bluuebunny/crossref_metadata_2025_split huggingface dataset:
Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal β-Class Carbonic Anhydrase
Authors: Smith Kerry S. , Ingram-Smith Cheryl , Ferry James G. 
Published: July 2002

ABSTRACT

The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon
Methanobacterium thermoautotrophicum.
Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the
k
cat
/

K
m

value depends only on the rate constants for the CO
2
hydration step, whereas
k
cat
also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol.
182:
6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem.
276:
10299-10305, 2001). The
k
cat
/

K
m

values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO
2
hydration step. Parallel decreases in
k
cat
and
k
cat
/

K
m

values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the
k
cat
of the D34A variant was chemically rescued by replacement of 2-(
N
-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both
k
cat
and
k
cat
/

K
m

values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the
k
cat
of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.
Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from
Vibrio cholerae
Authors: Beaber John W. , Hochhut Bianca , Waldor Matthew K. 
Published: July 2002

ABSTRACT

SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian
Vibrio cholerae
. We have determined the 100-kb DNA sequence of SXT. This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources. We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision. SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid. More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility. Two SXT loci, designated
setC
and
setD
, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer. Another locus, designated
setR
, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.
Glycine Betaine Transmethylase Mutant of
Pseudomonas aeruginosa
Authors: Serra Ana L. , Mariscotti Javier F. , Barra José L. , Lucchesi Gloria I. , Domenech Carlos E. , Lisa Angela T. 
Published: July 2002

ABSTRACT

The gene for glycine betaine transmethylase (
gbt
) was identified in
Pseudomonas aeruginosa
strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of
P. aeruginosa
PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in
gbt
blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.
The MexR Repressor of themexAB-oprMMultidrug Efflux Operon inPseudomonas aeruginosa: Characterization of Mutations Compromising Activity
Authors: Adewoye Lateef , Sutherland Ainsley , Srikumar Ramakrishnan , Poole Keith 
Published: July 2002

ABSTRACTMutations inmexRyield a multidrug resistance phenotype innalBmutants ofPseudomonas aeruginosaas a result of derepression of themexAB-oprMmultidrug efflux operon. MexR produced by severalnalBstrains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.
Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain
Authors: Ohashi Tomoo , Hale Cynthia A. , de Boer Piet A. J. , Erickson Harold P. 
Published: July 2002

ABSTRACT
The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.
Mutant Analysis Shows that Alanine Racemases from
Pseudomonas aeruginosa
and
Escherichia coli
Are Dimeric
Authors: Strych Ulrich , Benedik Michael J. 
Published: July 2002

ABSTRACT

Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor
d
-alanine. We present evidence that the enzymes from
Pseudomonas aeruginosa
and
Escherichia coli
function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of
dadX
in
E. coli
suppresses bacterial growth in a dominant negative fashion.
Rapid Detection of
Leishmania infantum
Infection in Dogs: Comparative Study Using an Immunochromatographic Dipstick Test, Enzyme-Linked Immunosorbent Assay, and PCR
Authors: Reithinger Richard , Quinnell Rupert J. , Alexander Bruce , Davies Clive R. 
Published: July 2002

ABSTRACT

Current zoonotic visceral leishmaniasis (ZVL) control programs in Brazil include the culling of
Leishmania infantum-
infected reservoir dogs, a strategy that has failed to prevent a rise of canine and human ZVL cases over the past decade. One of the main reasons this strategy has failed is because of a long delay between sample collection, sample analysis, and control implementation. A rapid, sensitive, and specific diagnostic tool would be highly desirable, because it would allow control interventions to be implemented in situ. We compared an immunochromatographic dipstick test to enzyme-linked immunosorbent assay (ELISA) and PCR for detecting
L. infantum
infections in dogs from an area of ZVL endemicity in Brazil. The dipstick test was shown to have 61 to 75% specificity and 72 to 77% sensitivity, compared to 100% specificity for both ELISA and PCR and 71 to 88% and 51 to 64% sensitivity for ELISA and PCR, respectively. Of the field samples tested, 92 of 175 (53%), 65 of 175 (37%), and 47 of 175 (27%) were positive by dipstick, ELISA, and PCR, respectively. The positive and negative predictive values for the tested dipstick were 58 to 77% and 75%, respectively. Efforts should be made to develop a more specific dipstick test for diagnosis of leishmaniasis, because they may ultimately prove more cost-effective than currently used diagnostic tests when used in mass-screening surveys.
Real-Time PCR for Quantification of Human Herpesvirus 6 DNA from Lymph Nodes and Saliva
Authors: Collot S. , Petit B. , Bordessoule D. , Alain S. , Touati M. , Denis F. , Ranger-Rogez S. 
Published: July 2002

ABSTRACT

A real-time quantitative PCR assay has been developed to measure human herpesvirus 6 (HHV-6) DNA in biological specimens. The assay sensitivity was 10 copies of DNA per well, with a linear dynamic range of 10 to 10
7
copies of HHV-6 DNA. Intra- and interassay variations were, respectively, 0.88 and 0.8% for samples containing 10
2
DNA copies, 0.99 and 0.96% for samples containing 10
4
copies, and 0.76 and 0.9% for samples containing 10
6
copies. Among 34 saliva samples from healthy subjects, 26 were found to contain HHV-6 DNA (76.5%; median, 23,870 copies/ml), and following a single freeze-thaw cycle, 25 of the same samples were found to be positive for HHV-6 DNA, although at a statistically significantly lower concentration (median, 3,497 copies/ml). The assay enabled detection of HHV-6 DNA in lymph node biopsies from patients with Hodgkin's disease (HD) (13 of 37 patients [35.1%]), B-cell neoplasms (8 of 36 patients [22.2%]), and T- or NK-cell neoplasms (3 of 13 patients [23.1%]), with concentrations ranging from 100 to 864,640 HHV-6 copies per μg of DNA (HHV-6B being found in every case except two). All HD patients infected with HHV-6 presented clinically with the nodular sclerosis subtype of HD. The real-time quantitative PCR assay developed here was simple to perform and was sensitive over a wide range of HHV-6 concentrations. It therefore appears to be of potential value in clinical investigation or diagnosis of HHV-6 infection.
Detection of
Tropheryma whipplei
DNA in Feces by PCR Using a Target Capture Method
Authors: Maibach Romana C. , Dutly Fabrizio , Altwegg Martin 
Published: July 2002

ABSTRACT

Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting
Tropheryma whipplei
DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a
T. whipplei
-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of
T. whipplei
DNA and one not requiring invasive sampling procedures.
Comparison of an Assay Using Signal Amplification of the Heat-Dissociated p24 Antigen with the Roche Monitor Human Immunodeficiency Virus RNA Assay
Authors: Pascual Alvaro , Cachafeiro Ada , Funk Michele L. , Fiscus Susan A. 
Published: July 2002

ABSTRACT

We compared an assay using signal amplification of a heat-dissociated p24 antigen (HDAg) with the Roche Monitor human immunodeficiency virus (HIV) RNA assay. The two assays gave comparable results when 130 specimens from 130 patients were tested (
r
= 0.60,
P
< 0.0001). The HDAg assay was almost as sensitive (85%) as the Roche HIV RNA kit (95%), just as specific (25 negative results from 25 HIV seronegative volunteers [100%]), less variable (mean log standard deviation of 0.07 compared to 0.11 when eight specimens were tested three or four times), and less expensive (reagent and labor costs, $8 versus $75). The assay appeared to be useful for monitoring established patients (
n
= 17) and identifying seroconverters (
n
= 4). HIV subtypes A to F were all recognized. This assay should be useful for monitoring patients in resource-poor countries and for monitoring vaccine recipients.
Helicobacter marmotae
sp. nov. Isolated from Livers of Woodchucks and Intestines of Cats
Authors: Fox James G. , Shen Zeli , Xu Shilu , Feng Yan , Dangler Charles A. , Dewhirst Floyd E. , Paster Bruce J. , Cullen John M. 
Published: July 2002

ABSTRACT

Woodchucks (
Marmota monax
) have a high incidence of hepatocellular carcinoma (HCC) associated with chronic infection with woodchuck hepatitis virus (WHV) and serve as a model of hepatitis B virus-associated HCC in humans.
Helicobacter hepaticus
, an enterohepatic helicobacter in mice, is known to cause hepatocellular adenomas and carcinomas in susceptible mouse strains. In long-term chemical bioassays conducted with B6C3F
1
mice,
H
.
hepaticus
has been regarded as a confounding factor because of its tumor-promoting activity. In order to determine if woodchucks harbor a
Helicobacter
sp. that might play a role in potentiating hepatic inflammation or neoplasia, a study was undertaken to determine whether woodchucks' livers were infected with a
Helicobacter
sp. Frozen liver samples from 20 (17 WHV-infected and 3 noninfected) woodchucks, 10 with WHV-associated hepatic tumors and 10 without tumors, were cultured by microaerobic techniques and analyzed by using genus- and species-specific helicobacter PCR primers. A 1,200-bp
Helicobacter
sp.-specific sequence was amplified from 14 liver samples. Southern hybridization confirmed the specific identity of the PCR products. Nine of the 10 livers with tumors had positive
Helicobacter
sp. identified by PCR, whereas 5 of the 10 livers without tumors were positive. By use of 16S rRNA species-specific primers for
H
.
marmotae
, two additional liver samples from the nontumor group had positive PCR amplicons confirmed by Southern hybridization. A urease-, catalase-, and oxidase-positive bacterium was isolated from one liver sample from a liver tumor-positive woodchuck. By 16S rRNA analysis and biochemical and phenotypic characteristics, the organism was classified as a novel
Helicobacter
sp. Subsequently, four additional bacterial strains isolated from feces of cats and characterized by biochemical, phenotypic, and 16S rRNA analysis were determined to be identical to the woodchuck isolate. We propose the name
Helicobacter marmotae
sp. nov. for these organisms. Further studies are required to ascertain if this novel
Helicobacter
sp. plays a tumor promotion role in hepadnavirus-associated tumors in woodchucks or causes enterohepatic disease in cats.
Detection of Epstein-Barr Virus Genomes in Peripheral Blood B Cells from Solid-Organ Transplant Recipients by Fluorescence In Situ Hybridization
Authors: Rose Camille , Green Michael , Webber Steven , Kingsley Lawrence , Day Roger , Watkins Simon , Reyes Jorges , Rowe David 
Published: July 2002

ABSTRACTResolution of Epstein-Barr Virus (EBV) infection in pediatric solid-organ transplant recipients often leads to an asymptomatic carrier state characterized by a persistently elevated circulating EBV load that is 2 to 4 orders of magnitude greater than the load typical of healthy latently infected individuals. Elevated EBV loads in immunosuppressed individuals are associated with an increased risk for development of posttransplant lymphoproliferative disease. We have performed fluorescence in situ hybridization (FISH) studies with peripheral blood B cells from carriers of persistent EBV loads in order to directly quantitate the number of EBV genomes per infected cell. Patients were assigned to two groups on the basis of the level of the persistent load (low-load carriers, 8 to 200 genomes/105peripheral blood lymphocytes; high-load carriers, >200 genomes/105peripheral blood lymphocytes). FISH analysis revealed that the low-load carriers predominantly had circulating virus-infected cells harboring one or two genome copies/cell. High-load carriers also had cells harboring one or two genome copies/cell; in addition, however, they carried a distinct population of cells with high numbers of viral genome copies. The increased viral loads correlated with an increase in the frequency of cells containing high numbers of viral genomes. We conclude that low-load carriers possess EBV-infected cells that are in a state similar to normal latency, whereas high-load carriers possess two populations of virus-positive B cells, one of which carries an increased number of viral genomes per cell and is not typical of normal latency.
Differentiation of Porcine
Brachyspira
Species by a Novel
nox
PCR-Based Restriction Fragment Length Polymorphism Analysis
Authors: Rohde Judith , Rothkamp Anja , Gerlach Gerald F. 
Published: July 2002

ABSTRACT

A novel PCR-based restriction fragment length polymorphism analysis of the
Brachyspira nox
gene was developed. The restriction patterns for
Brachyspira hyodysenteriae
,
B
.
pilosicoli
,
B
.
intermedia
,
B
.
murdochii
, and
B
.
innocens
were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust.
Virulence Genes in
Helicobacter pylori
Strains from West Bengal Residents with Overt
H. pylori
-Associated Disease and Healthy Volunteers
Authors: Chattopadhyay Santanu , Datta Simanti , Chowdhury Abhijit , Chowdhury Sujit , Mukhopadhyay Asish K. , Rajendran K. , Bhattacharya S. K. , Berg Douglas E. , Nair G. Balakrish 
Published: July 2002

ABSTRACT

We compared putative molecular markers of virulence (
vacA
,
cagA
, and
iceA
) of
Helicobacter pylori
strains isolated from 52 adult duodenal ulcer patients from West Bengal, India, with those of
H
.
pylori
strains isolated from 48 adult healthy volunteers from the same region. On the basis of genotyping by PCR, we conclude that the
H. pylori
strains isolated from the two study groups were indistinguishable and that there are geographic variations in the association of certain putative
H. pylori
virulence genes with clinical status.
Escalating Association of
Vibrio cholerae
O139 with Cholera Outbreaks in India
Authors: Sinha Sutapa , Chakraborty Rupa , De Keya , Khan Asis , Datta Simanti , Ramamurthy T. , Bhattacharya S. K. , Takeda Yoshifumi , Nair G. Balakrish 
Published: July 2002

ABSTRACT

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only
V. cholerae
O1 biotype ElTor or
V. cholerae
O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of
V. cholerae
O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of
V. cholerae
O139.
Laboratory Contamination of Specimens with Quality Control Strains of Vancomycin-Resistant Enterococci in Ontario
Authors: Katz K. C. , McGeer A. , Low D. E. , Willey B. M. 
Published: July 2002

ABSTRACT

Cross-contamination with laboratory control strains of vancomycin-resistant
Enterococcus faecalis
was documented in 15 clinical specimens from nine clinical microbiology laboratories in Ontario, Canada. Laboratories should be alert to the possibility of contamination of specimens with vancomycin-resistant enterococci from the laboratory environment. Molecular typing of strains may assist in elucidating the source of such contamination.
Trichuris vulpis
Recovered from a Patient with Chronic Diarrhea and Five Dogs
Authors: Dunn James J. , Columbus Sharon T. , Aldeen William E. , Davis Mark , Carroll Karen C. 
Published: July 2002

ABSTRACT

We report a case of human infection with the whipworm of dogs,
Trichuris vulpis
, in a woman with duodenal ulcer disease, chronic diarrhea, and close contact with dogs. Morphologically,
T. vulpis
ova resemble those of the human whipworm (
T. trichiura
) but are nearly twice their size.
Species-Specific Receptor Recognition by a Minor-Group Human Rhinovirus (HRV): HRV Serotype 1A Distinguishes between the Murine and the Human Low-Density Lipoprotein Receptor
Authors: Reithmayer Manuela , Reischl Andrea , Snyers Luc , Blaas Dieter 
Published: July 2002

ABSTRACT
Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.