H. D. M. , Eley A.
Published: July 2002

Elementary bodies (EBs) of the obligate intracellular bacterium
Chlamydia trachomatis
are responsible for the first step of attachment to host cells. We have studied the effects of EBs on human sperm protein tyrosine phosphorylation, which is important to sperm function. Indirect immunofluorescence using antiphosphotyrosine antibodies showed that serovar E, but not LGV, caused increased tyrosine phosphorylation which was localized to the sperm tail region. Immunoblotting revealed that serovar E caused a marked increase in tyrosine phosphorylation of 80- and 95-kDa sperm proteins, whereas serovar LGV caused increased phosphorylation of only the 80-kDa moiety. Considering the importance of tyrosine phosphorylation for sperm capacitation and other aspects of sperm function, we conclude that EBs may affect these events.
BorreliaSpirochetes Upregulate Release and Activation of Matrix Metalloproteinase Gelatinase B (MMP-9) and Collagenase 1 (MMP-1) in Human Cells
Authors: Gebbia Joseph A. , Coleman James L. , Benach Jorge L
Published: July 2002

Borrelia burgdorferi, the spirochetal agent of Lyme disease, stimulated human peripheral blood monocytes to release pro-matrix metalloproteinase-9 (gelatinase B; pro-MMP-9) and active matrix metalloproteinase-1 (collagenase-1; MMP-1). Human neutrophils also released pro-MMP-9 and a 130-kDa protein with gelatinolytic activity in response to liveB. burgdorferi. In addition, U937 cells and human keratinocyte cells were also stimulated to release pro-MMP-9 under the same conditions. However, human umbilical vein endothelial cells (HUVECs) released pro-MMP-9 and pro-MMP-2 in a constitutive manner and were not influenced by live spirochetes. MMPs produced by human monocytes also enhanced the penetration ofB. burgdorferithrough extracellular matrix component barriers in vitro. Plasmin stabilized on the surface of the Lyme disease spirochete was shown to activate pro-MMP-9 to its active form. This active form was also observed in the plasma of mice infected with a relapsing fever borrelia. These results suggest that borreliae can upregulate MMPs and possibly mediate an activation cascade initiated by plasmin bound to the microbial surface. MMPs may play a role in dissemination of the Lyme disease spirochete and in the pathogenesis ofBorreliainfection.
Identification of
Legionella pneumophila
Genes Important for Infection of Amoebas by Signature-Tagged Mutagenesis
Authors: Polesky Andrea H. , Ross Julianna T. D. , Falkow Stanley , Tompkins Lucy S.
Published: July 2002

Legionella pneumophila
is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for
Legionella
infections. Signature-tagged mutagenesis was employed to identify
Legionella pneumophila
genes necessary for survival in the amoeba
Acanthamoeba castellanii
. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in
Hartmannella vermiformis
, an amoeba linked to hospital outbreaks of
Legionella
pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known
L. pneumophila
genes,
lspK
and
aroB
. The other four were in novel genes. One gene has similarity to a cytochrome
c
-type biogenesis protein of
Pseudomonas fluorescens
. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in
Vibrio cholera
. The third is similar to
traA
of
Rhizobium
sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new
L. pneumophila
virulence genes.
Polarized Entry of Uropathogenic Afa/Dr Diffusely AdheringEscherichia coliStrain IH11128 into Human Epithelial Cells: Evidence for α5β1Integrin Recognition and Subsequent Internalization through a Pathway Involving Caveolae and Dynamic Unstable Microtubules
Authors: Guignot Julie , Bernet-Camard Marie-Françoise , Poüs Christian , Plançon Laure , Le Bouguenec Chantal , Servin Alain L.
Published: July 2002

Afa/Dr diffusely adheringEscherichia colistrain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the α5β1integrin and a pathway involving caveolae and dynamic microtubules (MTs). IH11128 invasion within HeLa cells was dramatically decreased after the cells were treated with the cholesterol-extracting drug methyl-β-cyclodextrin or the caveola-disrupting drug filipin. Disassembly of the dynamically unstable MT network by the compound 201-F resulted in a total abolition of IH11128 entry. In apically infected polarized fully differentiated Caco-2/TC7 cells, no IH11128 entry was observed. The entry of bacteria into apically IH11128-infected fully differentiated Caco-2/TC7 cells was greatly enhanced by treating cells with Ca2+-free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated polarized Caco-2/TC7 cells grown on inverted inserts mounted in chamber culture showed a highly significant level of intracellular IH11128 bacteria compared with cells subjected to the apical route of infection. No expression of CD55 and CD66e, the receptors for the Afa/Dr adhesins, was found at the basolateral domains of these cells. Consistent with the hypothesis that a cell-to-cell adhesion molecule acts as a receptor for polarized IH11128 entry, an antibody blockade using anti-α5β1integrin polyclonal antibody completely abolished bacterial entry. Experiments conducted with the laboratory strainE. coliK-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, demonstrated that thedraoperon is involved in polarized entry of IH11128 bacteria. Examined as a function of cell differentiation, the number of internalized bacteria decreased dramatically beyond cell confluency. Surviving intracellular IH11128 bacteria residing intracellularly had no effect on the functional differentiation of Caco-2/TC7 cells.
The
Haemophilus influenzae
Hia Adhesin Is an Autotransporter Protein That Remains Uncleaved at the C Terminus and Fully Cell Associated
Authors: St. Geme Joseph W. , Cutter David
Published: July 2002

Nontypeable
Haemophilus influenzae
is a gram-negative commensal organism that is commonly associated with localized respiratory tract disease. The pathogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells. Hia is the major adhesin expressed by a subset of nontypeable
H. influenzae
strains and promotes efficient adherence to a variety of human epithelial cell lines. Based on previous work, Hia is transported to the surface of
Escherichia coli
transformants and is capable of mediating
E. coli
adherence without the assistance of other
H. influenzae
proteins. In the present study, we examined the mechanism of Hia secretion. PhoA fusions, deletional mutagenesis, and N-terminal amino acid sequencing established that the signal for Hia export from the cytoplasm resides in the first 49 amino acids, including a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelectron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure analysis predicted that the C terminus of Hia forms a β-barrel with a central hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter proteins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hia is the prototype of an important subfamily of autotransporter proteins.
InvB Is a Type III Secretion Chaperone Specific for SspA
Authors: Bronstein Philip A. , Miao Edward A. , Miller Samuel I.
Published: July 2002

A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within
Salmonella
pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of
sspA
but that its absence results in reduced SspA protein levels in
Salmonella enterica
serovar Typhimurium.
Identification and Characterization of the Nitrobenzene Catabolic Plasmids pNB1 and pNB2 in
Pseudomonas putida
HS12
Authors: Park Hee-Sung , Kim Hak-Sung
Published: July 2002

Pseudomonas putida
HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated
nbz
, are located on two plasmids, pNB1 and pNB2. The genes
nbzA
,
nbzC
,
nbzD
, and
nbzE
, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the
nbzB
gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in
Escherichia coli
strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes
nbzA
and
nbzB
are located on the 1.1-kb
Sma
I-
Sna
BI fragment of pNB1 and the 1.0-kb
Ssp
I-
Sph
I fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes
nbzC
,
nbzD
, and
nbzE
, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb
Sna
BI-
Sma
I fragment of pNB1 and clustered in the order
nbzC-nbzD-nbzE
as an operon. The
nbzCDE
genes, which are transcribed in the opposite direction of the
nbzA
gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.
DsbA and DsbC Affect Extracellular Enzyme Formation in
Pseudomonas aeruginosa
Authors: Urban Andreas , Leipelt Martina , Eggert Thorsten , Jaeger Karl-Erich
Published: July 2002

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in
Pseudomonas aeruginosa
were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either
dsbA
or
dsbC
or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The
dsbA
mutant no longer produced these enzymes, whereas the lipase activity was doubled in the
dsbC
mutant. Also, extracellar lipase production was severely reduced in a
P. aeruginosa dsbA
mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene
lipA
was constitutively expressed in
trans
in a
lipA dsbA
double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.
Constitutive Expression of
Escherichia coli tat
Genes Indicates an Important Role for the Twin-Arginine Translocase during Aerobic and Anaerobic Growth
Authors: Jack Rachael L. , Sargent Frank , Berks Ben C. , Sawers Gary , Palmer Tracy
Published: July 2002

The transcription start sites for the
tatABCD
and
tatE
loci, encoding components of the Tat (twin-arginine translocase) protein export pathway, have been identified. Expression studies indicate that the
tatABCD
and
tatE
transcription units are expressed constitutively. Translational fusion experiments suggest that TatA is synthesized at a much higher level than the other Tat proteins.
VibD and VibH Are Required for Late Steps in Vibriobactin Biosynthesis in
Vibrio cholerae
Authors: Wyckoff Elizabeth E. , Smith Stacey L. , Payne Shelley M.
Published: July 2002

Vibrio cholerae
synthesizes the catechol siderophore vibriobactin. In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes.
vibD
encodes a phosphopantetheinyl transferase, and
vibH
encodes a novel nonribosomal peptide synthase. Both VibD and VibH are required for vibriobactin biosynthesis.
In Vivo Titration of Mitomycin C Action by Four
Escherichia coli
Genomic Regions on Multicopy Plasmids
Authors: Wei Yan , Vollmer Amy C. , LaRossa Robert A.
Published: July 2002

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type
Escherichia coli
. MMC resistance is caused by the presence of any of four distinct
E. coli
genes (
mdfA, gyrl, rob
, and
sdiA
) on high-copy-number vectors.
mdfA
encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The
gyrI
(also called
sbmC
) gene product inhibits DNA gyrase activity in vitro, while the
rob
protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of
ftsQAZ
genes involved in cell division.
Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of
Escherichia coli
RNA Polymerase during σ
70
Binding
Authors: Anthony Larry C. , Dombkowski Alan A. , Burgess Richard R.
Published: July 2002

RNA polymerase of
Escherichia coli
is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ
70
both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ
70
. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ
70
. We also show that the locked coiled-coil is capable of inducing a conformational change within σ
70
that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ
70
or upon recognition of the −10 nontemplate strand of DNA.
Construction and Characterization of a
cheA
Mutant of
Treponema denticola
Authors: Lux Renate , Sim Jee-Hyun , Tsai Jon P. , Shi Wenyuan
Published: July 2002

The
Treponema denticola cheA
gene, encoding the central kinase of the general chemotaxis pathway, was analyzed for its role in chemotaxis and tissue penetration. The
cheA
gene was interrupted by insertion of an
ermF-ermAM
gene cassette. Reverse transcription-PCR confirmed that the other downstream chemotaxis genes within the same operon (
cheW
,
cheX
, and
cheY
) were still expressed in the
cheA
mutant strain. Lack of
cheA
resulted in decreased swarming on soft-agar swarm plates and failure to respond chemotactically to a mixture of nutrients. Behavioral analyses using video microscopy revealed that the
cheA
mutant exhibited coordinated cell movement. The cellular reversal frequency, however, was severely reduced, indicating that CheA in
T. denticola
mainly controls cellular reversal and that active chemotaxis signaling input is not required for coordination of flagellar rotation at both cell poles.
Multilocus Genotyping Indicates that the Ability To Invade the Bloodstream Is Widespread among
Candida albicans
Isolates
Authors: Luu Lien N. , Cowen Leah E. , Sirjusingh Caroline , Kohn Linda M. , Anderson James B.
Published: July 2002

Multilocus genotyping was used to compare populations of
Candida albicans
from oral mucosa and blood. No significant differences in allele frequencies between the two samples were detected, and in a dendrogram of genotypic similarities, genotypes from both types of samples were finely interspersed. This is evidence for widespread distribution of invasive potential.
Clinical Relevance of the
babA2
Genotype of
Helicobacter pylori
in Japanese Clinical Isolates
Authors: Mizushima Takuji , Sugiyama Toshiro , Komatsu Yoshito , Ishizuka Jun , Kato Mototsgu , Asaka Masahiro
Published: July 2002

Genotypic variation of
Helicobacter pylori
is speculated to associate with different clinical outcomes. In Western countries, the gene encoding blood group antigen-binding adhesin (BabA),
babA2
, is of high clinical relevance and is a useful marker to identify patients who are at higher risk for peptic ulceration and gastric adenocarcinoma, as are
vacA
and
cagA
. We investigated the presence of
babA2
and
cagA
in 179 Japanese clinical isolates by PCR and Southern blot analysis and looked for correlations with various clinical outcomes (nonulcer dyspepsia, duodenal ulcers, gastric ulcers gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma). The prevalence of the
babA2
genotype was 84.9% and that of the
cagA
genotype was 96.1%. There was no correlation between the
babA2
and
cagA
genotypes, and there was no association between the
babA2
or
cagA
status and clinical outcome. These results indicate that
babA2
status is not of high clinical relevance in Japan and that Japanese strains are different from those infecting Western populations.
Quantitative Detection of
Borrelia burgdorferi
in 2-Millimeter Skin Samples of Erythema Migrans Lesions: Correlation of Results with Clinical and Laboratory Findings
Authors: Liveris Dionysios , Wang Guiqing , Girao Gary , Byrne Daniel W. , Nowakowski John , McKenna Donna , Nadelman Robert , Wormser Gary P. , Schwartz Ira
Published: July 2002

Variability of disease manifestations has been noted in patients with Lyme disease. A contributing factor to this variation may be the number of spirochetes present in infected patients. We evaluated clinical and laboratory findings for patients with erythema migrans with regard to the number of
Borrelia burgdorferi
organisms detected by quantitative PCR (qPCR) in 2-mm skin biopsy specimens.
B. burgdorferi
was detected in 80% (40 of 50) of the specimens tested; the mean number of spirochetes in these specimens ranged over 3 orders of magnitude (10 to 11,000 spirochetes per 2-mm biopsy specimen). Larger numbers of spirochetes were significantly associated with a shorter duration of the erythema migrans skin lesion (
P
= 0.020), smaller skin lesions (
P
= 0.020), and infection with a specific genotype of
B. burgdorferi
(
P
= 0.008) but not with the number or severity of symptoms. Skin culture positivity was significantly associated with skin lesions containing larger numbers of spirochetes (
P
= 0.019).
Coronavirus-Induced Demyelination Occurs in the Absence of Inducible Nitric Oxide Synthase
Authors: Wu Gregory F. , Pewe Lecia , Perlman Stanley
Published: July 2002

Demyelination induced by mouse hepatitis virus (MHV), strain JHM, is in large part immune mediated, but little is known about the mechanisms involved in this process. Previous results suggest that inducible nitric oxide synthase (NOS2) contributes transiently to MHV-induced demyelination. Herein, we show that equivalent amounts of demyelination were evident at day 12 after MHV infection in mice genetically deficient in NOS2 (NOS2−/−) and in C57BL/6 mice. Furthermore, using an established adoptive transfer model and pharmacological inhibitors of NOS2 function, we could demonstrate no effect on MHV-induced demyelination. These results indicate that NOS2 function is not required for demyelination in mice infected with MHV.
Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints
Authors: Lamartina Stefania , Ciliberto Gennaro , Toniatti Carlo
Published: July 2002

The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (
trs
). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS and
trs
homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1
trs
only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1
trs
sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.