bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Characterization of
linR
, Involved in Regulation of the Downstream Pathway for γ-Hexachlorocyclohexane Degradation in
Sphingomonas paucimobilis
UT26
Authors: Miyauchi Keisuke , Lee Haeng-Seog , Fukuda Masao , Takagi Masamichi , Nagata Yuji 
Published: July 2002

ABSTRACT

In
Sphingomonas paucimobilis
UT26, LinD and LinE activities, which are responsible for the degradation of γ-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the
linE
gene was determined, and an open reading frame (ORF) was found in divergent orientation from
linE
. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it
linR
. The fragment containing the putative LTTR recognition sequence (a palindromic TN
11
A sequence), which exists immediately upstream of
linE
, was ligated with the reporter gene
lacZ
and was inserted into the plasmid expressing LinR under the control of the
lac
promoter. When the resultant plasmid was introduced into
Escherichia coli
, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in
linR
, revealed that the expression of the
linD
and
linE
genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the
linR
gene is directly involved in the inducible expression of the
linD
and
linE
genes.
Influence of Host Factors on Immunoglobulin G Concentration in Oral Fluid Specimens
Authors: Granade Timothy C. , Phillips Susan K. , Kitson-Piggott Wendy , Gomez Perry , Mahabir Bisram , Oleander Herbert , George J. Richard , Baggs James , Parekh Bharat 
Published: July 2002

ABSTRACT

The influence of host factors (tobacco use, dentition, bleeding gums, oral rinsing, nasal medications, and time since the last meal) on immunoglobulin G (IgG) concentration in oral fluids (OF) was determined by univariate and multivariate analysis. Significant differences in IgG concentration were found to be associated with human immunodeficiency virus (HIV) status (HIV antibody positive, +16.60 μg/ml,
P
= 0.0001), sex (female, +1.23 μg/ml,
P
= 0.004), dentition (+2.83 μg/ml, edentulous versus dentulous,
P
= 0.0001), bleeding gums (+6.35 μg/ml,
P
= 0.0001), and time since the last meal (+3.55 μg/ml, >6 h,
P
= 0.0001). These factors could impact diagnostic methods that rely on the immunoglobulin concentration in OF specimens.
Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis
Authors: Berner Reinhard , Fürll Birgitt , Stelter Felix , Dröse Jana , Müller Hans-Peter , Schütt Christine 
Published: July 2002

ABSTRACTNo data on lipopolysaccharide-binding protein (LBP) in newborns with sepsis have been available up to now. We therefore determined levels of LBP and soluble CD14 (sCD14) in plasma of healthy and septic neonates in order to evaluate their potential diagnostic role. The study included prospectively collected patient samples of two recently published studies on cytokine expression in neonatal sepsis. Twenty-nine septic patients were enrolled in the present analysis. Samples—either cord blood or peripheral blood—from patients admitted within the first 24 h of life for suspicion of sepsis and cord blood samples of a control group of 40 healthy mature infants delivered spontaneously were analyzed. For seven patients of the septic group, a second sample collected between 24 and 48 h of life was available. Levels of sCD14 and LBP in plasma were determined by an enzyme immunoassay using recombinant CD14 and LBP as standards. LBP and sCD14 were correlated to cytokine plasma levels. In septic neonates, LBP (median, 36.6 versus 7.8 μg/ml;P< 0.001) and sCD14 (median, 0.42 versus 0.28 μg/ml;P< 0.001) levels were highly elevated when compared to those of healthy neonates and strongly correlated to granulocyte colony-stimulating factor (G-CSF), interleukin-1β (IL-1β), IL-6, and IL-8 levels. LBP levels in septic neonates analyzed between 24 and 48 h of life even increased when compared to samples obtained at or shortly after delivery (median, 36.6 versus 60 μg/ml;P= 0.038). In summary, levels of LBP in plasma of neonates with early-onset sepsis are significantly elevated; the elevated plasma levels seem to persist for more than 24 h, which could provide the clinician with a prolonged time period to identify the newborn with bacterial sepsis.
Metalloprotease Is Not Essential for
Vibrio vulnificus
Virulence in Mice
Authors: Shao Chung-Ping , Hor Lien-I 
Published: July 2002

ABSTRACT

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of
Vibrio vulnificus
, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of
Vibrio vulnificus
by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.
Temperature-Regulated Protein Synthesis by
Leptospira interrogans
Authors: Nally Jarlath E. , Timoney John F. , Stevenson Brian 
Published: July 2002

ABSTRACT

Leptospira interrogans
is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in
L. interrogans
. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with
L. interrogans
serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.
Cellular and Humoral Immune Responses toBorrelia burgdorferiAntigens in Patients with Culture-Positive Early Lyme Disease
Authors: Vaz Austin , Glickstein Lisa , Field Jodie A. , McHugh Gail , Sikand Vijay K. , Damle Nitin , Steere Allen C. 
Published: July 2002

ABSTRACTWe determined cellular and humoral immune responses toBorrelia burgdorferilysate and to recombinant flagellin (FlaB), OspC, and OspA in acute- and convalescent-phase samples from 39 culture-positive patients with erythema migrans and in 20 healthy control subjects. During the acute illness, a median of 4 days after the onset of erythema migrans, 51% of the patients had proliferative cellular responses and 72% had antibody responses to at least one of the borrelial antigens tested. During convalescence, at the conclusion of antibiotic therapy, 64% of the patients had proliferative cellular reactivity and 95% had antibody reactivity with at least one of the spirochetal antigens tested. In both acute- and convalescent-phase samples, cellular immune responses were found as frequently to OspA as to OspC and FlaB. Although antibody responses were also frequently seen to OspC and FlaB, only a few patients had marginal antibody reactivity with OspA. The percentage of patients with proliferative responses was similar in those with clinical evidence of localized or disseminated infection, whereas humoral reactivity was found more often in those with disseminated disease. We conclude that cellular and humoral responses toB. burgdorferiantigens are often found among patients with early Lyme disease. In contrast with the other antigens tested, cellular but not humoral reactivity was often found with OspA.
Counter-Protective Role for Interleukin-5 during AcuteToxoplasma gondiiInfection
Authors: Nickdel M. B. , Roberts F. , Brombacher F. , Alexander J. , Roberts C. W. 
Published: July 2002

ABSTRACTThe role of interleukin-5 (IL-5) duringToxoplasma gondiiinfection was investigated by comparing disease progression in IL-5 gene deficient (IL-5−/−) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5−/− mice infected orally withT. gondiiwere less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5−/− mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5−/− mice produced levels of IL-12, interferon-γ (IFN-γ), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated withToxoplasmalysate antigen. However, peak serum IL-12 and IFN-γ levels (at days 6 and 8, respectively) were significantly higher in IL-5−/− mice than in WT mice. In addition, WT mice but not IL-5−/− mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration ofNω-nitro-l-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5−/− and WT mice, indicating a protective role for nitric oxide during the early stages of oralT. gondiiinfection. In comparison with oral infection, no difference in mortality was observed between IL-5−/− and WT mice following intraperitoneal infection withT. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection withT. gondiiwhich is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-γ levels.
Prolonged Survival and Cytoplasmic pH Homeostasis of
Helicobacter pylori
at pH 1
Authors: Stingl Kerstin , Uhlemann Eva-Maria , Deckers-Hebestreit Gabriele , Schmid Roland , Bakker Evert P. , Altendorf Karlheinz 
Published: July 2002

ABSTRACT

In the presence of urea,
Helicobacter pylori
survived for at least 3 h at pH 1. Under these conditions, the cells maintained their cytoplasmic pH at 5.8. De novo protein synthesis during acid shock was not essential for survival of
H. pylori
at pH 1.
Complete Nucleotide Sequence and Analysis of the Locus of Enterocyte Effacement from Rabbit Diarrheagenic
Escherichia coli
RDEC-1
Authors: Zhu Chengru , Agin Tonia S. , Elliott Simon J. , Johnson Laura A. , Thate Timothy E. , Kaper James B. , Boedeker Edgar C. 
Published: July 2002

ABSTRACT

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic
Escherichia coli
strain RDEC-1 (O15:H−) and compared it with those from human enteropathogenic
E. coli
(EPEC, O127:H6) and enterohemorrhagic
E. coli
(EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at
selC
and is flanked by an IS2 element and the
lifA
toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC.
orf3
and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of
LEE1, LEE2, LEE3, tir
, and
LEE4
operons are highly conserved among the LEEs, although the upstream regions varied considerably for
tir
and the crucial
LEE1
promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.
Identification of Continuous B-Cell Epitopes on the Protein Moiety of the 58-Kilodalton Cell Wall Mannoprotein ofCandida albicansBelonging to a Family of Immunodominant Fungal Antigens
Authors: Viudes Angel , Perea Sofia , Lopez-Ribot Jose L. 
Published: July 2002

ABSTRACTThe 58-kiloDalton mannoprotein (mp58) on the surface ofCandida albicansis highly immunogenic, is expressed by allC. albicansisolates tested, and elicits strong antibody responses during candidiasis. It belongs to a family of immunodominant fungal antigens with representatives also in different species ofAspergillus. The amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) was used to synthesize a complete set of overlapping dodecapeptides (overlap, 7; offset, 5) covalently attached to the surface of derivatized polyethylene pins. The pin-coupled peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by a number of antiserum preparations containing anti-mp58 antibodies. This comprehensive epitope-scanning study revealed the presence of multiple immunoreactive continuous B-cell epitopes within the protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein (encompassing amino acid residues 16 to 50 and 286 to 299, respectively) and four internal regions spanning amino acids at positions 66 to 92, 121 to 142, 148 to 192, and 211 to 232. Further delineation of epitopic regions and identification of the boundaries of the antigenic sites was performed upon ELISA testing with a second Pepset consisting of completely overlapping 8-mer peptides spanning these reactive regions in the protein moiety of mp58. The highly reactive epitopic region at the C terminus of the protein was further evaluated using both window net and replacement net analyses. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein was immunogenic when injected into mice after being coupled to a carrier protein. Moreover, antibodies in the resulting sera specifically recognized the homologus mp58 in ELISAs and immunoblot assays. Delineation of the antibody responses to mp58 could provide the basis for the development of novel immunity-based prophylactic, therapeutic, and diagnostic techniques for the management of candidiasis.
Fas-Mediated Apoptosis of Neutrophils in Sera of Patients with Infection
Authors: Nwakoby Izuchukwu E. , Reddy Krishna , Patel Puja , Shah Neena , Sharma Saroj , Bhaskaran Madhu , Gibbons Nora , Kapasi Aditi A. , Singhal Pravin C. 
Published: July 2002

ABSTRACTIn the presence of infection, neutropenia is considered to be a marker of poor prognosis; conversely, neutrophilia may not be a determinant of a better prognosis. Since apoptotic neutrophils are compromised functionally, we evaluated the effect of infection on neutrophil apoptosis. The rate of apoptosis was greater for neutrophils isolated from patients with infection than for healthy controls.Escherichia colidid not directly modulate the rate of neutrophil apoptosis. However, sera from infected patients promoted (P< 0.001) neutrophil apoptosis. Interestingly, the sera of patients with different types of infection (gram negative, gram positive, or culture negative) exerted a more or less identical response on neutrophil apoptosis. Sera of infected patients showed a fivefold greater content of FasL compared to controls. Moreover, anti-FasL antibody partly attenuated the infected-serum-induced neutrophil apoptosis. In in vitro studies,E. colienhanced monocyte FasL expression. Moreover, conditioned media prepared from activated macrophages from control mice showed enhanced apoptosis of human as well as mouse neutrophils. On the contrary, conditioned media prepared from activated macrophages isolated from FasL-deficient mice induced only a mild degree of neutrophil apoptosis. These results suggest that neutrophils in patients with infection undergo apoptosis at an accelerated rate. Infection not only promoted monocyte expression of FasL but also increased FasL content of the serum. Because the functional status of apoptotic cells is compromised, a significant number of neutrophils may not be participating in the body's defense. Since neutrophils play the most important role in innate immunity, their compromised status in the presence of infection may transfer the host defense burden from an innate response to acquired immunity. The present study provides some insight into the lack of correlation between neutrophilia and the outcome of infection.
Lysine 2,3-Aminomutase from
Clostridium subterminale
SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene
kamA
in
Escherichia coli
Authors: Ruzicka Frank J. , Lieder Kafryn W. , Frey Perry A. 
Published: July 2002

ABSTRACT

Lysine 2,3-aminomutase (KAM, EC
5.4.3.2
.) catalyzes the interconversion of
l
-lysine and
l
-β-lysine, the first step in lysine degradation in
Clostridium subterminale
SB4. KAM requires
S
-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5′-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene
kamA
for
C. subterminale
SB4 KAM and conditions for its expression in
Escherichia coli
. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and
C. subterminale
SB4 chromosomal DNA to produce a portion of
kamA
containing 1,029 base pairs of the gene. The complete gene was obtained from a genomic library of
C. subterminale
SB4 chromosomal DNA by use of DNA probe analysis based on the 1,029-base pair fragment. The full-length gene consisted of 1,251 base pairs specifying a protein of 47,030 Da, in reasonable agreement with 47,173 Da obtained by electrospray mass spectrometry of the purified enzyme. N- and C-terminal amino acid analysis of KAM and its cyanogen bromide peptides firmly correlated its amino acid sequence with the nucleotide sequence of
kamA
. A survey of bacterial genome databases identified seven homologs with 31 to 72% sequence identity to KAM, none of which were known enzymes. An
E. coli
expression system consisting of pET 23a(+) plus
kamA
yielded unsatisfactory expression and bacterial growth. Codon usage in
kamA
includes the use of AGA for all 29 arginine residues. AGA is rarely used in
E. coli
, and arginine clusters at positions 4 and 5, 25 and 27, and 134, 135, and 136 apparently compound the barrier to expression. Coexpression of
E. coli argU
dramatically enhanced both cell growth and expression of KAM. Purified recombinant KAM is equivalent to that purified from
C. subterminale
SB4.
Regulation of the
rulAB
Mutagenic DNA Repair Operon of
Pseudomonas syringae
by UV-B (290 to 320 Nanometers) Radiation and Analysis of
rulAB
-Mediated Mutability In Vitro and In Planta
Authors: Kim Jae J. , Sundin George W. 
Published: July 2002

ABSTRACT

The effects of the
rulAB
operon of
Pseudomonas syringae
on mutagenic DNA repair and the transcriptional regulation of
rulAB
following irradiation with UV-B wavelengths were determined. For a
rulB
::Km insertional mutant constructed in
P. syringae
pv. syringae B86-17, sensitivity to UV-B irradiation increased and UV mutability decreased by 12- to 14-fold.
rulAB
-induced UV mutability was also tracked in phyllosphere populations of B86-17 for up to 5 days following plant inoculation. UV mutability to rifampin resistance (Rif
r
) was detected at all sampling points at levels which were significantly greater than in nonirradiated controls. In
P. aeruginosa
PAO1, the cloned
rulAB
determinant on pJJK17 conferred a 30-fold increase in survival and a 200-fold increase in mutability following a UV-B dose of 1,900 J m
−2
. In comparative studies using defined genetic constructs, we determined that
rulAB
restored mutability to the
Escherichia coli umuDC
deletion mutant RW120 at a level between those of its homologs
mucAB
and
umuDC
. Analyses using a
rulAB
::
inaZ
transcriptional fusion in
Pseudomonas fluorescens
Pf5 showed that
rulAB
was rapidly induced after UV-B irradiation, with expression levels peaking at 4 h. At the highest UV-B dose administered, transcriptional activity of the
rulAB
promoter was elevated as much as 261-fold compared to that of a nonirradiated control. The importance of
rulAB
for survival of
P. syringae
in its phyllosphere habitat, coupled with its wide distribution among a broad range of
P. syringae
genotypes, suggests that this determinant would be appropriate for continued investigations into the ecological ramifications of mutagenic DNA repair.