bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Iron-Binding Protein, Dpr, fromStreptococcus mutansPrevents Iron-Dependent Hydroxyl Radical Formation In Vitro
Authors: Yamamoto Yuji , Poole Leslie B. , Hantgan Roy R. , Kamio Yoshiyuki 
Published: July 2002

ABSTRACTThedprgene is an antioxidant gene which was isolated from theStreptococcus mutanschromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant ofEscherichia coli, and it was proven to play an indispensable role in oxygen tolerance inS. mutans. Here, we purified the 20-kDadprgene product, Dpr, from a crude extract ofS. mutansas an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. UnlikeE. coliDps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 μM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein inS. mutansand may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.
Biosynthesis Pathway of ADP-
l
-
glycero
-β-
d
-
manno
-Heptose in
Escherichia coli
Authors: Kneidinger Bernd , Marolda Cristina , Graninger Michael , Zamyatina Alla , McArthur Fiona , Kosma Paul , Valvano Miguel A. , Messner Paul 
Published: July 2002

ABSTRACT

The steps involved in the biosynthesis of the ADP-
l
-
glycero
-β-
d
-
manno-
heptose (ADP-
l
-β-
d
-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-
d
-β-
d
-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene,
gmhB
(formerly
yaeD
), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-
d
-β-
d
-heptose. Our results demonstrate that the synthesis of ADP-
d
-β-
d
-heptose in
Escherichia coli
requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional
d
-β-
d
-heptose 7-phosphate kinase/
d
-β-
d
-heptose 1-phosphate adenylyltransferase), and GmhB (
d
,
d
-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-
l
-β-
d
-heptose precursor utilized in the assembly of inner core LPS.
Characterization of an ADP-Ribosyltransferase Toxin (AexT) from
Aeromonas salmonicida
subsp.
salmonicida
Authors: Braun Martin , Stuber Katja , Schlatter Yvonne , Wahli Thomas , Kuhnert Peter , Frey Joachim 
Published: July 2002

ABSTRACT

An ADP-ribosylating toxin named
Aeromonas salmonicida
exoenzyme T (AexT) in
A. salmonicida
subsp.
salmonicida
, the etiological agent of furunculosis in fish, was characterized. Gene
aexT
, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of
Pseudomonas aeruginosa
and to the YopE cytotoxin of different
Yersinia
species. The
aexT
gene was detected in all of the 12
A. salmonicida
subsp.
salmonicida
strains tested but was absent from all other
Aeromonas
species. Recombinant AexT produced in
Escherichia coli
possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by
A. salmonicida
subsp.
salmonicida
and cross-reacted with ExoS and ExoT of
P. aeruginosa
. AexT toxin could be detected in a wild type (wt) strain of
A. salmonicida
subsp.
salmonicida
freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when
A. salmonicida
subsp.
salmonicida
was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt
A. salmonicida
subsp.
salmonicida
, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An
aexT
mutant which was constructed from the wt strain with an insertionally inactivated
aexT
gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of
A. salmonicida
subsp.
salmonicida
for RTG-2 fish cells.
Immunodiagnosis of
Ehrlichia canis
Infection with Recombinant Proteins
Authors: McBride Jere W. , Corstvet Richard E. , Breitschwerdt Edward B. , Walker David H. 
Published: July 2002

ABSTRACT

Ehrlichia canis
causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive
E. canis
proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive
E. canis
surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in
E. chaffeensis
DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with
E. chaffeensis
as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for
E. canis
, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for
E. canis
infections.
Genotyping
Encephalitozoon cuniculi
by Multilocus Analyses of Genes with Repetitive Sequences
Authors: Xiao Lihua , Li Lixia , Visvesvara Govinda S. , Moura Hercules , Didier Elizabeth S. , Lal Altaf A. 
Published: July 2002

ABSTRACT

Encephalitozoon cuniculi
infects a wide range of mammalian hosts. Three genotypes based on the number of GTTT repeats in the internal transcribed spacer (ITS) of the rRNA have been described, of which genotypes I and III have been identified in humans. In this study, the genetic diversity of
E. cuniculi
was examined at the polar tube protein (PTP) and spore wall protein I (SWP-1) loci. Nucleotide sequence analysis of the PTP gene divided 11
E. cuniculi
isolates into three genotypes in congruence with the result of analysis of the ITS of the rRNA gene. The three PTP genotypes differed from one another by the copy number of the 78-bp central repeat as well as point mutations. These
E. cuniculi
isolates also differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some
E. cuniculi
isolates had heterogeneous copies of the SWP-1 gene with various numbers of repeats. Intragenotypic variation was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate
E. cuniculi
in clinical samples.
Change of Major Genotype of Enterovirus 71 in Outbreaks of Hand-Foot-and-Mouth Disease in Taiwan between 1998 and 2000
Authors: Wang Jen-Ren , Tuan Yen-Chang , Tsai Huey-Pin , Yan Jing-Jou , Liu Ching-Chuan , Su Ih-Jen 
Published: July 2002

ABSTRACT
Two outbreaks of hand-foot-and-mouth disease (HFMD) occurred in Taiwan between 1998 and 2000. Enteroviruses were isolated from a total of 1,892 patients in this laboratory during this period. Of the virus isolates, enterovirus 71 (EV71) was diagnosed in 44.4% of the patients (132 of 297) in 1998, 2% (13 of 646) in 1999, and 20.5% (195 of 949) in 2000. Genetic analyses of the 5′-untranslated and VP1 regions of EV71 isolates by reverse transcription-PCR and sequencing were performed to understand the diversity of EV71 in these outbreaks of HFMD. Most EV71 isolates from the 1998 epidemic belonged to genotype C, while only one-tenth of the isolates were genotype B. Interestingly, all EV71 isolates tested from 1999 to 2000 belonged to genotype B. This study indicated that two genogroups of EV71 capable of inducing severe clinical illness have been circulating in Taiwan. Furthermore, the predominant EV71 genotypes responsible for each of the two major HFMD outbreaks within the 3-year period in Taiwan were different.
Carriage Frequency, Intensity of Carriage, and Strains of Oral Yeast Species Vary in the Progression to Oral Candidiasis in Human Immunodeficiency Virus-Positive Individuals
Authors: Vargas Kaaren G. , Joly Sophie 
Published: July 2002

ABSTRACTCandidasamples were taken over a period of 2 years from 54 human immunodeficiency virus (HIV)-positive asymptomatic subjects to evaluate changes in yeast carriage, intensity of carriage, and genotype over time. Overall, we found that HIV-positive patients with CD4+-cell counts of between 200 and 400/μl had significantly more yeast colonization than healthy control subjects. Of the 54 patients, 11 developed thrush. We found that intensity of carriage in these 11 patients increased significantly in the progression from asymptomatic yeast carrier to an episode of oral thrush. Also, the most common yeast species isolated wasCandidaalbicans; however, we did see a number of patients harboring multiple species at the same time. Using theC.albicans-specific probe Ca3, we found that 54% (n= 6) of the 11 patients who developed thrush maintained genetically similar strains throughout the study period, with minor genetic variations in all patients except one. Forty-six percent of these patients had either multiple strains throughout the study period (n= 2), strain replacement (n= 1), or species replacement (n= 2). Of the patients who had multiple strains, one (I4) was infected by two different strains ofCandidadubliniensisdistinguished by a recently developed species-specific probe. These results suggest that commensal strains colonizing HIV-positive individuals can undergo alterations prior to producing an episode of thrush.
Environmental Occurrence ofMadurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan
Authors: Ahmed Abdalla , Adelmann Daniel , Fahal Ahmed , Verbrugh Henri , van Belkum Alex , de Hoog Sybren 
Published: July 2002

ABSTRACTMadurella mycetomatisis the main causative agent of human eumycetoma, a severe debilitating disease endemic in Sudan. It has been suggested that eumycetoma has a soil-borne or thorn prick-mediated origin. For this reason, efforts were undertaken to cultureM. mycetomatisfrom soil samples (n= 43) and thorn collections (n= 35) derived from areas in which it is endemic. However, ribosomal sequencing data revealed that the black fungi obtained all belonged to other fungal species. In addition, we performed PCR-mediated detection followed by restriction fragment length polymorphism (RFLP) analysis for the identification ofM. mycetomatisDNA from the environmental samples as well as biopsies from patients with mycetoma. In the case of the Sudanese soil samples, 17 out of 74 (23%) samples were positive forM. mycetomatisDNA. Among the thorn collections, 1 out of 22 (5%) was positive in the PCR. All PCR RFLP patterns clearly indicated the presence ofM. mycetomatis. In contrast, 15 Dutch and English control soil samples were all negative. Clinically and environmentally obtained fungal PCR products share the same PCR RFLP patterns, suggesting identity, at least at the species level. These observations support the hypothesis that eumycetoma is primarily environmentally acquired and suggest thatM. mycetomatisneeds special conditions for growth, as direct isolation from the environment seems to be impossible.
Fatal Case of
Campylobacter lari
Prosthetic Joint Infection and Bacteremia in an Immunocompetent Patient
Authors: Werno Anja M. , Klena John D. , Shaw Geoffrey M. , Murdoch David R. 
Published: July 2002

ABSTRACT

Campylobacter lari
is an infrequent cause of intestinal and extraintestinal infection in humans. We report a case of
C. lari
prosthetic joint infection and bacteremia in an 81-year-old immunocompetent man. The infection was associated with septic shock and fatal outcome.
C. lari
may cause severe disease, even in an immunocompetent host.
Relatively Alcohol-Resistant Mycobacteria Are Emerging Pathogens in Patients Receiving Acupuncture Treatment
Authors: Woo Patrick C. Y. , Leung Kit-Wah , Wong Samson S. Y. , Chong Ken T. K. , Cheung Elim Y. L. , Yuen Kwok-Yung 
Published: July 2002

ABSTRACT

Acupuncture has been gaining popularity as a form of alternative medicine. In the past, only blood-borne viruses and anecdotal reports of bacterial infections have been associated with acupuncture. We report on four patients with mycobacterial infections complicating acupuncture who were encountered in a 2-year period. All had clinical and/or radiological lesions at acupuncture point- and meridian-specific locations. There was no other history of trauma or other clinical foci of infections, and the chest radiographs were normal. Histological studies of biopsy specimens of all four patients showed changes compatible with chronic inflammation, with granulomatous inflammation present in three patients and acid-fast bacilli present in two. Conventional biochemical tests and whole-cell fatty acid analysis for identification were inconclusive for all four nonpigmented mycobacteria recovered from tissue biopsies. 16S rRNA gene sequencing showed that the strains from two patients were
Mycobacterium chelonae
and that those from the other two were
Mycobacterium nonchromogenicum
. Alcohol resistance assay using the quantitative suspension test revealed that all four strains showed prolonged survival in 75% alcohol compared to other skin flora. Mycobacterial infections transmitted by acupuncture are an emerging problem. A high index of suspicion is essential to recognize this clinical syndrome, and strict implementation of proper infection control guidelines for acupuncture is mandatory.
Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat
A/D
Junction Element
Authors: Haberman Rebecca P. , McCown Thomas J. , Samulski Richard Jude 
Published: July 2002

ABSTRACT

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the “off” state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other
cis
-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the
A/D
elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.
Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly
Authors: Sanchez Veronica , Greis Kenneth D. , Sztul Elizabeth , Britt William J. 
Published: July 2002

ABSTRACT

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment,
cis
or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the
trans
-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.
Human Herpesvirus 8 LANA Interacts with Proteins of the mSin3 Corepressor Complex and Negatively Regulates Epstein-Barr Virus Gene Expression in Dually Infected PEL Cells
Authors: Krithivas Anita , Young David B. , Liao Gangling , Greene Deborah , Hayward S. Diane 
Published: July 2002

ABSTRACT

The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione
S
-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5×Gal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.
Genotypic, Phenotypic, and Modeling Studies of a Deletion in the β3-β4 Region of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Gene That Is Associated with Resistance to Nucleoside Reverse Transcriptase Inhibitors
Authors: Winters Mark A. , Coolley Kristi L. , Cheng Peng , Girard Yvette A. , Hamdan Hasnah , Kovari Ladislau C. , Merigan Thomas C. 
Published: July 2002

ABSTRACT
Point mutations and inserts in the β3-β4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the β3-β4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the β3-β4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.