bluuebunny/crossref_metadata_2025 huggingface dataset:
Redundancy in Periplasmic Binding Protein-Dependent Transport Systems for Trehalose, Sucrose, and Maltose in
Sinorhizobium meliloti
Authors: Jensen John Beck , Peters N. Kent , Bhuvaneswari T. V.
Published: July 2002

We have identified a cluster of six genes involved in trehalose transport and utilization (
thu
) in
Sinorhizobium meliloti
. Four of these genes,
thuE
, -
F
, -
G
, and -
K
, were found to encode components of a binding protein-dependent trehalose/maltose/sucrose ABC transporter. Their deduced gene products comprise a trehalose/maltose-binding protein (ThuE), two integral membrane proteins (ThuF and ThuG), and an ATP-binding protein (ThuK). In addition, a putative regulatory protein (ThuR) was found divergently transcribed from the
thuEFGK
operon. When the
thuE
locus was inactivated by gene replacement, the resulting
S. meliloti
strain was impaired in its ability to grow on trehalose, and a significant retardation in growth was seen on maltose as well. The wild type and the
thuE
mutant were indistinguishable for growth on glucose and sucrose. This suggested a possible overlap in function of the
thuEFGK
operon with the
aglEFGAK
operon, which was identified as a binding protein-dependent ATP-binding transport system for sucrose, maltose, and trehalose. The

K
m

s for trehalose transport were 8 ± 1 nM and 55 ± 5 nM in the uninduced and induced cultures, respectively. Transport and growth experiments using mutants impaired in either or both of these transport systems show that these systems form the major transport systems for trehalose, maltose, and sucrose. By using a
thuE
′
-lacZ
fusion, we show that
thuE
is induced only by trehalose and not by cellobiose, glucose, maltopentaose, maltose, mannitol, or sucrose, suggesting that the
thuEFGK
system is primarily targeted toward trehalose. The
aglEFGAK
operon, on the other hand, is induced primarily by sucrose and to a lesser extent by trehalose. Tests for root colonization, nodulation, and nitrogen fixation suggest that uptake of disaccharides can be critical for colonization of alfalfa roots but is not important for nodulation and nitrogen fixation per se.
Methylation of Inorganic and Organic Selenium by the Bacterial Thiopurine Methyltransferase
Authors: Ranjard Lionel , Prigent-Combaret Claire , Nazaret Sylvie , Cournoyer Benoit
Published: July 2002

Escherichia colicells expressing thetpmgene encoding the bacterial thiopurine methyltransferase (bTPMT) are shown to methylate selenite and (methyl)selenocysteine into dimethylselenide (DMSe) and dimethyldiselenide (DMDSe).E. colicells expressingtpmfrom a gene library cosmid clone (harboring aPseudomonas syringaeinsert of about 20 kb) also methylated selenate into DMSe and DMDSe. bTPMT is the first methyltransferase shown to be involved in the methylation of these selenium derivatives.
The Highly Conserved TldD and TldE Proteins of
Escherichia coli
Are Involved in Microcin B17 Processing and in CcdA Degradation
Authors: Allali Noureddine , Afif Hassan , Couturier Martine , Van Melderen Laurence
Published: July 2002

Microcin B17 (MccB17) is a peptide antibiotic produced by
Escherichia coli
strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal
tldE
(
pmbA
) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently,
tldE
as well as
tldD
mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the
ccd
poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the
ccd
system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.
Physical Map of the Linear Chromosome of
Streptomyces hygroscopicus
10-22 Deduced by Analysis of Overlapping Large Chromosomal Deletions
Authors: Pang Xiuhua , Zhou Xiufen , Sun Yuhui , Deng Zixin
Published: July 2002

The chromosomal DNA of
Streptomyces hygroscopicus
10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with
Ase
I gave 11 fragments with a total length of ca. 7.36 Mb. The
Ase
I sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.
Regulation of Purine Hydroxylase and Xanthine Dehydrogenase from
Clostridium purinolyticum
in Response to Purines, Selenium, and Molybdenum
Authors: Self William T.
Published: July 2002

The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by
Clostridium purinolyticum
was unexpected. In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH. PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously. In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates. The levels of PH protein in extracts were determined by immunoblot assay. The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium. PH levels were highest in the cells cultured in the presence of either adenine or purine. XDH activity increased dramatically in cells grown with either xanthine or uric acid. The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients.
Serum Interleukin-6 (IL-6), IL-10, Tumor Necrosis Factor (TNF) Alpha, Soluble Type II TNF Receptor, and Transforming Growth Factor Beta Levels in Human Immunodeficiency Virus Type 1-Infected Individuals withMycobacterium aviumComplex Disease
Authors: Havlir Diane V. , Torriani Francesca J. , Schrier Rachel D. , Huang Jimmy Y. , Lederman Michael M. , Chervenak Keith A. , Boom W. Henry
Published: July 2002

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons withMycobacterium aviumcomplex (MAC) bacteremia, the levels of IL-1α (interleukin-1α), IL-6, IL-10, tumor necrosis factor alpha (TNF-α), soluble type II TNF receptor (sTNF-RII), and transforming growth factorβ(TGF-β) in serum were measured in two cohorts of HIV-1-infected persons with MAC bacteremia. The first cohort was part of a MAC prophylaxis study. Patients with bacteremia were matched with controls without bacteremia. Elevated IL-6, IL-10, TNF-α, sTNF-RII, and TGF-β levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1α was not detected. No differences in cytokine levels in serum were noted at baseline and at the time of bacteremia between patients with MAC and controls. In the second cohort, subjects had serum samples collected at the time of MAC bacteremia and thereafter while on macrolide therapy. Serum samples at time of bacteremia were collected from HIV-1-infected persons at a time when neither highly active antiretroviral therapy (HAART) nor MAC prophylaxis was used routinely. MAC treatment resulted in decreased levels of IL-6 and TNF-α in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-α by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-α in serum in HIV-1-infected persons who are not on HAART.
Comparison of Three Transport Systems (Starplex StarSwab II, the New Copan Vi-Pak Amies Agar Gel Collection and Transport Swabs, and BBL Port-A-Cul) for Maintenance of Anaerobic and Fastidious Aerobic Organisms
Authors: Hindiyeh Musa , Acevedo Victoria , Carroll Karen C.
Published: July 2002

Of utmost importance in evaluations of clinical samples for infectious agents is proper specimen transport to the clinical laboratory. In the present study we compared three transport systems (the new Starplex StarSwab II, the new Copan Vi-Pak Amies Agar Gel collection and transport swabs, and the BBL Port-A-Cul) for survival of anaerobic and fastidious aerobic bacteria. The new Copan Vi-Pak system has been modified by nitrogen gas flushing to keep an ideal low E
h
condition and to prevent oxidation of the transport medium. The Copan Vi-Pak system outperformed the other two swabs evaluated by maintaining the viabilities of both anaerobic and fastidious aerobic bacteria for 24 h for the majority of the organisms evaluated. This time period should be sufficient for transport of specimens to the clinical microbiology laboratory without compromising organism recovery.
PCR for Specific Detection of H7 Flagellar Variant of
fliC
among Extraintestinal Pathogenic
Escherichia coli
Authors: Johnson James R. , Stell Adam L.
Published: July 2002

A newly developed PCR-based assay for the H7 variant of the
Escherichia coli
flagellin gene,
fliC
, was 100% sensitive and specific in comparison with serology and probe hybridization. It revealed broad conservation of the H7
fliC
variant among phylogenetically diverse lineages of extraintestinal pathogenic
E. coli
(ExPEC) and superseded serotyping for certain isolates with ambiguous or non-H7 serotyping results. The H7 primers functioned well when incorporated into a multiplex PCR assay for diverse virulence-associated genes of ExPEC.
Optimal Inoculation Methods and Quality Control for the NCCLS Oxacillin Agar Screen Test for Detection of Oxacillin Resistance in
Staphylococcus aureus
Authors: Swenson Jana M. , Spargo Jean , Tenover Fred C. , Ferraro Mary Jane
Published: July 2002

To define more precisely the inoculation methods to be used in the oxacillin screen test for
Staphylococcus aureus
, we tested agar screen plates prepared in house with 6 μg of oxacillin/ml and 4% NaCl using the four different inoculation methods that would most likely be used by clinical laboratories. The organisms selected for testing were 19 heteroresistant
mecA
-producing strains and 41 non-
mecA
-producing strains for which oxacillin MICs were near the susceptible breakpoint. The inoculation method that was preferred by all four readers and that resulted in the best combination of sensitivity and specificity was a 1-μl loopful of a 0.5 McFarland suspension. A second objective of the study was to then use this method to inoculate plates from five different manufacturers of commercially prepared media. Although all commercial media performed with acceptable sensitivity compared to the reference lot, one of the commercial lots demonstrated a lack of specificity. Those lots of oxacillin screen medium that fail to grow heteroresistant strains can be detected by using
S. aureus
ATCC 43300 as a positive control in the test and by using transmitted light to carefully examine the plates for any growth. However, lack of specificity with commercial lots may be difficult to detect using any of the current quality control organisms.
Selection of a Teicoplanin-Resistant
Enterococcus faecium
Mutant during an Outbreak Caused by Vancomycin-Resistant Enterococci with the VanB Phenotype
Authors: Kawalec Magdalena , Gniadkowski Marek , Ke˛dzierska Jolanta , Skotnicki Aleksander , Fiett Janusz , Hryniewicz Waleria
Published: July 2002

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant
Enterococcus faecalis
(VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant
Enterococcus faecium
(VREM) strains. Two similar restriction fragment length polymorphism types of the
vanB
gene cluster characterized VRES and VREM isolates, and they both contained the same
vanB2
variant of the
vanB
gene. Two vancomycin-susceptible
E. faecium
(VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of
vanB
gene cluster copies. Most probably they reflected the dynamic evolution of an
E. faecium
strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its
vanB
gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the
vanB
gene cluster, including the regulatory genes
vanR

B

and -
S

B

, and the vancomycin-inducible promoter
P

YB

. Expression of the resistance genes
vanH

B

, -
B
, and -
X

B

was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.
Multidrug-Resistant
Trichosporon asahii
Infection of Nongranulocytopenic Patients in Three Intensive Care Units
Authors: Wolf Dana G. , Falk Rama , Hacham Moshe , Theelen Bart , Boekhout Teun , Scorzetti Gloria , Shapiro Mervyn , Block Colin , Salkin Ira F. , Polacheck Itzhack
Published: July 2002

Trichosporon asahii
(
Trichosporon beigelii
) infections are rare but have been associated with a wide spectrum of clinical manifestations, ranging from superficial involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients. We report on the recent recovery of
T. asahii
isolates with reduced susceptibility in vitro to amphotericin B (AMB), flucytosine, and azoles from six nongranulocytopenic patients who exhibited risk factors and who developed either superficial infections (four individuals) or invasive infections (two individuals) while in intensive care units. The latter two patients responded clinically and microbiologically to AMB treatment. All six isolates were closely related according to random amplified polymorphic DNA studies and showed 71% similarity by amplified fragment length polymorphism analysis, suggesting a common nosocomial origin. We also review the literature pertaining to
T. asahii
infections and discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus.
Protocol for Detection of Biofilms on Needleless Connectors Attached to Central Venous Catheters
Authors: Donlan R. M. , Murga R. , Bell M. , Toscano C. M. , Carr J. H. , Novicki T. J. , Zuckerman C. , Corey L. C. , Miller J. M.
Published: July 2002

Central venous catheter needleless connectors (NCs) have been shown to develop microbial contamination. A protocol was developed for the collection, processing, and examination of NCs to detect and measure biofilms on these devices. Sixty-three percent of 24 NCs collected from a bone marrow transplant center contained biofilms comprised primarily of coagulase-negative staphylococci.
Comparative Pathogenesis of Infection of Pigs with Hepatitis E Viruses Recovered from a Pig and a Human
Authors: Halbur P. G. , Kasorndorkbua C. , Gilbert C. , Guenette D. , Potters M. B. , Purcell R. H. , Emerson S. U. , Toth T. E. , Meng X. J.
Published: July 2002

Specific-pathogen-free pigs were inoculated with one of two hepatitis E viruses (HEV) (one recovered from a pig and the other from a human) to study the relative pathogenesis of the two viruses in swine. Fifty-four pigs were randomly assigned to three groups. Seventeen pigs in group 1 served as uninoculated controls, 18 pigs in group 2 were intravenously inoculated with the swine HEV recovered from a pig in the United States, and 19 pigs in group 3 were intravenously inoculated with the US-2 strain of human HEV recovered from a hepatitis patient in the United States. Two to four pigs from each group were necropsied at 3, 7, 14, 20, 27, or 55 days postinoculation (DPI). Evidence of clinical disease or elevation of liver enzymes or bilirubin was not found in pigs from any of the three groups. Enlarged hepatic and mesenteric lymph nodes were observed in both HEV-inoculated groups. Multifocal lymphoplasmacytic hepatitis was observed in 9 of 17, 15 of 18, and 16 of 19 pigs in groups 1 to 3, respectively. Focal hepatocellular necrosis was observed in 5 of 17, 10 of 18, and 13 of 19 pigs in groups 1 to 3, respectively. Hepatitis lesions were very mild in group 1 pigs, mild to moderate in group 2 pigs, and moderate to severe in group 3 pigs. Hepatic inflammation and hepatocellular necrosis peaked in severity at 20 DPI and were still moderately severe at 55 DPI in the group inoculated with human HEV. Hepatitis lesions were absent or nearly resolved by 55 DPI in the swine-HEV-inoculated pigs. All HEV-inoculated pigs seroconverted to anti-HEV immunoglobulin G. HEV RNA was detected by reverse transcriptase PCR in feces, liver tissue, and bile of pigs in both HEV-inoculated groups from 3 to 27 DPI. Based on evaluation of microscopic lesions, the US-2 strain of human HEV induced more severe and persistent hepatic lesions in pigs than did swine HEV. Pig livers or cells from the livers of HEV-infected pigs may represent a risk for transmission of HEV from pigs to human xenograft recipients. Since HEV was shed in the feces of infected pigs, exposure to feces from infected pigs represents a risk for transmission of HEV, and pigs should be considered a reservoir for HEV.
Persistence of a Distinct
Corynebacterium diphtheriae
Clonal Group within Two Communities in the United States and Canada Where Diphtheria Is Endemic
Authors: Marston Chung K. , Jamieson Frances , Cahoon Fred , Lesiak Gail , Golaz Anne , Reeves Mike , Popovic Tanja
Published: July 2002

Molecular characterization of 53 U.S. and Canadian
Corynebacterium diphtheriae
isolates by multilocus enzyme electrophoresis, ribotyping, and random amplified polymorphic DNA showed that strains with distinct molecular subtypes have persisted in the United States and Canada for at least 25 years. These strains are endemic rather than imported from countries with current endemic or epidemic diphtheria.
Bacteremia by
Dermabacter hominis
, a Rare Pathogen
Authors: Gómez-Garcés José Luis , Oteo Jesús , Garcı́a Guadalupe , Aracil Belén , Alós Juan Ignacio , Funke Guido
Published: July 2002

Dermabacter hominis
is a gram-positive, catalase-positive, glucose-fermenting rod, which, as it grows forms small greyish-white colonies with a characteristic pungent odor. Previously known as coryneform Centers for Disease Control and Prevention groups 3 and 5, it was catalogued as
D. hominis
in 1994. Various strains isolated in blood cultures, abscesses, or wounds in the 1970s were retrospectively characterized in referral centers as
D. hominis
. In this report we describe two patients with severe underlying pathology who developed bacteremias by
D. hominis
within the context of their clinical pictures.
Experimental Paratuberculosis in Calves following Inoculation with a Rabbit Isolate of
Mycobacterium avium
subsp.
paratuberculosis
Authors: Beard P. M. , Stevenson K. , Pirie A. , Rudge K. , Buxton D. , Rhind S. M. , Sinclair M. C. , Wildblood L. A. , Jones D. G. , Sharp J. M.
Published: July 2002

The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of
Mycobacterium avium
subsp.
paratuberculosis
recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of
M. avium
subsp.
paratuberculosis
infection. Similar results were obtained from a group of calves infected with a bovine isolate of
M. avium
subsp.
paratuberculosis
. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of
M. avium
subsp.
paratuberculosis
should therefore be considered when formulating control plans for the disease.
Murine Monoclonal Antibodies against
Escherichia coli
O4 Lipopolysaccharide and H5 Flagellin
Authors: Rivera-Betancourt Mildred , Keen James E.
Published: July 2002

Two murine monoclonal antibodies (MAb), 2C5-F10 and 8D1-H10, reactive with
Escherichia coli
O4 and H5 antigens, respectively, were generated and characterized. Enzyme immunoassays and immunoblots demonstrated that MAb 2C5-F10 reacted specifically with lipopolysaccharide O antigen of
E. coli
O4 isolates, while MAb 8D1-H10 reacted with
E. coli
strains expressing H5 flagella.
Peritoneal Dialysis-Associated Peritonitis Caused by
Dermabacter hominis
Authors: Radtke Andreas , Bergh Kåre , Øien Cecilia M. , Bevanger Lars S.
Published: July 2002

Dermabacter hominis
was the cause of a peritoneal dialysis-associated peritonitis.
D. hominis
was identified by phenotypic criteria and by sequencing the 16S rRNA gene. Clinical cure was achieved with cefuroxime treatment despite the isolate's reduced susceptibility to this drug (MIC, 12 mg/liter) on in vitro testing. The successful treatment was probably due to the high concentrations attained by intraperitoneal administration of the drug.
Evaluation of a Rapid PCR-Based Epidemiological Typing Method for Routine Studies of
Mycobacterium tuberculosis
Authors: Yates Malcolm D. , Drobniewski Francis A. , Wilson Stuart M.
Published: July 2002

Restriction fragment length polymorphism (RFLP) based on the insertion sequence IS
6110
is used to investigate episodes of suspected transmission of infection of tuberculosis but usually takes a number of weeks from receipt of request to obtain a result. Often investigations would benefit from a more rapid method, possibly one containing an amplification step. The method employed uses a simple DNA extraction followed by a PCR step involving a single primer. Restriction enzyme analysis was performed when the patterns obtained from the PCR products were indistinguishable, especially when only single similar-size bands were obtained. The isolates used were strains of
Mycobacterium tuberculosis
submitted for epidemiological investigations as part of (i) possible contact-outbreak (22 episodes involving between 2 and 20 patients), (ii) possible incidents of laboratory cross-contamination (21 episodes), and (iii) possible change in drug resistance pattern or a case of reinfection (1 patient). The PCR products giving similar patterns were then subjected to restriction enzyme analysis. In conclusion it has been shown that this method is rapid, with results within 1 to 2 days of the request being received; is reproducible; and gives the same results as does RFLP. The restriction enzyme analysis stage has improved the efficiency of the technique.
New Simple and Rapid Test for Culture Confirmation of
Mycobacterium tuberculosis
Complex: a Multicenter Study
Authors: Hasegawa Naoki , Miura Takao , Ishii Koudou , Yamaguchi Kazuhiro , Lindner Thomas H. , Merritt Samuel , Matthews Janis D. , Siddiqi Salman H.
Published: July 2002

Mycobacterial antigen MPB64 has been identified as a
Mycobacterium tuberculoisis
complex-specific secretory protein since 1984. Recently, a simple culture confirmation test for
M. tuberculosis
complex has been developed by using lateral flow immunochromatographic assay (ICA) to detect MPB64 with anti-MPB64 monoclonal antibody. The current multicenter study evaluated the performance of an ICA slide test for MPB64 antigen in the clinical setting. Primary positive cultures from clinical specimens, as well as stock cultures, were tested. Approximately 100 μl of positive liquid culture medium or suspension made from colonies on solid medium was placed into the test well of the plastic slide devise, and the test was read after 15 min. No processing or instrumentation was required. A total of 304 mycobacterial isolates consisting of
M. tuberculosis
complex (171 isolates) and mycobacteria other than
M. tuberculosis
(MOTT) complex (133 isolates) belonging to 18 different species were tested. Growth in liquid media (Mycobacteria Growth Indicator Tube [MGIT] and Radiometric 12B), as well as in solid (Löwenstein-Jensen and Middlebrook 7H10 agar) media, was evaluated. Results were compared with those obtained with nucleic acid-based and/or high-pressure liquid chromatography identification. All MOTT were found to be negative on the ICA slide with no cross-reaction. All
M. tuberculosis
and
M. africanum
cultures were found to be positive, whereas the results of
M. bovis
and
M. bovis
BCG cultures were variable since some of the BCG strains are known to lack MPB64 antigen production. The results did not change with prolonged storage of cultures. This low-tech rapid test with high sensitivity and specificity could provide an alternative to currently available identification methods, particularly for recently introduced nonradiometric liquid culture systems such as MGIT.
Ribosomal DNA-Directed PCR for Identification of
Achromobacter
(
Alcaligenes
)
xylosoxidans
Recovered from Sputum Samples from Cystic Fibrosis Patients
Authors: Liu Lixia , Coenye Tom , Burns Jane L. , Whitby Paul W. , Stull Terrence L. , LiPuma John J.
Published: July 2002

The opportunistic human pathogen
Achromobacter
(
Alcaligenes
)
xylosoxidans
has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of
A. xylosoxidans
as
Burkholderia cepacia
complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of
A. xylosoxidans
, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47
A. xylosoxidans
and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of
A. xylosoxidans
, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.
Simultaneous Differentiation and Typing of
Entamoeba histolytica
and
Entamoeba dispar
Authors: Zaki Mehreen , Meelu Parool , Sun Wei , Clark C. Graham
Published: July 2002

Sequences corresponding to some of the polymorphic loci previously reported from
Entamoeba histolytica
have been detected in
Entamoeba dispar
. Comparison of nucleotide sequences of two loci between
E. dispar
strain SAW760 and
E. histolytica
strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained, primer pairs aimed at amplifying species-specific products were designed and tested on a variety of
E. histolytica
and
E. dispar
samples. Amplification results were in complete agreement with the original species classification in all cases, and the PCR products displayed discernible size and pattern variations among the isolates.
Evaluation of Eight Rapid Screening Tests for Acute Leptospirosis in Hawaii
Authors: Effler Paul V. , Bogard April K. , Domen Harry Y. , Katz Alan R. , Higa Henry Y. , Sasaki David M.
Published: July 2002

Leptospirosis is a major public health problem throughout the world. Clinical recognition of leptospirosis is challenging, and the definitive serologic diagnostic assay, the microscopic agglutination test, is time-consuming and difficult to conduct. Various serologic screening tests have been developed, but their performance among ill persons in the United States has not been established. Eight screening tests were compared using 379 serum samples obtained in 1998 and 1999 from a series of 236 patients (33 with confirmed infection). The median number of days between illness onset and specimen collection was 9. The overall sensitivity, by specimen, for each test was as follows: indirect hemagglutination assay (MRL Diagnostics, Cypress, Calif.), 29%; INDX Leptospira Dip-S-Tick (PanBio InDx, Inc., Baltimore, Md.), 52%; Biognost IgM IFA test (Bios GmbH Labordiagnostik, Gräfelfing, Germany), 40%; Biolisa IgM ELISA (Bios GmbH, Labordiagnostik), 48%; Leptospira IgM ELISA (PanBio Pty Ltd., Brisbane, Australia), 36%; SERION ELISA classic Leptospira (Institut Virion•Serion GmbH, Würzburg, Germany), 48%; LEPTO Dipstick(Organon-Teknika, Ltd., Amsterdam, The Netherlands), 34%; Biosave latex agglutination test (LATEX; Bios GmbH Labordiagnostik), 86%. Test specificity ranged from 85 to 100% among all tests except LATEX, for which the specificity was significantly lower, at 10%. Test sensitivity was particularly low (<25%) for all tests (except LATEX) on specimens collected during the first week of illness. This is the most comprehensive field trial of leptospirosis screening tests reported to date. The data indicate that immunoglobulin M detection tests have limited utility for diagnosing leptospirosis during the initial evaluation of patients seen in Hawaii, a time when important therapeutic decisions are made. Improved leptospirosis screening tests are needed.
AMF-1/Gps2 Binds p300 and Enhances Its Interaction with Papillomavirus E2 Proteins
Authors: Peng Yu-Cai , Breiding David E. , Sverdrup Francis , Richard James , Androphy Elliot J.
Published: July 2002

The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208–7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.
Respiratory Syncytial Virus G and/or SH Glycoproteins Modify CC and CXC Chemokine mRNA Expression in the BALB/c Mouse
Authors: Tripp Ralph A. , Jones Les , Anderson Larry J.
Published: July 2002

Chemokine mRNA expression by pulmonary leukocytes following infection of BALB/c mice with two strains of respiratory syncytial virus (RSV) and one strain of parainfluenza virus type 3 (PIV-3) was determined. The results suggest that RSV G and/or SH proteins inhibit early MIP-1α, MIP-1β, MIP-2, MCP-1, and IP-10 mRNA expression. TCA-3 mRNA expression was found to be increased during PIV-3 infection.
Novel Mouse Type D Endogenous Proviruses and ETn Elements Share Long Terminal Repeat and Internal Sequences
Authors: Mager Dixie L. , Freeman J. Douglas
Published: July 2002

The repetitive ETn (early transposon) family of sequences represents an active “mobile mutagen” in the mouse genome. The presence of long terminal repeats (LTRs) and other diagnostic features indicate that ETns are retrotransposons but they contain no long open reading frames or documented similarity to the genes of known retroviruses or other retroelements. Thus, the mechanisms responsible for the mobility of this family have been unknown. In this study, we used computer searches to detect a small region of previously unrecognized type D retroviralpolhomology within ETn elements. This small region was used to isolate two mouse endogenous proviral elements withgag,pro, andpolgenes similar to simian type D viruses. This new family of mouse endogenous proviruses, termed MusD, is present in several hundred copies in the genome. Interestingly, the MusD LTRs, 3′ internal region, and the 5′ region expected to contain the packaging signal are very closely related to members of the ETn subfamily that have recently transposed. Analysis of different mouse strains indicates that MusD elements predate the existence of the mobile subfamily of ETns. These findings indicate that the ETn family was likely created via recombination events resulting in a near complete substitution of MusD coding sequences with unrelated DNA. Furthermore, these results suggest that ETn transcripts retrotranspose using proteins provided by MusD proviruses.
Processing of α-Globin and ICP0 mRNA in Cells Infected with Herpes Simplex Virus Type 1 ICP27 Mutants
Authors: Ellison Kimberly S. , Rice Stephen A. , Verity Robert , Smiley James R.
Published: July 2002

Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of viral gene expression that modulates RNA splicing, polyadenylation, and nuclear export. We have previously reported that ICP27 causes the cytoplasmic accumulation of unspliced α-globin pre-mRNA. Here we examined the effects of a series of ICP27 mutations that alter important functional regions of the protein on the processing and nuclear transport of α-globin and HSV ICP0 RNA. The results demonstrate that ICP27 mutants that are impaired for growth in noncomplementing cells, including mutants in the N- and C-terminal regions, are defective in the accumulation of α-globin pre-mRNA. Unexpectedly, several mutants that are competent to repress the expression of reporter genes in transient transfection assays failed to accumulate unspliced RNA, implying that different mechanisms are responsible for transrepression and pre-mRNA accumulation. Several mutants caused a marked increase in the length and heterogeneity of the α-globin mRNA poly(A) tail, suggesting that ICP27 may directly or indirectly affect the regulation of poly(A) polymerase. ICP27 was also required for the accumulation of multiple ICP0 intron-bearing transcripts, but this effect displayed a mutational sensitivity profile different from that of accumulation of unspliced α-globin RNA. Moreover, unlike spliced and unspliced α-globin RNAs, which were efficiently exported to the cytoplasm, spliced and intron-containing ICP0 transcripts were predominantly nuclear in localization, and ICP27 was not required for nuclear retention of the spliced message. We propose that these transcript- and ICP27 allele-specific differences may be explained by the presence of a strong
cis
-acting ICP27 response element in the α-globin transcript.
Upregulation of Tyrosine Kinase TKT by the Epstein-Barr Virus Transactivator Zta
Authors: Lu Jean , Chen Shao-Yin , Chua Huey-Huey , Liu Yu-Sheng , Huang Yu-Tzu , Chang Yao , Chen Jen-Yang , Sheen Tzung-Shiahn , Tsai Ching-Hwa
Published: July 2002

The Zta protein is a key transactivator involved in initiating the Epstein-Barr virus (EBV) lytic cascade. In addition to transactivating many viral genes, Zta has the capacity to influence host cellular signals by binding to promoter regions or by interacting with several important cellular factors. Based on the observation that tyrosine kinases play central roles in determining the fate of cells, a kinase display assay was used to investigate whether cells expressing Zta have an altered pattern of kinase expression. The assay revealed that TRK-related tyrosine kinase (TKT) is expressed at significant levels in Zta transfectants but not in control cells. Additional evidence was obtained from Northern and Western blotting. Importantly, the upregulation of phosphorylated TKT and TKT downstream effector matrix metalloproteinase 1 in Zta transfectants hinted that TKT might initiate a signaling cascade in Zta-expressing cells. In addition, deletion analysis of the Zta protein revealed that the transactivation and dimerization domains were both essential for the upregulation of TKT transcription. Moreover, correlation of expression levels of Zta and TKT transcripts in nasopharyngeal carcinoma biopsy specimens was clearly demonstrated by quantitative PCR (Q-PCR), which provides the first evidence for an effect of Zta on cellular gene expression in vivo. These findings offer insight into the virus-cell interactions and may help us elucidate the role of EBV in tumorigenesis.
Differential Dynamics of CD4+and CD8+T-Lymphocyte Proliferation and Activation in Acute Simian Immunodeficiency Virus Infection
Authors: Kaur Amitinder , Hale Corrina L. , Ramanujan Saroja , Jain Rakesh K. , Johnson R. Paul
Published: July 2002

Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67+lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67+CD8+T lymphocytes and a 2- to 3-fold increase in Ki-67+CD3−CD8+natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67+CD4+T lymphocytes during acute infection, although transient increases in Ki-67−and Ki-67+CD4+T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4+T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67+CD8+T lymphocytes were phenotypically CD45RA−CD49dhiFashiCD25−CD69−CD28−HLA-DR−and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8+T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4+and CD8+T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.
Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping
Authors: Ahola Tero , den Boon Johan A. , Ahlquist Paul
Published: July 2002

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast
Saccharomyces cerevisiae
, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene
XRN1
, encoding the major exonuclease that degrades uncapped mRNAs.
XRN1
deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.
Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid Clones
Authors: Ehsani Mariam E. , Abraha Tshge W. , Netherland-Snell Cecile , Mueller Niklaus , Taylor Meghan M. , Holwerda Barry
Published: July 2002

We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.
Direct Inhibitory Effect of Rotavirus NSP4(114-135) Peptide on the Na
+
-
d
-Glucose Symporter of Rabbit Intestinal Brush Border Membrane
Authors: Halaihel Nabil , Liévin Vanessa , Ball Judith M. , Estes Mary K. , Alvarado Francisco , Vasseur Monique
Published: July 2002

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of
d
-glucose and of
l
-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na
+
-
d
-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na
+
-
l
-leucine symport about equally and partially via a different transport mechanism, in that Na
+
behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.
Induction of Human Immunodeficiency Virus (HIV)-Specific CD8 T-Cell Responses byListeria monocytogenesand a HyperattenuatedListeriaStrain Engineered To Express HIV Antigens
Authors: Friedman Rachel S. , Frankel Fred R. , Xu Zhan , Lieberman Judy
Published: July 2002

Induction of cell-mediated immunity may be essential for an effective AIDS vaccine.Listeria monocytogenesis an attractive bacterial vector to elicit T-cell immunity to human immunodeficiency virus (HIV) because it specifically infects monocytes, key antigen-presenting cells, and because natural infection originates at the mucosa. Immunization with recombinantL. monocytogeneshas been shown to protect mice from lymphocytic choriomeningitis virus, influenza virus, and tumor inoculation.L. monocytogenesexpressing HIVgagelicits sustained high levels of Gag-specific cytotoxic T lymphocytes (CTLs) in mice. We have examined the ability ofListeriato infect human monocytes and present HIV antigens to CD8 T lymphocytes of HIV-infected donors to induce a secondary T-cell immune response. Using this in vitro vaccination protocol, we show thatL. monocytogenesexpressing the HIV-1gaggene efficiently provides a strong stimulus for Gag-specific CTLs in HIV-infected donor peripheral blood mononuclear cells.Listeriaexpressing Nef also elicits a secondary in vitro anti-Nef CTL response. SinceL. monocytogenesis a pathogen, before it can be seriously considered as a human vaccine vector, safety concerns must be addressed. We therefore have produced a highly attenuated strain ofL. monocytogenesthat requiresd-alanine for viability. The recombinant bacteria are attenuated at least 105-fold. We show that when these hyperattenuated bacteria are engineered to express HIV-1 Gag, they are at least as efficient at stimulating Gag-specific human CTLs in vitro as wild-type recombinants. These results suggest that attenuatedListeriais an attractive candidate vaccine vector to induce T-cell immunity to HIV in humans.