bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationships between morphological and chemical characteristics of perennial ryegrass varieties and intake by sheep under continuous stocking management
Authors: Orr R. J. , Cook J. E. , Champion R. A. , Rook A. J.
Published: November 2004

Plots of five intermediate‐heading varieties of perennial ryegrass (Lolium perenne L.) [AberDove, Belramo and Glen (diploid); Twins (tetraploid); and AberExcel (tetraploid hybrid)] were continuously stocked with sheep to maintain a target sward surface height of 40–50 mm. Daily dry matter (DM) intake was significantly different (F‐value = 0·032) between the varieties, with the tetraploid hybrid AberExcel having the highest values for daily DM intake and intake rate during eating. Amongst the diploid varieties, intake rate tended to be higher for sheep grazing Glen. The varieties comprised a wide range in potential growth habit, from the relatively prostrate, highly tillered Glen to the more‐erect AberExcel and there were differences between them in the vertical distribution of leaves within the sward canopy. The leaves of AberExcel weighed 3·6 mg DM cm−2 leaf area in contrast to the other varieties (4·3–5·3 mg DM cm−2 leaf area) resulting in a high leaf area index (LAI) in relation to the green leaf mass. Intake rate was not significantly correlated with extended tiller and sheath tube lengths, partition of herbage mass, number of tillers per square metre or LAI. However, canonical variates analysis showed that there were significant differences between the varieties for the morphological and chemical factors examined. Other factors also need to be explored to explain these differences in ingestive behaviour in order to identify plant traits that are correlated with herbage intake rate. These are needed for varieties destined for grazing use, both during the breeding programme and their subsequent evaluation.
Genetic diversity studies in farm animals – a survey
Authors: Baumung R. , Simianer H. , Hoffmann I.
Published: November 2004

During the past decade a large number of genetic diversity studies in domestic livestock based on microsatellite loci was carried out all over the world. A survey among research groups reveals that in 50% of 87 projects more than eight breeds are investigated. Breeds are chosen mainly because of their long history of isolation, unique phenotypic qualities or an evolution within a unique environment. In half of the projects the average planned sample size per breed is higher than 50. Small ruminants are investigated in the majority of projects. The most preferred type of sample is blood and the most preferred markers are microsatellite loci. To combine results from different diversity studies, the adherence to recommendations, e.g. which loci to genotype or required sample sizes per breed, is necessary. The so called International Society for Animal Genetics (ISAG)/FAO recommendations, which were developed for that purpose, are well known, but the use of recommended microsatellite loci varies between species. The highest acceptance was found in pig, and the lowest in chicken.
Effects of a structured health education programme by a diabetic education nurse on cardiovascular risk factors in Chinese Type 2 diabetic patients: a 1‐year prospective randomized study
Authors: Ko G. T. C. , Li J. K. Y. , Kan E. C. Y. , Lo M. K. W.
Published: November 2004

Aims  To assess the effect of regular diabetic health education on cardiovascular risk factors in Chinese Type 2 diabetic patients.

Methods  This was a 1‐year prospective randomized study. One hundred and eighty Type 2 diabetic subjects were recruited from three regional diabetic centres in Hong Kong. Ninety received additional structured reinforcement of diabetic health education by a trained nurse after the doctors’ consultations every 3 months (intervention group). The others received the same medical care except no nursing reinforcement (control group). Outcome measures included fasting plasma glucose, HbA1c, body mass index, waist circumference, blood pressure and lipid profiles, which were assessed before the study and after 1 year.

Results  Two of the controls defaulted follow‐up. The intervention group and controls had similar age and sex distribution. At the end of study, the intervention group had reducted their waist circumference, diastolic blood pressure, HbA1c, total cholesterol and low‐density lipoprotein cholesterol levels. The controls had reduced their total cholesterol and low‐density lipoprotein cholesterol levels. Other cardiovascular risk factors were not significantly changed in the controls. Addition of drugs and/or dosage increment of anti‐diabetic drugs, lipid‐lowering agents and anti‐hypertensive agents were similar between the two groups.

Conclusions  Regular structured reinforcement with diabetic health education is useful. It helps to control more successfully some of the cardiovascular risk factors in Chinese Type 2 diabetic patients.
Exploring the idiotypes of insulin antibodies as markers for remission in Type 1 diabetes
Authors: Devendra D. , Galloway T. S. , Horton S. J. , Wilkin T. J.
Published: November 2004

Aims  Complete or partial remission can occur in newly diagnosed Type 1 diabetes patients. We created idiotype‐specific reagents to explore the idiotypes of insulin antibodies (IA) in a patient in remission, and to compare with a patient who was not.

Methods  Phage display was used to create a library of phagotopes specific to insulin binding in four sera. Sera from a Type 1 diabetes subject deemed to have undergone remission were taken at diagnosis and again during remission. Sera from a non‐remitter were taken at diagnosis and after 3 months on insulin. Phagotopes from the four sera were randomly selected and tested for insulin specificity in a radiobinding assay by using sera from remitters and non‐remitters.

Results  IA‐binding phagotope selected from serum during remission displaced insulin binding in all nine IA+ remitters and all 10 IA+ non‐remitters. IA‐binding phagotope selected from the non‐remission patient (3 months after insulin therapy) displaced insulin binding in 8/9 IA+ remitters and 8/10 IA+ non‐remitters. The consensus peptide sequences adduced from the phages were identical for both these phagotopes. Phagotopes derived from insulin autoantibody‐positive individuals at diagnosis were unable to displace insulin binding in the IA+ sera 3 months later, whether in remission or not.

Conclusions  We have established the principle of using phage display in the investigation of insulin antibodies during remission in Type 1 diabetes. The immunological characteristics of IA 3 months after the introduction of insulin treatment were different from those at diagnosis of Type 1 diabetes (IAA). Using phage display technology, it was not possible to distinguish insulin antibodies according to remission status.
Emotional specificity of startle potentiation during the early stages of picture viewing
Authors: Stanley James , Knight Robert G.
Published: November 2004

Potentiation of the startle blink reflex shortly after picture onset has only been reported in a sample of high animal‐fear participants during fear‐evoking stimuli. The experiment tested the emotional specificity of startle reflex modification at early (300 ms) and late (2–5 s) stages of picture viewing in an unselected sample (n=55). Participants viewed two negative picture categories, threat and disgust contents, in addition to neutral and positive pictures. Blink potentiation occurred for threat contents at both probe times, relative to neutral responses. Disgust contents only potentiated blinks relative to positive content responses. These results are inconsistent with a basic, prepulse interpretation of early startle modification and suggest that affective modification can be observed shortly after picture onset, depending on the specific emotional content of the picture.
Cardiovascular reactivity and adaptation to recurrent psychological stress: Replication and extension
Authors: Kelsey Robert M. , Soderlund Kathleen , Arthur Carlotta M.
Published: November 2004

We analyzed the effects of evaluative observation and baseline duration on cardiovascular reactivity and adaptation to recurrent psychological stress. Cardiovascular reactivity to mental arithmetic stress was assessed in college men and women (N=224) during two pretest tasks, a test task, and a posttest task. Participants were assigned randomly in a 2 × 2 design to manipulations of baseline duration before the test task (4 min vs. 12 min) and evaluative observation during the test task (observed vs. control). Repeated exposure to stress attenuated cardiac but not vascular reactivity. Evaluative observation disrupted cardiac adaptation, resulting in a resurgence of β‐adrenergic cardiac reactivity during the test task. Cardiac adaptation resumed fully during the posttest task. Baseline duration had no effect on reactivity. The results replicate and extend previous work, and support the dual process theory of habituation and sensitization.
Clinical findings and diagnostic value of post‐traumatic thoracic radiographs in dogs and cats with blunt trauma
Authors: Sigrist Nadja E. , Doherr Marcus G. , Spreng David E.
Published: November 2004

Objective: To characterize the clinical findings in dogs and cats that sustained blunt trauma and to compare clinical respiratory examination results with post‐traumatic thoracic radiography findings.

Design: Retrospective clinical study.

Setting: University small animal teaching hospital.

Animals, interventions and measurements: Case records of 63 dogs and 96 cats presenting with a history of blunt trauma and thoracic radiographs between September 2001 and May 2003 were examined. Clinical signs of respiratory distress (respiratory rate (RR), pulmonary auscultation) and outcome were compared with radiographic signs of blunt trauma.

Results: Forty‐nine percent of dogs and 63.5% of cats had radiographic signs attributed to thoracic trauma. Twenty‐two percent of dogs and 28% of cats had normal radiographs. Abnormal auscultation results were significantly associated with radiographic signs of thoracic trauma, radiography score and presence and degree of contusions. Seventy‐two percent of animals with no other injuries showed signs of thoracic trauma on chest radiographs. No correlation was found between the radiographic findings and outcome, whereas the trauma score at presentation was significantly associated with outcome and with signs of chest trauma but not with the radiography score.

Conclusion: Thoracic trauma is encountered in many blunt trauma patients. The RR of animals with blunt trauma is not useful in predicting thoracic injury, whereas abnormal chest auscultation results are indicative of chest abnormalities. Thorough chest auscultation is, therefore, mandatory in all trauma animals and might help in the assessment of necessity of chest radiographs.
Effect of sample handling on venous PCO2, pH, bicarbonate, and base excess measured with a point‐of‐care analyzer
Authors: Richey Meghan T. , McGrath Charles J. , Portillo Erin , Scott Mary , Claypool Larry
Published: November 2004

Objective:The purpose of this study was to determine the effect of timing of analysis, collection tube type and repeated opening of sample tubes on venous PCO2, pH, HCO3, and base excess (BE) results.

Design:Prospective experimental study, paired sample analysis.

Setting:Veterinary Medical Teaching Hospital.

Animals:Twenty dogs.

Interventions:Jugular venous blood samples.

Measurements and main results:PCO2, pH, HCO3, and BE were determined immediately following collection (control) and at selected times up to 30 minutes after placement in either screw top or vacuum heparin collection tubes. A different set of screw top and vacuum heparin collection tubes were sampled repeatedly over time for up to 15 minutes.

In the screw top delayed analysis group, only pH changed significantly at one time point. PCO2decreased significantly in all other groups and resulted in a significant reciprocal pH change in the vacuum tubes with either delayed single analysis or repeated sampling. HCO3and BE declined significantly in multi‐sampled vacuum tubes and HCO3also decreased significantly in multi‐sampled screw top tubes.

Conclusions:Analysis of acid–base status is optimally performed on freshly drawn blood. However, when it is anticipated there will be a delay in analysis of samples kept at room temperature, the use of 2.0 mL plastic screw top heparin anticoagulant tubes may result in fewer pre‐analytical errors than 3.5 mL glass vacuum tubes.
Application of Genetic Algorithms in the Engine Technology Selection Process
Authors: Roth Bryce , Patel Chirag
Published: November 2004

The objective of this paper is to demonstrate the application of genetic algorithms to the engine technology selection process. The “technology identification, evaluation, and selection” method is discussed in conjunction with genetic algorithm optimization as a technique to quickly evaluate the impact of various technologies and select the subset with the highest potential payoff. Techniques used to model various aspects of engine technologies are described, with emphasis on technology constraints and their impact on the combinatorial optimization of technologies. Challenges include objective function formulation and development of models to deal with incompatibilities among different technologies. Typical results are presented for an 80-technology optimization using various visualization techniques to assist in easy interpretation of genetic algorithm results. Finally, several ideas for future development of these methods are briefly explored.
Fuzzy Approaches for Searching Optimal Component Matching Point in Gas Turbine Performance Simulation
Authors: Kong Changduk , Ki Jayoung , Kang Myoungcheol
Published: November 2004

In performance simulation of gas turbine, some iteration processes for searching the operating point, which should be matched through gas path components, would be needed. Therefore if the engine is very complicated, the efficiency of convergence is getting lower due to increasing the number of routines and iterations for matching procedure. Furthermore, there may be some problems in numerical calculation such as the tendency of divergence, instability, and increase of calculation time. In the traditional matching methods, after some variable parameters are initially assumed and then calculated, the calculation is iterated to converge within an appropriate error range by expressing the new variables with the previous calculation error function. Even though these traditional methods show comparatively reasonable results in performance simulation, there may be some unsatisfied results in particular cases. Moreover, in this case it can be more difficult in more precise calculation due to simplification by using various assumptions. Therefore in order to search the optimal matching point in performance simulation of gas turbine efficiently and quickly, the fuzzy approaches were applied. In performance simulation using the fuzzy logic, it was found that more effective engine operating points were found and calculation time was considerably reduced less than the traditional methods. Furthermore, MATLAB was used to apply the fuzzy logic easily as well as to make user-friendly circumstance in performance analysis.
Fault Detection and Identification in an IM270 Gas Turbine Using Measurements for Engine Control
Authors: Kurosaki Masahiro , Morioka Tadashi , Ebina Kosuke , Maruyama Masatoshi , Yasuda Tomoshige , Endoh Makoto
Published: November 2004

A unique fault detection and identification algorithm using measurements for engine control use is presented. The algorithm detects an engine fault and identifies the associated component, using a gas path analysis technique with a detailed nonlinear engine model. The algorithm is intended to detect steplike changes in component performance rather than gradual change of all components. Component performance deviation (efficiency and flow rate) is represented by a magnitude and a phase. The phase is selected to minimize the error of evaluation matrices. Then the magnitude is computed. By utilizing operational data of the IM270 engine, the compressor and the turbine performance deviation was quantitatively identified.
CaNdt80 Is Involved in Drug Resistance in
Candida albicans
by Regulating
CDR1
Authors: Chen Chia-Geun , Yang Yun-Liang , Shih Hsin-I , Su Chia-Li , Lo Hsiu-Jung
Published: November 2004

Overexpression of
CDR1
, an efflux pump, is one of the major mechanisms contributing to drug resistance in
Candida albicans. CDR1 p-lacZ
was constructed and transformed into a
Saccharomyces cerevisiae
strain so that the
lacZ
gene could be used as the reporter to monitor the activity of the
CDR1
promoter. Overexpression of
CaNDT80
, the
C. albicans
homolog of
S. cerevisiae NDT80
, increases the β-galactosidase activity of the
CDR1 p-lacZ
construct in
S. cerevisiae
. Furthermore, mutations in
CaNDT80
abolish the induction of
CDR1
expression by antifungal agents in
C. albicans
. Consistently, the
Candt80
/
Candt80
mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in
C. albicans
whose function has been identified.
Intranasal Interleukin-12 Treatment Promotes Antimicrobial Clearance and Survival in PulmonaryFrancisella tularensissubsp.novicidaInfection
Authors: Pammit Michael A. , Budhavarapu Varija N. , Raulie Erin K. , Klose Karl E. , Teale Judy M. , Arulanandam Bernard P.
Published: November 2004

Francisella tularensisis a highly virulent facultative intracellular bacterium and is considered a potential biological warfare agent. Inhalation tularemia can lead to the development of bronchopneumonia, which is frequently fatal without medical intervention. Treatment strategies that directly target the respiratory mucosa may extend the efficacy of therapy, particularly for the medical management of acute aerosol exposure. To this end, we describe an intranasal (i.n.) strategy for the treatment of pulmonaryFrancisellainfection in mice that uses a combinatorial approach with the conventional antibiotic gentamicin and interleukin 12 (IL-12). The i.n. administration of IL-12 alone promoted bacterial clearance and extended the time to death but did not prevent mortality against lethal pulmonary challenge withFrancisella tularensissubsp.novicida.However, i.n. treatment with gentamicin and IL-12 therapeutically at 8 and 24 h after challenge markedly enhanced the rate of survival (70 to 100%) against pulmonary infection compared to the rates of survival for animals treated with antibiotic alone (17%) or IL-12 alone (0%). A delay in combinatorial therapy over a span of 4 days progressively decreased the efficacy of this treatment regimen. This combinatorial treatment was shown to be highly dependent upon the induction of endogenous gamma interferon and may also involve the activation of natural killer cells. Together, these findings suggest that IL-12 may be a potent adjunct for chemotherapy to enhance drug effectiveness against pulmonaryFrancisellainfection.
Potent and Long-Acting Dimeric Inhibitors of Influenza Virus Neuraminidase Are Effective at a Once-Weekly Dosing Regimen
Authors: Macdonald Simon J. F. , Watson Keith G. , Cameron Rachel , Chalmers David K. , Demaine Derek A. , Fenton Rob J. , Gower David , Hamblin J. Nicole , Hamilton Stephanie , Hart Graham J. , Inglis Graham G. A. , Jin Betty , Jones Haydn T. , McConnell Darryl B. , Mason Andy M. , Nguyen Van , Owens Ian J. , Parry Nigel , Reece Phillip A. , Shanahan Stephen E. , Smith Donna , Wu Wen-Yang , Tucker Simon P.
Published: November 2004

Dimeric derivatives (compounds 7 to 9) of the influenza virus neuraminidase inhibitor zanamivir (compound 2), which have linking groups of 14 to 18 atoms in length, are approximately 100-fold more potent inhibitors of influenza virus replication in vitro and in vivo than zanamivir. The observed optimum linker length of 18 to 22 Å, together with observations that the dimers cause aggregation of isolated neuraminidase tetramers and whole virus, indicate that the dimers benefit from multivalent binding via intertetramer and intervirion linkages. The outstanding long-lasting protective activities shown by compounds 8 and 9 in mouse influenza infectivity experiments and the extremely long residence times observed in the lungs of rats suggest that a single low dose of a dimer would provide effective treatment and prophylaxis for influenza virus infections.
Rapid, Simple In Vivo Screen for New Drugs Active against
Mycobacterium tuberculosis
Authors: Nikonenko Boris V. , Samala Rowena , Einck Leo , Nacy Carol A.
Published: November 2004

We evaluated the use of a simple and easy-to-obtain potential marker of tuberculosis (TB) drug efficacy, body weight, and correlated weight loss or gain with the number of CFU of
Mycobacterium tuberculosis
in lungs and spleens of infected mice. C3H mice were infected intravenously with 10
6
CFU of virulent
M. tuberculosis
H37Rv, and body weight was evaluated for several weeks after infection. At day 20, infected untreated mice consistently lost more than 25% of their body weight. Chemotherapy with selected orally active anti-TB drugs was initiated 7 days following infection and continued for 13 days. Drugs that were administered daily by gavage included isoniazid (INH), ethambutol (EMB), rifampin (RIF), and moxifloxacin (MXF). At the most effective doses, each of these drugs inhibited bacterial growth and abolished infection-induced body weight loss. Chemotherapy with 1/10 the standard dose of INH determined in accepted long-term murine models of TB also prevented body weight loss, while chemotherapy with 1/10 the standard dose of RIF did not. With only 2 weeks of chemotherapy, we observed a good reverse correlation between CFU in lung or spleen and body weight of mice. The simple measurement of weight in TB-infected drug-treated mice required only a weight balance, and go/no-go drug efficacy data was available on day 20 without the necessity of prolonged drug treatment and long (3 weeks or more) in vitro culture times to obtain organ CFU values.
Penicillin-Binding Protein 2 Is Essential for Expression of High-Level Vancomycin Resistance and Cell Wall Synthesis in Vancomycin- Resistant
Staphylococcus aureus
Carrying the Enterococcal
vanA
Gene Complex
Authors: Severin Anatoly , Wu Shang Wei , Tabei Keiko , Tomasz Alexander
Published: November 2004

A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in
Staphylococcus aureus
. The transcription of
pbp2
of the highly vancomycin- and oxacillin-resistant strain COLVA
200
and its mutant derivative with inactivated
mecA
were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-
d
-thiogalactopyranoside inducer was determined. The results indicate that
mecA—
the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of
mecA
, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant
S. aureus
(A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem.
279
:3398-3407, 2004) is penicillin binding protein 2.
TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1
Authors: Andries Koen , Azijn Hilde , Thielemans Theo , Ludovici Donald , Kukla Michael , Heeres Jan , Janssen Paul , De Corte Bart , Vingerhoets Johan , Pauwels Rudi , de Béthune Marie-Pierre
Published: November 2004

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC
50
] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC
50
= 3.5 μM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of α
1
-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC
50
of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC
50
< 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.
Clinically Relevant Interpretation of Genotype and Relationship to Plasma Drug Concentrations for Resistance to Saquinavir-Ritonavir in Human Immunodeficiency Virus Type 1 Protease Inhibitor-Experienced Patients
Authors: Marcelin Anne-Geneviève , Dalban Cécile , Peytavin Gilles , Lamotte Claire , Agher Rachid , Delaugerre Constance , Wirden Marc , Conan Françoise , Dantin Sylvie , Katlama Christine , Costagliola Dominique , Calvez Vincent
Published: November 2004

It has been shown that virological protease inhibitor (PI) resistance mutations present at the initiation of saquinavir (SQV) plus ritonavir (RTV) therapy in PI-experienced patients are the strongest predictors of virological response. But most of the current resistance algorithms are adapted for unboosted SQV regimens. We applied a stepwise methodology for the development and validation of a clinically relevant genotypic resistance score for an SQV (800 mg twice per day [b.i.d.]) plus RTV (100 mg b.i.d.)-containing regimen. PI-experienced patients treated by this regimen achieved a human immunodeficiency virus plasma viral load (VL) of <200 copies/ml at months 3 to 5 for 41.7% of subjects. Adjusted in a multivariate analysis, taking into account all the confounding factors, such as the nucleoside used, five mutations were combined in a resistance score associated with a reduced virological response to an SQV-plus-RTV regimen: L24I, I62V, V82A/F/T/S, I84V, and L90IM. Patients with isolates harboring 0 to 1 mutation among the score achieved −2.20 log
10
and −1.23 log
10
copies/ml of VL reduction, respectively, while it was −0.27 log
10
copies/ml for those with at least two mutations, classifying the isolates as “no evidence of resistance” (0 or 1 mutation) or “resistance ” (≥2 mutations). The minimum concentration in plasma (
C
min
) of SQV alone was not associated with the virological response. However, the combination of the SQV
C
min
and the genotypic score, expressed as the genotypic inhibitory quotient, was predictive of the virological response, suggesting that the interpretation of SQV concentrations in plasma should be done only in the context of the resistance index provided by viral genotype for PI-experienced patients.
Chalcomycin Biosynthesis Gene Cluster from
Streptomyces bikiniensis
: Novel Features of an Unusual Ketolide Produced through Expression of the
chm
Polyketide Synthase in
Streptomyces fradiae
Authors: Ward Shannon L. , Hu Zhihao , Schirmer Andreas , Reid Ralph , Revill W. Peter , Reeves Christopher D. , Petrakovsky Oleg V. , Dong Steven D. , Katz Leonard
Published: November 2004

Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium
Streptomyces bikiniensis
, contains a 2,3-
trans
double bond and the neutral sugar
d
-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides. Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from
S. bikiniensis
with very high identity to a unique ketosynthase domain of the tylosin PKS. The resulting amplimers were used to identify two overlapping cosmids encompassing the
chm
PKS. Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it. The
chm
PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin. Expression of PKS in the heterologous host
Streptomyces fradiae
, from which the
tyl
genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-
trans
enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the
chm
PKS. The production of a 3-keto macrolide from the
chm
PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-
trans
double bond in chalcomycin. From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-
d
-chalcose was proposed.
Biochemical Characterization of the THIN-B Metallo-β-Lactamase of
Janthinobacterium lividum
Authors: Docquier Jean-Denis , Lopizzo Teresa , Liberatori Sabrina , Prenna Manuela , Thaller Maria Cristina , Frère Jean-Marie , Rossolini Gian Maria
Published: November 2004

The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species
Janthinobacterium lividum
, was overproduced in
Escherichia coli
by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.
Molecular and Kinetic Comparison of the Novel Extended-Spectrum β-Lactamases CTX-M-25 and CTX-M-26
Authors: Munday Craig J. , Boyd David A. , Brenwald Nigel , Miller Mark , Andrews Jennifer M. , Wise Richard , Mulvey Michael R. , Hawkey Peter M.
Published: November 2004

CTX-M-25 is a novel extended-spectrum β-lactamase isolated from a single Canadian
Escherichia coli
isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of β-lactamase inhibitors. The
bla
CTX-M-25
gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The
bla
CTX-M-26
gene was detected on a 100-kb plasmid isolated from a
Klebsiella pneumoniae
strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the
bla
CTX-M-25
-harboring plasmid. Both CTX-M genes were located downstream of IS
Ecp1
, although the copy upstream of
bla
CTX-M-25
was disrupted by IS
50-A
. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (
k
cat
values, 33 and 0.005 s
−1
for CTX-M-25 and CTX-M-26, respectively).
Neuraminidase Inhibitor-Rimantadine Combinations Exert Additive and Synergistic Anti-Influenza Virus Effects in MDCK Cells
Authors: Govorkova Elena A. , Fang Hong-Bin , Tan Ming , Webster Robert G.
Published: November 2004

There is insufficient information about combination therapy with approved anti-influenza agents. We tested combinations that paired a neuraminidase (NA) inhibitor (zanamivir, oseltamivir carboxylate, or peramivir) with rimantadine against infection of MDCK cells with H1N1 and H3N2 subtypes of influenza A virus and characterized their mode of interaction. When reduction of extracellular virus was analyzed by individual regression models and three-dimensional representations of the data, all three combinations showed additive and synergistic effects with no cytotoxicity. Maximum synergy against A/New Caledonia/20/99 (H1N1) virus infection was observed with <2.5 μM rimantadine paired with low concentrations of NA inhibitors. All combinations reduced the extracellular yield of A/Panama/2007/99 (H3N2) influenza virus synergistically. However, our findings were different for the cell-associated virus yield. At some drug concentrations, the yield of cell-associated virus was inhibited antagonistically. Therefore, the method of analysis can be a crucial factor in evaluating the interactions of drugs with different mechanisms. We hypothesize that assays based on cell-associated virus yield may underestimate the efficacies of drug combinations that include an NA inhibitor. Taken together, our results suggest that regimens that combine NA inhibitors and rimantadine exert synergistic anti-influenza effects in vitro. These findings provide baseline information for therapeutic testing of the drug combinations in vivo.
Polymorphism in
Plasmodium falciparum
Drug Transporter Proteins and Reversal of In Vitro Chloroquine Resistance by a 9,10-Dihydroethanoanthracene Derivative
Authors: Millet Julie , Alibert Sandrine , Torrentino-Madamet Marylin , Rogier Christophe , Santelli-Rouvier Christiane , Bigot Patricia , Mosnier Joel , Baret Eric , Barbe Jacques , Parzy Daniel , Pradines Bruno
Published: November 2004

BG958 reverses resistance in chloroquine-resistant isolates from different countries. Five mutations in the
Plasmodium falciparum crt
(
pfcrt
) gene resulting in the amino acid changes K76T, M74I, N75E, A220S, and R371I are systematically identified in resistance-reversed Asian, African, and Brazilian parasites which possess the
pfcrt
(CIET) haplotype. In combination with BG958, the activity of chloroquine is increased in parasites with the N86Y mutation in
pfmdr1
.
Glucocorticoids Increase In Vitro and In Vivo Activities of Antibiotics against
Chlamydophila pneumoniae
Authors: Caronzolo Dario , Lucini Valeria , Pannacci Marilou , Grosso Silvia , Colleoni Federica , Fraschini Franco , Scaglione Francesco
Published: November 2004

The in vitro and in vivo antichlamydial activities of dexamethasone and beclomethasone alone and in combination with an antibiotic were tested. In vitro, dexamethasone and beclomethasone decreased the number of inclusion-forming units versus the control number (
P
< 0.001). The combination of glucocorticoids with azithromycin, telithromycin, or levofloxacin was more active than antibiotics used alone (
P
< 0.001). The combination, tested in a murine
Chlamydophila pneumoniae
infection model, produced similar results.
Effects of a Number of Classes of 50S Inhibitors on Stop Codon Readthrough during Protein Synthesis
Authors: Thompson Jill , Pratt Catherine A. , Dahlberg Albert E.
Published: November 2004

The effect of a number of antibiotics on stop codon readthrough during protein synthesis in
Escherichia coli
was examined. Inhibitors which bind close to the entrance of the peptide exit tunnel on the 50S ribosomal subunit promote substantial levels of readthrough, presumably by disrupting the mechanism of peptide release.
Chromosome-Encoded CTX-M-3 from
Kluyvera ascorbata
: a Possible Origin of Plasmid-Borne CTX-M-1-Derived Cefotaximases
Authors: Rodríguez María Margarita , Power Pablo , Radice Marcela , Vay Carlos , Famiglietti Angela , Galleni Moreno , Ayala Juan A. , Gutkind Gabriel
Published: November 2004

A gene identical to plasmid-borne
bla
CTX-M-3
is present in the chromosome of one
Kluyvera ascorbata
strain. It is associated with a structure including an inverted repeat right and an open reading frame 477-like gene probably involved in the mobilization of
bla
CTX-M-3
. Two other
K. ascorbata
strains rendered the previously described
bla
KLUA-9
gene.
A Subinhibitory Concentration of Clarithromycin Inhibits
Mycobacterium avium
Biofilm Formation
Authors: Carter George , Young Lowell S. , Bermudez Luiz E.
Published: November 2004

Mycobacterium avium
causes disseminated infection in immunosuppressed individuals and lung infection in patients with chronic lung diseases.
M. avium
forms biofilm in the environment and possibly in human airways. Antibiotics with activity against the bacterium could inhibit biofilm formation. Clarithromycin inhibits biofilm formation but has no activity against established biofilm.
In Vitro Interactions between Antifungals and Immunosuppressants against
Aspergillus fumigatus
Isolates from Transplant and Nontransplant Patients
Authors: Steinbach William J. , Singh Nina , Miller Jackie L. , Benjamin Daniel K. , Schell Wiley A. , Heitman Joseph , Perfect John R.
Published: November 2004

We performed in vitro antifungal checkerboard testing on 12
Aspergillus fumigatus
clinical isolates (6 transplant recipients and 6 nontransplant patients) with three antifungal agents (amphotericin B, voriconazole, and caspofungin) and three immunosuppressants (FK506, cyclosporine, and rapamycin). We were not able to detect a difference in calcineurin inhibitor antifungal activity against isolates from transplant recipients and nontransplant patients.
Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein
Authors: Casaday Rebecca J. , Bailey Justin R. , Kalb Suzanne R. , Brignole Edward J. , Loveland Amy N. , Cotter Robert J. , Gibson Wade
Published: November 2004

Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream “priming” phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.
Insertion of a Classical Nuclear Import Signal into the Matrix Domain of the Rous Sarcoma Virus Gag Protein Interferes with Virus Replication
Authors: Garbitt Rachel A. , Bone Karen R. , Parent Leslie J.
Published: November 2004

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a “classical ” nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the
gag
gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.
In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques
Authors: Ambrose Zandrea , Boltz Valerie , Palmer Sarah , Coffin John M. , Hughes Stephen H. , KewalRamani Vineet N.
Published: November 2004

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV
mne
: SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV
mne
contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV
mne
. Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV
mne
particles, the RT-SHIV
mne
virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV
mne
and SIV-RT-YY particles. HIV-1 and RT-SHIV
mne
were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV
mne
. Classical NNRTI resis tance mutations were selected in RT-SHIV
mne
after in vitro drug treatment and were monitored in a sensitive allele-specific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV
mne
may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.
Antiviral Effects of Human Immunodeficiency Virus Type 1-Specific Small Interfering RNAs against Targets Conserved in Select Neurotropic Viral Strains
Authors: Dave Rajnish S. , Pomerantz Roger J.
Published: November 2004

RNA interference, a natural biological phenomenon mediated by small interfering RNAs (siRNAs), has been demonstrated in recent studies to be an effective strategy against human immunodeficiency virus type 1 (HIV-1). In the present study, we used 21-bp chemically synthesized siRNA duplexes whose sequences were derived from the gp41 gene,nef,tat, andrevregions of viral RNA. These sequences are conserved in select neurotropic strains of HIV-1 (JR-FL, JR-CSF, and YU-2). The designed siRNAs exerted a potent antiviral effect on these HIV-1 strains. The antiviral effect was mediated at the RNA level (as observed by the down-regulation of the HIV-1-specific spliced transcript generating a 1.2-kbp reverse transcription [RT]-PCR product) as well as viral assembly on the cell membrane. Spliced transcripts (apart from the most abundant transcript generating a 1.2-kbp RT-PCR product) arising from an unspliced precursor likely contributed, albeit to a lesser extent, to the antiviral effect. The resultant progeny viruses had infectivities similar to that of input virus. We therefore conclude that these siRNAs interfere with the processing of the unspliced transcripts for the gp41 gene,tat,rev, andnef, eventually affecting viral assembly and leading to the overall inhibition of viral production. Apart from using the gp41 gene as a target, the conservation of each of these targets in the above-mentioned viral strains, as well as several primary isolates, would enable these siRNAs to be used as potent antiviral tools for investigations with cells derived from the central nervous system in order to evaluate their therapeutic potential and assess their utility in inhibiting HIV-1 neuropathogenesis and neuroinvasion.
Intrahepatic Gene Expression during Chronic Hepatitis C Virus Infection in Chimpanzees
Authors: Bigger Catherine B. , Guerra Bernadette , Brasky Kathleen M. , Hubbard Gene , Beard Michael R. , Luxon Bruce A. , Lemon Stanley M. , Lanford Robert E.
Published: November 2004

Hepatitis C virus (HCV) infections represent a global health problem and are a major contributor to end-stage liver disease including cirrhosis and hepatocellular carcinoma. An improved understanding of the parameters involved in disease progression is needed to develop better therapies and diagnostic markers of disease manifestation. To better understand the dynamics of host gene expression resulting from persistent virus infection, DNA microarray analyses were conducted on livers from 10 chimpanzees persistently infected with HCV. A total of 162 genes were differentially regulated in chronically infected animals compared to uninfected controls. Many genes exhibited a remarkable consistency in changes in expression in the 10 chronically infected animals. A second method of analysis identified 971 genes altered in expression during chronic infection at a 99% confidence level. As with acute-resolving HCV infections, many interferon (IFN)-stimulated genes (ISGs) were transcriptionally elevated, suggesting an ongoing response to IFN and/or double-stranded RNA which is amplified in downstream ISG expression. Thus, persistent infection with HCV results in a complex and partially predictable pattern of gene expression, although the underlying mechanisms regulating the different pathways are not well defined. A single genotype 3-infected animal was available for analysis, and this animal exhibited reduced levels of ISG expression compared to levels of expression with genotype 1 infections and increased expression of a number of genes potentially involved in steatosis. Gene expression data in concert with other observations from HCV infections permit speculation on the regulation of specific aspects of HCV infection.