bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a Gene Cluster in
Klebsiella pneumoniae
Which Includes
citX
, a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group
Authors: Schneider Karin , Kästner Christopher N. , Meyer Margareta , Wessel Mirja , Dimroth Peter , Bott Michael 
Published: July 2002

ABSTRACT

The biosynthesis of the 2′-(5"-phosphoribosyl)-3′-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2′-(5"-triphosphoribosyl)-3′-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In
Escherichia coli
, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster,
citCDEFXG
. In the homologous
citCDEFG
operon of
Klebsiella pneumoniae
,
citX
is missing. A search for
K. pneumoniae citX
led to the identification of a second genome region involved in citrate fermentation which comprised the
citWX
genes and the divergent
citYZ
genes. The
citX
gene was confirmed to encode holo-citrate lyase synthase, whereas
citW
was shown to encode a citrate carrier, the third one identified in this species. The
citYZ
genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed ≥40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in
K. pneumoniae
. A
citZ
insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.
Detection of Antibodies to a Pathogenic Mycoplasma in American Alligators (Alligator mississippiensis), Broad-Nosed Caimans (Caiman latirostris), and Siamese Crocodiles (Crocodylus siamensis)
Authors: Brown D. R. , Schumacher I. M. , Nogueira M. F. , Richey L. J. , Zacher L. A. , Schoeb T. R. , Vliet K. A. , Bennett R. A. , Jacobson E. R. , Brown M. B. 
Published: July 2002

ABSTRACTAn epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995.Mycoplasma alligatorissp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled forM. alligatorisas the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response toM. alligatorisexposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P< 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P< 0.05) in the relatively closely related broad-nosed caimanCaiman latirostrisand the relatively distantly related Siamese crocodileCrocodylus siamensisfollowing experimental inoculation withM. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogenM. alligatorisamong captive, repatriated, and wild crocodilian species.
Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies toNeospora caninumin Cattle
Authors: Baszler Timothy V. , Adams Scott , Vander-Schalie John , Mathison Bruce A. , Kostovic Miladin 
Published: July 2002

ABSTRACTA previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody toNeospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing nativeN. caninumantigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a “gold standard” set of 184 cow sera defined by fetal histopathology andN. caninumimmunohistochemistry and by maternalN. caninumindirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknownN. caninumstatus. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknownN. caninumstatus revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modifiedN. caninumcELISA provided a simple, rapid, and versatile method to accurately identifyN. caninuminfection status in cattle using a single cutoff value.
Comparison of Methods for Identification of
Mycobacterium abscessus
and
M. chelonae
Isolates
Authors: Yakrus Mitchell A. , Hernandez S. Moises , Floyd Margaret M. , Sikes David , Butler W. Ray , Metchock Beverly 
Published: July 2002

ABSTRACT

Mycobacterium abscessus
and
Mycobacterium chelonae
are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either
M. abscessus
or
M. chelonae
that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be
M. abscessus
and 29 were identified as
M. chelonae
. Eight isolates originally submitted as
M. chelonae
were identified as
M. abscessus
, and one isolate submitted as
M. abscessus
was found to be
M. chelonae
. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of
M. abscessus
exhibited resistance to tobramycin (MIC of 8 to ≥16 μg/ml), while all isolates of
M. chelonae
were susceptible to this drug (MIC of ≤4 μg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of
M. abscessus
and
M. chelonae
.
Identification of
Mycobacterium avium
subsp.
paratuberculosis
in Biopsy Specimens from Patients with Crohn's Disease Identified by In Situ Hybridization
Authors: Sechi Leonardo A. , Manuela Mura , Francesco Tanda , Amelia Lissia , Antonello Solinas , Giovanni Fadda , Stefania Zanetti 
Published: July 2002

ABSTRACT

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient
Mycobacterium avium
subsp.
paratuberculosis
in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS
900
as a probe.
Molecular and Phenotypic Characterization of Potentially New
Shigella dysenteriae
Serotype
Authors: Coimbra Roney S. , Lenormand Pascal , Grimont Francine , Bouvet Philippe , Matsushita Shigeru , Grimont Patrick A. D. 
Published: July 2002

ABSTRACT

From September 1997 to November 1998, the French National Center for
Salmonella
and
Shigella
received 22
Shigella
isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established
Shigella
serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new
Shigella dysenteriae
serotype (represented by strain 96–204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96–204, showed biochemical reactions typical of
S. dysenteriae
and gave positive results in a PCR assay for detection of the plasmid
ipaH
gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to
S. dysenteriae
serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other
Shigella
or
Escherichia coli
serotypes.
Comparison of Quantitative and Qualitative PCR Assays for Cytomegalovirus DNA in Plasma
Authors: Caliendo Angela M. , Schuurman Rob , Yen-Lieberman Belinda , Spector Stephen A. , Andersen Janet , Manjiry R. , Crumpacker Clyde , Lurain Nell S. , Erice Alejo 
Published: July 2002

ABSTRACT
We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.
Precore Stop Mutant in HBeAg-Positive Patients with Chronic Hepatitis B: Clinical Characteristics and Correlation with the Course of HBeAg-to-Anti-HBe Seroconversion
Authors: Chu Chia-Ming , Yeh Chau-Ting , Lee Ching-Song , Sheen I-Shyan , Liaw Yun-Fan 
Published: July 2002

ABSTRACTThis study aimed to investigate the ratios of precore stop mutant (codon 28; TGG to TAG) to total viremia in 53 HBeAg-positive patients with chronic hepatitis B by amplification-created restriction site assays along the course of HBeAg-to-anti-HBe seroconversion. At baseline, 11% had exclusive wild-type hepatitis B virus (HBV), 15% had exclusively precore mutant, and 74% had mixed viral strains. Precore mutant ratios correlated little with age, sex, or HBV DNA levels (allP> 0.1), but correlated modestly with alanine aminotransferase (ALT) levels (P= 0.05). The intervals from presentation to anti-HBe seroconversion correlated significantly with ALT and precore mutant ratios in univariate analysis but with only precore mutant ratios in multivariate analysis (P= 0.003). Precore mutant ratios at baseline were significantly higher (P< 0.001) in six patients with persistent high viremia and ALT elevation after anti-HBe seroconversion (group 1) than in 47 with remission (group 2). All group 1 patients had exclusive precore mutant after anti-HBe seroconversion, as did only 14 (30%) of the group 2 patients (P= 0.003). Among group 2 patients, precore mutant ratios at baseline or after anti-HBe seroconversion showed no significant difference between 34 patients with sustained remission and 13 with relapse. Cirrhosis developed in 50% (5 of 10) of patients with precore mutant ratios >50% at baseline but only in 12% (5 of 43) of those with precore mutant ratios of <50% at baseline (P< 0.05). In conclusion, precore mutant of variable ratios was frequently detected in HBeAg-positive patients with chronic hepatitis B. Precore mutant ratios tended to correlate with ALT levels and anti-HBe seroconversion, but high precore mutant ratios were associated with persistent hepatitis after anti-HBe seroconversion and increased risk of cirrhosis.
Comparative Evaluation of the VERSANT HCV RNA 3.0, QUANTIPLEX HCV RNA 2.0, and COBAS AMPLICOR HCV MONITOR Version 2.0 Assays for Quantification of Hepatitis C Virus RNA in Serum
Authors: Germer Jeffrey J. , Heimgartner Paul J. , Ilstrup Duane M. , Harmsen W. Scott , Jenkins Greg D. , Patel Robin 
Published: July 2002

ABSTRACT

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (
P
= 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (
P
≤ 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (
P
≤ 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.
Application of a 5′ Nuclease Assay for Detection ofLawsonia intracellularisin Fecal Samples from Pigs
Authors: Lindecrona R. H. , Jensen T. K. , Andersen P. H. , Møller K. 
Published: July 2002

ABSTRACTA 5′ nuclease assay was developed to detectLawsonia intracellularisin porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5′ nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of immunohistochemistry (IM) on ileal sections of the same animals. There was 91% agreement between the results of IM and the 5′ nuclease assay. In the 5′ nuclease assay, 111 (54%) of the pigs tested positive forL. intracellularisinfection, with a mean cycle threshold (Ct) value of 27.2, whereas 98 (48%) of the pigs tested positive by IM. On average, the Ct and ΔRn values for the positive samples were 27.2 (standard deviation [SD], 3.7) and 1.6 (SD, 0.7), respectively. A Ct value of 27.2 corresponds to a fecal excretion of approximately 107L. intracellulariscells per g of feces. Furthermore, a total of 40 fecal samples derived from a herd known to be free from infection withL. intracellularisall tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1L. intracellulariscell per PCR tube. In conclusion, the 5′ nuclease assay that has been developed represents an applicable fast method for detection ofL. intracellularisin fecal samples, with a sensitivity and specificity comparable to those of IM.
Human Lymphocyte Apoptosis after Exposure to Influenza A Virus
Authors: Nichols Joan E. , Niles Jean A. , Roberts Norbert J. 
Published: July 2002

ABSTRACTInfection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3+, CD4+, CD8+, and CD19+lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.
Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype
Authors: Boot Hein J. , ter Huurne A. Agnes H. M. , Hoekman Arjan J. W. , Peeters Ben P. H. , Gielkens Arno L. J. 
Published: July 2002

ABSTRACT
Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.
Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity
Authors: Nixdorf Ralf , Klupp Barbara G. , Karger Axel , Mettenleiter Thomas C. 
Published: July 2002

ABSTRACT

Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) are the most highly conserved glycoproteins within the family
Herpesviridae
and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral cell-to-cell spread. These processes, though related, appear to be distinct, and thus it was interesting to analyze whether they require different functions of gB. To this end, we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracytoplasmic α-helical domain encompassing putative YQRL and dileucine internalization signals, (ii) two predicted intracytoplasmic α-helical domains, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmic domain and the transmembrane anchor region. Confocal laser scanning microscopy showed that gB derivatives lacking at least the last 29 amino acids (aa) localize close to the plasma membrane, while the full-length protein accumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB
−
) demonstrated that the 29-aa truncated form lacking the putative internalization signals and the C-terminal α-helical domain (gB-008) was efficiently incorporated into PrV-gB
−
virions and efficiently complemented infectivity and cell-to-cell spread. Moreover, gB-008 exhibited an enhanced fusogenic activity. In contrast, gB proteins lacking both α-helical domains (gB-007), the complete intracytoplasmic domain, or the intracytoplasmic domain and transmembrane anchor were only inefficiently or not at all incorporated into PrV-gB
−
virions and did not complement infectivity. However, gB-007 was able to mediate cell-to-cell spread of PrV-gB
−
. Similar phenotypes were observed when virus recombinants expressing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not required for gB incorporation into virions nor for its function in either entry or cell-to-cell spread. Moreover, they indicate different requirements for gB in these membrane fusion processes.