bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiple Pathways of Spx (YjbD) Proteolysis inBacillus subtilis
Authors: Nakano Shunji , Zheng Guolu , Nakano Michiko M. , Zuber Peter 
Published: July 2002

ABSTRACTATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways. Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development ofBacillus subtilis. Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation. Thespx(formallyyjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred byclpPandclpX. The level of Spx in wild-type cells grown in competence medium is low, and that inclpPmutants is high. This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from theclpPmutation. In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx. Western blot analysis showed that Spx accumulated inclpXmutant to the same level as that observed in theclpPmutant. In contrast, a very low concentration of Spx was detected in aclpCmutant. An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH. However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx. Transcription ofspx, as measured by expression ofspx-lacZ, was slightly increased by theclpXmutation. To exclude a possible effect ofclpXandclpPonspxtranscription, thespxgene was placed under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible Pspacpromoter. In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx. Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP. The putative proteolysis by ClpXP might require another adapter protein. Spx probably is degraded by ClpCP under as yet unidentified conditions. This study suggests that the level of Spx is tightly controlled by two different ClpP proteases.
Genetic Variability of
Yersinia pestis
Isolates as Predicted by PCR-Based IS
100
Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (
glpD
)
Authors: Motin Vladimir L. , Georgescu Anca M. , Elliott Jeffrey M. , Hu Ping , Worsham Patricia L. , Ott Linda L. , Slezak Tomas R. , Sokhansanj Bahrad A. , Regala Warren M. , Brubaker Robert R. , Garcia Emilio 
Published: July 2002

ABSTRACT

A PCR-based genotyping system that detects divergence of IS
100
locations within the
Yersinia pestis
genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS
100
and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in
Y. pestis
suggested that a 93-bp deletion within the
glpD
gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The
glpD
gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the
glpD
version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different
Yersinia
strains in nature. The relevance of this observation on the emergence of epidemic
Y. pestis
strains is discussed.
RecA Protein from the Extremely Radioresistant Bacterium
Deinococcus radiodurans
: Expression, Purification, and Characterization
Authors: Kim Jong-Il , Sharma Ajay K. , Abbott Stephen N. , Wood Elizabeth A. , Dwyer David W. , Jambura Aaron , Minton Kenneth W. , Inman Ross B. , Daly Michael J. , Cox Michael M. 
Published: July 2002

ABSTRACT

The RecA protein of
Deinococcus radiodurans
(RecA
Dr
) is essential for the extreme radiation resistance of this organism. The RecA
Dr
protein has been cloned and expressed in
Escherichia coli
and purified from this host. In some respects, the RecA
Dr
protein and the
E. coli
RecA (RecA
Ec
) proteins are close functional homologues. RecA
Dr
forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA
Ec
. The RecA
Dr
protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the
E. coli
SSB protein. As is the case with the
E. coli
RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA
Dr
protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA
Dr
protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA
Dr
protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA
Dr
protein binds much better to duplex DNA than the RecA
Ec
protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in
Deinococcus
.
Protective Role of
tolC
in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate
Authors: Shyu J. Bruce H. , Lies Douglas P. , Newman Dianne K. 
Published: July 2002

ABSTRACT

Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer. Here we provide genetic evidence that AQDS enters
Shewanella oneidensis
strain MR-1 and causes cell death if it accumulates past a critical concentration. A
tolC
homolog protects the cell from toxicity by mediating the efflux of AQDS. Electron transfer to AQDS appears to be independent of the
tolC
pathway, however, and requires the outer membrane protein encoded by
mtrB
. We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics.
Genetic Diversity of
Mycobacterium africanum
Clinical Isolates Based on IS
6110
-Restriction Fragment Length Polymorphism Analysis, Spoligotyping, and Variable Number of Tandem DNA Repeats
Authors: Viana-Niero Cristina , Gutierrez Cristina , Sola Christophe , Filliol Ingrid , Boulahbal Fadila , Vincent Véronique , Rastogi Nalin 
Published: July 2002

ABSTRACT

A collection of 105 clinical isolates originally identified as
Mycobacterium africanum
were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the
M. tuberculosis
complex, whereas genotypic characterization was based on IS
6110
-restriction fragment length polymorphism (IS
6110
-RFLP) analysis, IS
1081
-RFLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyping), variable number of tandem DNA repeats (VNTR), and the polymorphism of the
oxyR
,
pncA
, and
mtp
40 loci. The results obtained showed that a majority of
M. africanum
isolates were characterized by a specific spoligotyping pattern that was intermediate between those of
M. tuberculosis
and
M. bovis
, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respectively. A tentative
M. africanum
-specific spoligotyping signature appeared to be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as the polymorphism of
oxyR
and
pncA
, a total of 24 isolates were excluded from the final study (19 were identified as
M. tuberculosis
, 2 were identified as
M. canetti
, and 3 were identified as
M. bovis
). The remaining 81
M. africanum
isolates were efficiently subtyped in three distinct subtypes (A1 to A3) by IS
6110
-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of the A2 subtype but showed significant variations for subtypes A1 and A3. A phylogenetic tree based on a selection of isolates representing the three subtypes using VNTR and spoligotyping alone or in combination confirmed the subtypes described as well as the heterogeneity of subtype A3.
Identification of Aichi Virus Infection by Measurement of Immunoglobulin Responses in an Enzyme-Linked Immunosorbent Assay
Authors: Yamashita Teruo , Ito Miyabi , Tsuzuki Hideaki , Sakae Kenji 
Published: July 2002

ABSTRACT
Using inhibitory enzyme-linked immunosorbent assay, seroconversions to Aichi virus were detected in 24 (42.9%) of 56 patients with gastroenteritis in six outbreaks. Virus-specific immunoglobulin M (IgM) was detected in convalescent-phase sera from 7 of 24 patients. Of the other 17 patients, 12 developed a significant increase in both IgA and IgG levels and 5 developed a significant increase in IgG alone.
Molecular Characterization of New Clinical Isolates of
Candida albicans
and
C. dubliniensis
in Japan: Analysis Reveals a New Genotype of
C. albicans
with Group I Intron
Authors: Tamura Miki , Watanabe Kayo , Mikami Yuzuru , Yazawa Katsukiyo , Nishimura Kazuko 
Published: July 2002

ABSTRACT

The genetic diversity of recent clinical isolates of
Candida albicans
in Japan was studied on the basis of amplified DNA band lengths determined with a specific PCR primer reported to have been designed to span a transposable intron region in the 25S rRNA gene. Our analyses of 301 clinical isolates of
C. albicans
showed that they could be classified into five genotypes: genotype A (172 isolates), genotype B (66 isolates), genotype C (56 isolates), genotype D (
C. dubliniensis
; 5 isolates), and a new genotype (designated genotype E; 2 isolates). The new genotype E was characterized to have a group I intron-like sequence, which is longer than hitherto reported ones and which has a nucleotide sequence length of 962 bp. Our analysis of the 962-bp sequence indicated that it is composed of an intron similar to that of
C. dubliniensis
of 621 bp with a 341-bp insertion. Analysis of the sequence of the internal transcribed spacer (ITS) region of the genotype E strain showed that its sequence is identical to those of strains of other genotypes, with only a few base substitution differences. Throughout the study, the possible horizontal transfer of the group I intron between
C. dubliniensis
and
C. albicans
was suggested. A high degree of correlation between the presence of a group I intron in
C. albicans
genotype E and susceptibility to the antifungal agent flucytosine was observed. The five isolates of
C. dubliniensis
examined in the present study showed genetic diversity when they were compared by randomly amplified polymorphic DNA fingerprinting pattern analysis, and this diversity was also confirmed by the analysis of ITS region sequences.
Persistent ICT Malaria P.f/P.v Panmalarial and HRP2 Antigen Reactivity after Treatment of
Plasmodium falciparum
Malaria Is Associated with Gametocytemia and Results in False-Positive Diagnoses of
Plasmodium vivax
in Convalescence
Authors: Tjitra Emiliana , Suprianto Sri , McBroom James , Currie Bart J. , Anstey Nicholas M. 
Published: July 2002

ABSTRACT

A problem with rapid
Plasmodium falciparum
-specific antigen histidine-rich protein 2 (HRP2) detection tests for malaria is the persistence of antigen in blood after the disappearance of asexual-stage parasitemia and clinical symptoms, resulting in false-positive (FP) test results following treatment. The ICT P.f/P.v immunochromatographic test detects both HRP2 and a panmalarial antigen (PMA) found in both
P. falciparum
and
Plasmodium vivax
. To examine posttreatment antigen persistence with this test and whether persistent sexual-stage forms (gametocytes) are a cause of FP tests after treatment, we compared serial antigen test results with microscopy results from patients symptomatic with
P. falciparum
malaria in Indonesia for 28 days following treatment with chloroquine (CQ;
n
= 66), sulfadoxine-pyrimethamine (SP;
n
= 36), and artesunate plus sulfadoxine-pyrimethamine (ART + SP;
n
= 15). Persistent FP antigenemia following SP treatment occurred in 29% (HRP2) and 42% (PMA) of the patients on day 7 and in 10% (HRP2) and 23% (PMA) on day 14. The high rates of persistent HRP2 and PMA antigenemia following CQ and SP treatment were strongly associated with the presence of gametocytemia, with the proportion with gametocytes on day 7 posttreatment being significantly greater in those with FP results than in those with true-negative PMA and HRP2 results. Gametocyte frequency on day 14 post-SP treatment was also greater in those with FP PMA results. Following SP treatment, PMA persisted longer than HRP2, giving an FP diagnosis of
P. vivax
in up to 16% of patients on day 14, with all FP
P. vivax
diagnoses having gametocytemia. In contrast, PMA was rapidly cleared following ART + SP treatment in association with rapid clearance of gametocytemia. Gametocytes appear to be an important cause of persistent posttreatment panmalarial antigenemia in areas of endemicity and may also contribute in part to persistent HRP2 antigenemia following treatment.
Novel Penicillin-, Cephalosporin-, and Macrolide-Resistant Clones of
Streptococcus pneumoniae
Serotypes 23F and 19F in Taiwan Which Differ from International Epidemic Clones
Authors: Chiou Christine C. , McEllistrem Mary Catherine 
Published: July 2002

ABSTRACT

A cluster (14 of 18) of
Streptococcus pneumoniae
serotype 23F isolates that were resistant to penicillin (PEN), cephalosporin, and macrolide was found in one day care center in Kaohsiung, Taiwan. We analyzed the 18 isolates by pulsed field gel electrophoresis (PFGE). All but one serotype 23F isolate demonstrated identical PFGE patterns, which were different from the established pattern of the internationally spread Spanish 23F clone. The three strains of serotype 19F also showed a uniform pattern. These data strongly suggest that two novel clones of PEN-, cephalosporin-, and macrolide-resistant
S. pneumoniae
serotypes 23F and 19F are present in Taiwan.
Development of a Species-Specific PCR Assay for Detection of
Leishmania donovani
in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis
Authors: Salotra Poonam , Sreenivas G. , Pogue Gregory P. , Lee Nancy , Nakhasi Hira L. , Ramesh V. , Negi N. S. 
Published: July 2002

ABSTRACT

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of
Leishmania donovani
in a species-specific manner among Old World leishmanias. With Indian strains and isolates of
L. donovani
the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz.,
Mycobacterium tuberculosis, Mycobacterium leprae
, and
Plasmodium
spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.
Novel Approach To Reduce the Hepatitis C Virus (HCV) Window Period: Clinical Evaluation of a New Enzyme-Linked Immunosorbent Assay for HCV Core Antigen
Authors: Icardi Giancarlo , Ansaldi Filippo , Bruzzone Bianca Marisa , Durando Paolo , Lee Stephen , de Luigi Carla , Crovari Pietro 
Published: July 2002

ABSTRACT
The window period in hepatitis C virus (HCV) infection is still a major problem in ensuring blood safety. HCV RNA detection by nucleic acid amplification technology-based tests has contributed to reduce the infectivity of blood products, but it is expensive, time-consuming and affected by a high prevalence of false-positive results. The aim of this study was to assess the performance of a newly developed enzyme immunoassay for the detection of HCV core antigen and its suitability for use in the screening of blood units in order to identify infecting samples that do not contain specific antibodies. For evaluation of laboratory performance, different samples were selected: to evaluate specificity, we tested 2,586 sera from blood donors, 500 general population samples, and 58 “difficult sera”. All samples were tested by two screening assays, and results were negative. To estimate clinical sensitivity, 103 HCV RNA-positive, anti-HCV-negative samples, 6 natural seroconversion panels, and 9 commercial seroconversion panels were tested. Intra- and interassay precision were determined on two HCV-RNA-positive, anti-HCV-negative sera. Seventeen (0.66%) blood donor samples, 2 (0.4%) general population samples, and 2 (3.44%) difficult sera were initially reactive; 3 sera were positive on repetition. These 21 samples tested by reverse transcription-PCR were negative. The clinical sensitivity calculated with seroconversion panels and seroconverted patient samples was very similar to PCR sensitivity: 95% of PCR-positive, antibody-negative samples contained detectable HCV antigen. Data on intra- and interassay precision showed dispersion indices with values of less than 10%. In conclusion, the HCV antigen assay showed high sensitivity and specificity and could become a useful means of improving the safety of blood and blood products.
Molecular Epidemiology of Astrovirus Infection in Barcelona, Spain
Authors: Guix Susana , Caballero Santiago , Villena Cristina , Bartolomé Rosa , Latorre Cristina , Rabella Nuria , Simó Maria , Bosch Albert , Pintó Rosa M. 
Published: July 2002

ABSTRACT
A 3-year study involving 2,347 gastroenteritis samples was conducted to determine the prevalence, time distribution, and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus-positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8, and HAstV-2. HAstV-5, HAstV-6, and HAstV-7 were not detected during these 3 years. From our serotype data for each age group, we observed that HAstV-1, HAstV-2, and HAstV-3 affected mostly children younger than 3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.
Variable-Number Tandem Repeat Typing ofMycobacterium tuberculosisIsolates with Low Copy Numbers of IS6110by Using Mycobacterial Interspersed Repetitive Units
Authors: Cowan Lauren Steinlein , Mosher Laura , Diem Lois , Massey Jeffrey P. , Crawford Jack T. 
Published: July 2002

ABSTRACTA study set of 180Mycobacterium tuberculosisandMycobacterium bovisisolates having low copy numbers of IS6110were genotyped using the recently introduced method based on the variable-number tandem repeats ofmycobacterialinterspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.