bluuebunny/crossref_metadata_2025_split huggingface dataset:
Directed Evolution of Toluene
ortho
-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation
Authors: Canada Keith A. , Iwashita Sachiyo , Shim Hojae , Wood Thomas K. 
Published: July 2002

ABSTRACT

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene
ortho
-monooxygenase (TOM) of
Burkholderia cepacia
G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in
Escherichia coli,
the variant TOM-Green degraded TCE (2.5 ± 0.3 versus 1.39 ± 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and
trans
-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ± 0.03 versus 0.029 ± 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ± 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the α-subunit of the hydroxylase, which appears to act as a smaller “gate” to the diiron active center. This hypothesis was supported by the ability of
E. coli
expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.
Effects ofEscherichia coliPhysiology on Growth of Phage T7 In Vivo and In Silico
Authors: You Lingchong , Suthers Patrick F. , Yin John 
Published: July 2002

ABSTRACTPhage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We establishedEscherichia coliat different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions.
Controlled Comparison of BacT/ALERT FAN Aerobic Medium and BACTEC Fungal Blood Culture Medium for Detection of Fungemia
Authors: McDonald L. Clifford , Weinstein Melvin P. , Fune Jose , Mirrett Stanley , Reimer Larry G. , Reller L. Barth 
Published: July 2002

ABSTRACT

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (
P
< 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (
P
< 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (
P
value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.
Identification of Enteropathogenic
Escherichia coli
in Simian Immunodeficiency Virus-Infected Infant and Adult Rhesus Macaques
Authors: Mansfield Keith G. , Lin Kuei-Chin , Newman Joseph , Schauer David , MacKey John , Lackner Andrew A. , Carville Angela 
Published: July 2002

ABSTRACT

Enteropathogenic
Escherichia coli
(EPEC) was recognized as a common opportunistic pathogen of simian immunodeficiency virus-infected rhesus macaques (
Macaca mulatta
) with AIDS. Retrospective analysis revealed that 27 of 96 (28.1%) animals with AIDS had features of EPEC infection, and EPEC was the most frequent pathogen of the gastrointestinal tract identified morphologically. In 7.3% of animals dying with AIDS, EPEC represented the sole opportunistic agent of the gastrointestinal tract at death. In 20.8% of cases, it was seen in combination with one or more gastrointestinal pathogens, including
Cryptosporidium parvum, Enterocytozoon bieneusi, Mycobacterium avium, Entamoeba histolytica, Balantidium coli, Strongyloides stercoralis
, cytomegalovirus, and adenovirus. Clinically, infection was associated with persistent diarrhea and wasting and was more frequent in animals that died at under 1 year of age (
P
< 0.001, Fisher exact test). The organism was associated with the characteristic attaching and effacing lesion in colonic tissue sections and produced a focal adherence pattern on a HEp-2 assay but was negative for Shiga toxin production as assessed by PCR and a HeLa cell cytotoxicity assay. A 2.6-kb fragment encompassing the intimin gene was amplified and sequenced and revealed 99.2% identity to sequences obtained from human isolates (GenBank
AF116899
) corresponding to the epsilon intimin subtype. Further investigations with rhesus macaques may offer opportunities to study the impact of EPEC on AIDS pathogenesis and gastrointestinal dysfunction.
Molecular Characterization of Invasive and Noninvasive
Campylobacter jejuni
and
Campylobacter coli
Isolates
Authors: Carvalho Alexandro C. T. , Ruiz-Palacios Guillermo M. , Ramos-Cervantes Pilar , Cervantes Luz-Elena , Jiang Xi , Pickering Larry K. 
Published: July 2002

ABSTRACT

Campylobacter jejuni
is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of
Campylobacter
diarrhea. Molecular characterization of invasive and noninvasive
Campylobacter
isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%)
Campylobacter
isolates (
P
= 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the
iam
locus was amplified in 85% of invasive and 20% of noninvasive strains (
P
= 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire
iam
locus showed a
Hin
dIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive
Campylobacter
strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the
iam
locus.
Group A Streptococcal Genotypes from Pediatric Throat Isolates in Rome, Italy
Authors: Dicuonzo Giordano , Gherardi Giovanni , Lorino Giulia , Angeletti Silvia , De Cesaris Marina , Fiscarelli Ersilia , Bessen Debra E. , Beall Bernard 
Published: July 2002

ABSTRACT

In a study assessing genetic diversity, 114 group A streptococcus (GAS) isolates were recovered from pediatric pharyngitis patients in Rome, Italy. These isolates comprised 22 different M protein gene (
emm
) sequence types, 14 of which were associated with a distinct serum opacity factor/fibronectin binding protein gene (
sof
) sequence type. Isolates with the same
emm
gene sequence type generally shared a highly conserved chromosomal macrorestriction profile. In three instances, isolates with dissimilar macrorestriction profiles had identical
emm
types; in each of these cases multilocus sequence typing revealed that isolates with the same
emm
type were clones having the same allelic profiles. Ninety-eight percent of the pharyngeal isolates had
emm
types previously found to be highly associated with
mga
locus gene patterns commonly found in pharyngeal GAS isolates.
Virulence Properties and Serotypes of Shiga Toxin-Producing
Escherichia coli
from Healthy Australian Slaughter-Age Sheep
Authors: Djordjevic Steven P. , Hornitzky Michael A. , Bailey Graham , Gill Paul , Vanselow Barbara , Walker Keith , Bettelheim Karl A. 
Published: July 2002

ABSTRACT

A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing
Escherichia coli
(STEC) virulence factors (
stx
1
,
stx
2
,
eaeA
, and
ehxA
). A subset was cultured for STEC isolates containing associated virulence factors (
eaeA
and/or
ehxA
), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing
stx
1
,
stx
2
, and
ehxA
were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H
−
(22 isolates [24.4%]), O5:H
−
(16 isolates [17.8%]), O128:H2 (11 isolates [12.2%]), O123:H
−
(8 isolates [8.9%]), and O85:H49 (5 isolates [5.6%]). Two isolates (2.2%) were typed as O157:H
−
. A total of 78 of 90 STEC isolates (86.7%) expressed Shiga toxin in Vero cell culture and 75 of 84
ehxA
-positive isolates (89.3%) expressed enterohemolysin on washed sheep blood agar.
eaeA
was observed in 11 of 90 (12.2%) ovine STEC isolates, including serotypes O5:H
−
, O84:H
−
, O85:H49, O123:H
−
O136:H40, and O157:H
−
. Although only 2 of 90 isolates were typed as O157:H
−
, the predominant serotypes recovered during this study have been recovered from human patients with clinical disease, albeit rarely.
Human Immunodeficiency Virus Type 1 Drug Resistance Testing: a Comparison of Three Sequence-Based Methods
Authors: Erali Maria , Page Sam , Reimer Larry G. , Hillyard David R. 
Published: July 2002

ABSTRACT
The use of genotypic assays for determining drug resistance in human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients is increasing. These tests lack standardization and validation. The aim of this study was to evaluate several tests used for the determination of HIV-1 drug resistance. Two genotypic tests, the Visible Genetics TruGene HIV-1 Genotyping Kit and the Applied Biosystems HIV Genotyping System, were compared using 22 clinical samples. Genotyping results were also obtained from an independent reference laboratory. The Visible Genetics and Applied Biosystems genotyping tests identified similar mutations when differences in the drug databases and reference strains were taken into account, and 19 of 21 samples were equivalent. The concordance between the two assays was 99% (249 of 252 mutation sites). Mutations identified by the reference laboratory varied the most among those identified by the three genotypic tests, possibly because of differences in the databases. The concordance of the reference laboratory results with the results of the other two assays was 80% (201 of 252). Samples with 500 to 750 HIV RNA copies/ml could be sequenced by the Visible Genetics and Applied Biosystems assays using 1 ml of input. The Visible Genetics and Applied Biosystems assays both generated an accurate sequence. However, the throughput of the Visible Genetics assay is more limited and may require additional instruments. The two assays differ technically but are similar in overall complexity. Data analysis in the two assays is straightforward, but only the reports provided by Visible Genetics contain information relating mutations to drug resistance. HIV drug resistance genotyping by sequencing is a complex technology which presents a challenge for analysis, interpretation, and reporting.
Use of Real-Time Quantitative PCR To Detect
Chlamydophila felis
Infection
Authors: Helps Chris , Reeves Nicky , Tasker Séverine , Harbour Dave 
Published: July 2002

ABSTRACT

A real-time PCR assay was developed to detect and quantify
Chlamydophila felis
infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.
Characterization of Multidrug-Resistant
Salmonella enterica
Serovar Typhimurium Isolated in Japan
Authors: Izumiya Hidemasa , Terajima Jun , Matsushita Shigeru , Tamura Kazumichi , Watanabe Haruo 
Published: July 2002

ABSTRACT

A total of 221 isolates of multidrug-resistant
Salmonella enterica
serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.
Epidemiology of Candidemia: 3-Year Results from the Emerging Infections and the Epidemiology of Iowa Organisms Study
Authors: Diekema D. J. , Messer S. A. , Brueggemann A. B. , Coffman S. L. , Doern G. V. , Herwaldt L. A. , Pfaller M. A. 
Published: July 2002

ABSTRACT

Bloodstream infections due to
Candida
species cause significant morbidity and mortality. Surveillance for candidemia is necessary to detect trends in species distribution and antifungal resistance. We performed prospective surveillance for candidemia at 16 hospitals in the State of Iowa from 1 July 1998 through 30 June 2001. Using U.S. Census Bureau and Iowa Hospital Association data to estimate a population denominator, we calculated the annual incidence of candidemia in Iowa to be 6.0 per 100,000 of population.
Candida albicans
was the most common species detected, but 43% of candidemias were due to species other than
C. albicans
. Overall, only 3% of
Candida
species were resistant to fluconazole. However,
Candida glabrata
was the most commonly isolated species other than
C. albicans
and demonstrated some resistance to azoles (fluconazole MIC at which 90% of the isolates tested are inhibited, 32 μg/ml; 10% resistant, 10% susceptible dose dependent).
C. glabrata
was more commonly isolated from older patients (
P
= 0.02) and caused over 25% of candidemias among persons 65 years of age or older. The investigational triazoles posaconazole, ravuconazole, and voriconazole had excellent in vitro activity overall against
Candida
species.
C. albicans
is the most important cause of candidemia and remains highly susceptible to available antifungal agents. However,
C. glabrata
has emerged as an important and potentially antifungal resistant cause of candidemia, particularly among the elderly.
Introduction of New Clones of Penicillin-Nonsusceptible
Streptococcus pneumoniae
in Hong Kong
Authors: Ip Margaret , Lyon Donald J. , Yung Raymond W. H. , Tsang Lily , Cheng Augustine F. B. 
Published: July 2002

ABSTRACT

Analysis of penicillin-nonsusceptible
Streptococcus pneumoniae
(PNSP) isolates in Hong Kong by use of a combination of antibiogram typing, serotyping, multilocus sequence typing, and pulsed-field gel electrophoresis indicated that the dissemination of PNSP was the result of the spread of international clones: variants of the Spain
23F
-1 or Spain
6B
-2 clones were the predominant PNSP isolates from 1994 to 1997 and remained so, but Taiwan
19F
-14 and Taiwan serotype 6B clones were disseminated in Hong Kong in 1999 and 2000. Concomitant changes in antibiotic susceptibility profiles, with the rate of susceptibility to chloramphenicol rising from 10% in the period from 1994 to 1997 to 31% (
P
< 0.001) in 1999 and 2000, were noted to accompany the shift of clones.
Rapid Identification ofPenicillium marneffeiby PCR-Based Detection of Specific Sequences on the rRNA Gene
Authors: Vanittanakom Nongnuch , Vanittanakom Pramote , Hay Roderick J. 
Published: July 2002

ABSTRACTAn emerging pathogenic dimorphic fungus,Penicillium marneffei, is one of the major causes of morbidity in patients with human immunodeficiency virus infection in Southeast Asia. A PCR-hybridization assay has been developed to identify this pathogen. This study describes the use of single and nested PCR methods for the rapid identification ofP. marneffei.Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA genes ofP. marneffei.The outer primers (RRF1 and RRH1) were fungus specific. The inner primers (Pm1 and Pm2) were specific forP. marneffeiand were used in nested or single PCR. The specific fragment of approximately 400-bp was amplified from both mold and yeast forms of 13P. marneffeihuman isolates, 12 bamboo rat isolates, and 1 soil isolate, but not from other fungi, bacteria, and human DNA. The amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The sensitivities of the single PCR and nested PCR were 1.0 pg/μl and 1.8 fg/μl, respectively. The assay is useful for rapid identification ofP. marneffeicultures. Very young culture ofP. marneffei(2-day-old filamentous colony, 2 mm in diameter) could be performed by this assay. The species was identified within 7 h (single PCR) or 10 h (nested PCR), compared to 4 to 7 days for confirmation of dimorphism. The application of these PCR methods for early diagnosis of the disease needs to be studied further.
Tumor-Specific, Replication-Competent Adenovirus Vectors Overexpressing the Adenovirus Death Protein
Authors: Doronin Konstantin , Toth Karoly , Kuppuswamy Mohan , Ward Peter , Tollefson Ann E. , Wold William S. M. 
Published: July 2002

ABSTRACTWe have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting theadpgene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutantdl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G0to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus,dl1101/1107, or wild-type Ad. They are also more efficient thandl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1−E3−replication-defective vector expressing β-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10−4PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected withdl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.
Human Papillomavirus Type 16 E6 Induces Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase
Authors: Kao Wynn H. , Beaudenon Sylvie L. , Talis Andrea L. , Huibregtse Jon M. , Howley Peter M. 
Published: July 2002

ABSTRACT
The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.