bluuebunny/crossref_metadata_2025 huggingface dataset:
Aspects of the productivity of forage legumes in Northern Europe
Authors: Halling M. A. , Topp C. F. E. , Doyle C. J.
Published: November 2004

A database on the productivity of red clover (Trifolium pratense), white clover (Trifolium repens), lucerne (Medicago sativa), lotus (Lotus corniculatus) and galega (Galega orientalis) was compiled. It contains 1852 observations for 330 trials at fifty‐three sites in eight north European countries for the period from 1977 to 1997. The database includes information on climatic and site characteristics, as well as agronomic data, collated into four smaller data sets. This has permitted four key agronomic issues to be examined, namely: (i) how yield for a given variety varies under different environmental conditions; (ii) how the relative performance of different species varies between sites; (iii) how persistence varies between species and sites; and (iv) what the nature of the relationship is between varieties, yield and sites within a species? Cluster analysis revealed that geographical location had a fairly strong influence on yield. Across sites for an individual variety, the cumulative day‐degrees during the regrowth period had the highest correlation with total yield. In terms of the relative performance of different forage legume species across sites, red clover and lucerne were estimated to yield about 2·5 t DM ha−1 more than white clover and there was a significant impact of cumulative day‐degrees during regrowth and the age of ley on the total yield. It was found that red clover had the lowest persistence index and lucerne the highest. As regards the interaction between yield, varieties and sites, the length of growth period and the age of the ley explained two‐thirds of the variation in total yield in red clover varieties of contrasting maturity types.
Effect of six‐weekly harvests on the yield, chemical composition and dry matter degradability of Panicum maximum and Stylosanthes hamata in Nigeria
Authors: Bamikole M. A. , Akinsoyinu A. O. , Ezenwa I. , Babayemi O. J. , Akinlade J. , Adewumi M. K.
Published: November 2004

The dry matter (DM) yield and degradability of 6‐week‐old harvests of tropical forages were measured over a season. The forages were nitrogen‐fertilized Guinea grass (Panicum maximum, NFG), unfertilized Guinea grass (UFG), Verano stylo (Stylosanthes hamata,VS), a Guinea grass–Verano stylo mixture (GSM) and Guinea grass in the grass–Verano stylo mixture (GGSM). Six‐week‐old forages were made possible through a cutting regime, which produced four harvests in the growing season. The DM yields of the forages differed significantly (P < 0·001) and showed a significant reduction (P < 0·01) across the season. Crude protein and neutral‐detergent fibre concentrations were significantly (P < 0·01) different between the forages but there was no difference between harvests. The DM degradability of the forages at all harvests were significantly (P < 0·001) different with differences in the soluble fraction (a), degradable fraction (b), potential degradability (PD) and effective degradability (ED), but rate of degradability (c) did not show any significant difference between the forages. Significant (P < 0·01) differences were found between harvests for b and PD, and for the interaction between forage and harvest for b, PD and ED but were not found for the a and c fractions. Both the PD and ED values of all the forages fell with advancing harvests. Although the 6‐week‐old harvests of forage were found not to influence the characteristic reduction in yield of tropical grasses over time, it is concluded that such a management system could be used to obtain forage of relatively high nutritive value during the growing season.
Milk protein loci polymorphism in taurine (Bos taurus) and zebu (Bos indicus) populations bred in hot climate
Authors: Ceriotti G. , Marletta D. , Caroli A. , Erhardt G.
Published: November 2004

The genetic variation at four milk protein loci αs1‐casein (CSN1S1), β‐casein (CSN2), κ‐casein (CSN3) and β‐lactoglobulin (LGB) was investigated in 358 animals belonging to seven populations well adapted to hot climatic conditions, reared in Africa and Italy. Bos taurus (Somba, Lagune and Modicana), Bos indicus (Sudanese Zebu Peul, Azaouak and Adamawa), and B. taurus × B. indicus (Borgou) were studied by adapting different molecular methodologies to evaluate their genetic variability. All loci were polymorphic and a new CSN2 synonymous variant, named A2′, was detected and characterized. Noteworthy differences in the distribution of alleles and haplotypes were observed between zebuine and taurine cattle showing that milk protein polymorphism is suited for discriminating B. taurus from B. indicus efficiently, and also taurine breeds. Milk protein loci, being positively selected loci, can also provide information about the occurrence of germplasm particularly useful for breeding strategies and production improvement.
Population parameters in Brazilian Thoroughbred
Authors: Taveira R. Z. , Mota M. D. S. , Oliveira H. N.
Published: November 2004

The information in this study has been provided by the Brazilian Association of Racehorse Breeders [Associação Brasileira dos Criadores do Cavalo de Corrida (ABCCC)]. It can be found in the files on the CD‐ROM developed by the ABCCC in 1999. A total of 5008 finishing time records related to 2545 winning horses that ran in the classical calendar on Brazilian hippodromes during 25 years (1974–98) were analysed. There were a total of 9949 horses on the relationship matrix. The variance components were estimated using the multiple‐trait derivate‐free restricted maximum likelihood (MTDFREML) program, for an animal model. Generation intervals were higher in the maternal side (10.91 years) than in the paternal one (10.41 years). The estimates for genetic, permanent environmental and phenotypic variances and heritability were 0.291, 0.161, 3.486 and 0.08, respectively. The phenotypic standard deviation for time in races was 1.86729 s. Genetic time trend on Thoroughbred races in Brazil was small and could be accelerated if selection considered the trait time effectively. With respect to the animal's country of birth, the results show that there has been an intense participation of foreign animals in breeding Brazilian Thoroughbreds.
Effect of insulin treatment on the body composition of Type 2 diabetic patients
Authors: Sallé A. , Guilloteau G. , Ryan M. , Bouhanick B , Ritz P.
Published: November 2004

Objective  Insulin is used commonly in Type 2 diabetes and is often accompanied by weight gain. The composition of this weight gain is poorly understood. Predominant increases in fat mass could increase cardiovascular risks. The aim of the study was to evaluate insulin‐induced body composition changes.

Research design and methods  Body weight and composition of 35 Type 2 diabetic patients during their first 6 months of insulin therapy was compared with those in 34 Type 2 diabetic individuals treated with insulin for at least 1 year prior to commencing the study. Body composition was determined by the simultaneous measurement of body water spaces and body density.

Results  Over 6 months, glycaemic control improved in the new treatment group only (HbA1c: 7.26 ± 0.81 vs. 9.66 ± 1.60%; P < 0.0001), remaining stable in the previously treated group (7.67 ± 1.25 vs. 7.76 ± 1.26%; P = NS). Weight significantly increased over time in the newly treated group (+1.7 kg; P = 0.04), but not in the previously treated group (−0.3 kg). It comprised of both fat (+0.85 kg) and fat‐free mass (+0.55 kg). Total body water remained unchanged. Using bioelectrical impedance analysis, the gain in fat mass was +2.2 kg; P = 0.048.

Conclusions  Over 6 months, insulin therapy leads to a weight gain of 1.7 kg because of an increase in both fat and fat‐free mass. When body composition is determined by bioelectrical impedance analysis, the results are biased by fluctuations in hydration.
Early parasympathetic neuropathy associated with elevated fasting plasma C‐peptide concentrations and late parasympathetic neuropathy with hyperglycaemia and other microvascular complications
Authors: Gottsäter A. , Kangro M. , Sundkvist G.
Published: November 2004

Aims  To examine the relationship between parasympathetic neuropathy, hyperinsulinaemia, glycaemic control (HbA1c), and future diabetic complications.

Methods  We assessed parasympathetic nerve function [expiration/inspiration (E/I) ratio], glomerular filtration rate (GFR), glycaemic control (HbA1c), fasting plasma (f‐p‐) C‐peptide in 82 Type 2 diabetic patients (age 61 ± 1 years) 5 and 12–15 years after diagnosis. Diabetic retinopathy was assessed 15 years after diagnosis.

Results  High HbA1c values in the first study were associated with retinopathy (with 8.6 ± 2.0 vs. without retinopathy 6.2 ± 1.9%; P < 0.0001) and disturbed parasympathetic nerve function (low E/I ratio; rs = −0.41; P = 0.0061) in the second study, as well as with deteriorations in GFR between the first and second study (rs = 0.62; P < 0.0001). Patients with parasympathetic neuropathy in the first study had significantly higher f‐p‐C‐peptide concentrations than those without 3 years (1.20 ± 0.43 vs. 0.86 ± 0.40 nmol/l; P = 0.0015) and 5 years (1.20 ± 0.44 vs. 0.82 ± 0.33 nmol/l; P < 0.0001), but not 15 years after diagnosis.

Conclusion  High HbA1c values 5 years after diagnosis of Type 2 diabetes were associated with retinopathy, disturbed parasympathetic nerve function, and deterioration in GFR 7–10 years later. Parasympathetic neuropathy 5 years after diagnosis was associated with high C‐peptide concentrations. Parasympathetic nerve function has to be considered when β‐cell function is evaluated. Hyperglycaemia is an important factor for the development of complications in Type 2 diabetes.
Prevalence of Diabetic Eye disease in Tayside, Scotland (P‐DETS) Study: methodology
Authors: Ellis J. D. , Leese G. , McAlpine R. , Cole A. , MacEwen C. J. , Baines P. S. , Crombie I. , Morris A. D. ,   for the DARTS/MEMO Collaboration
Published: November 2004

Aims  To describe the use of a validated diabetes register for sampling frame generation and assessment of the representative nature of participants in a fieldwork study of diabetic eye disease.

Methods  We performed an observational, cross‐sectional fieldwork study of diabetic retinal disease using reference standard eye examination. We sampled the entire diabetic population using the Diabetes Audit and Research in Tayside Study (DARTS) diabetes register.

Results  The study population comprised 4825 diabetic patients aged over 16 years registered with one of 166 general practitioners (GPs) in 41 practices in Tayside in October 1999. This represented 61.1% of the Tayside diabetic population (7903). A total of 586 (66%; 95% confidence interval 63, 70) patients were examined from a sampling frame of 882 living patients registered with a Tayside GP. Demographic and disease parameters recorded on the DARTS patient register allowed comparison between participants and non‐participants.

Conclusions  This study shows the clear benefit of using a complete diabetic population as a sampling frame. This allows potential selection bias and external validity to be evaluated using routine data sources. Studies performed and reported in this way will aid the critical appraisal process.
Association of simple anthropometric measures of obesity with visceral fat and the metabolic syndrome in male Caucasian and Indo‐Asian subjects
Authors: Valsamakis G. , Chetty R. , Anwar A. , Banerjee A. K. , Barnett A. , Kumar S.
Published: November 2004

Aims  The aims of this study were first, to investigate the relationship between simple anthropometric measures of obesity with visceral fat as assessed by a single slice magnetic resonance imaging (MRI)‐scan in patients attending a hospital clinic. Second, to determine which anthropometric measure best relates to the adverse metabolic profile of the metabolic syndrome.

Methods  Forty‐one male subjects [body mass index (BMI): 30.2 + 5.8 kg/m2, age: 50.3 + 13.6 years] were studied by MRI‐scan to measure visceral fat at L4/L5 level and to investigate its relationship with simple anthropometric measures. Second, we studied 83 male subjects to determine which anthropometric measure best predicts the metabolic complications (using the ATPIII criteria) of obesity in the setting of a hospital clinic.

Results  Waist circumference was the best anthropometric measurement that correlated with MRI‐visceral fat mass assessed at L4/L5 in 41 subjects who had an MRI scan (P = 0.0001, r2 = 0.36, beta = 0.56) amongst variables which also included age, BMI, sagittal diameter, diabetes and ethnicity. Stepwise multiple regression analysis showed sagittal diameter (P = 0.001, r2 = 0.4, beta = 0.406), age (P = 0.003, beta = 0.271) and waist circumference (P = 0.012, beta = 0.297) were the best predictors of the adverse metabolic profile of the metabolic syndrome in all 83 male subjects amongst BMI, waist–hip ratio (WHR), ethnicity and diabetes‐related factors.

Conclusions  Waist circumference is a simple anthropometric parameter that best correlates with single slice MRI‐scan, but sagittal diameter (measured using abdominal calipers) better predicts the adverse metabolic profile of the metabolic syndrome. Although there is considerable variation in abdominal fat topography between ethnic groups, and also within populations, sagittal diameter assessment is a technique that is simple and best predicts the metabolic syndrome.
The effect of erroneous responses on response monitoring in patients with major depressive disorder: A study with event‐related potentials
Authors: Ruchsow Martin , Herrnberger Bärbel , Wiesend Christiane , Grön Georg , Spitzer Manfred , Kiefer Markus
Published: November 2004

Perceived failure is reported to have detrimental effects on subsequent performance in patients with major depressive disorder. We investigated the error‐related negativity (ERN)/error negativity (Ne), an electrophysiological correlate of response monitoring, using a 64‐channel EEG. Sixteen patients with DSM‐IV major depressive disorder and 16 matched controls participated in an Eriksen flanker task with continuous performance feedback that signaled monetary reward. Compared to controls, patients with major depressive disorder showed a less negative ERN/Ne in error trials following error trials. This result might reflect impaired response monitoring processes in major depressive disorder resulting from an underactivity in a central reward pathway and/or a deficit in strategic reasoning.
Ischemia–reperfusion injury pathophysiology, part I
Authors: McMichael Maureen , Moore Rustin M.
Published: November 2004

Objective:To review the current scientific literature on ischemia–reperfusion (IR) injury in both human and veterinary medicine. To describe the normal antioxidant defense mechanisms, the pathophysiology of IR injury, and the role of neutrophils in IR injury.

Data sources:Data sources include scientific reviews and original research publications in both human and veterinary medicine.

Summary:IR injury is a complex pathophysiological process involving numerous pathways and body systems. Normal antioxidant defense mechanisms function to limit oxidative injury during times of health. Ischemia is the period that occurs before oxygenated blood is re‐introduced and the severity of injury has been shown to correlate with the magnitude and length of ischemia in dogs. During ischemia, there is a buildup of substances (i.e., xanthine oxidase, hypoxanthine, etc.) that, upon re‐introduction of oxygen, form reactive oxygen species (ROS). ROS, produced in large part upon reperfusion, can cause extensive damage to DNA, proteins, carbohydrates, and lipids. Although mammalian systems are endowed with abundant antioxidant defenses, the generation of large amounts of ROS can overwhelm these mechanisms leading to cell dysfunction and death. Neutrophils play a critical role in IR injury and may mediate the majority of mucosal and microvascular injury that occurs by releasing ROS and proteolytic enzymes. Although experimental studies have been carried out on cats, dogs, and horses there are few clinical studies on companion animals.

Conclusions:The pathophysiology of IR injury is complex and involves damage by ROS to all biological membranes. Neutrophils play a major role in IR injury and initiate and propogate much of the damage. This article is intended as a review of the pathophysiology of IR injury.
Ischemia–reperfusion injury: assessment and treatment, part II
Authors: McMichael Maureen
Published: November 2004

Objective:To review the current scientific literature on ischemia–reperfusion (IR) injury in both human and veterinary medicine and to describe the assessment of IR injury, the available testing methods, and the options available for treatment.

Data sources:Data sources include scientific reviews and original research publications in both human and veterinary medicine.

Summary:The assessment of IR injury includes measuring products formed by the reaction of reactive oxygen species (ROS) with biological membranes, measuring levels of endogenous antioxidants, and measuring ROS themselves. Testing depends on the laboratory used, the test method chosen, the sample submitted (i.e., plasma, urine, tissue, etc.), and the timing of the test in relation to sample collection. For this reason, testing is not standardized and pharmacological data on antioxidant effectiveness are not available. Antioxidants and drugs tested have included single agents as well as ‘cocktails’ consisting of several agents working at different key points in the injury cascade.

Conclusions:There are several new testing methods as well as new strategies for attempting to ameliorate the damage inflicted upon reperfusion and this article is intended as a review of the assessment and treatment of IR injury.
Suspected toxicosis after topical administration of minoxidil in 2 cats
Authors: DeClementi Camille , Bailey Keith L. , Goldstein Spencer C. , Orser Michael Scott
Published: November 2004

Objective: To discuss the clinical presentation and necropsy findings of 2 cats after topical administration of a minoxidil solution. Additionally, a potential management plan is offered for future cases.

Case summary: Two cats with dermal exposure to topical minoxidil solution were identified from the ASPCA Animal Poison Control Center (APCC) files. Both cats were presented with lethargy and dyspnea within 36 hours of exposure. The cats were hypothermic, and had pulmonary edema and pleural effusion present on thoracic radiography. Both cats died despite supportive care. Necropsy of both cats confirmed pleural effusion and pulmonary edema and indicated cardiac compromise.

New or unique information provided: Topical administration of minoxidil solution may cause life‐threatening pulmonary edema, pleural effusion, and cardiac dysfunction in the cat.
Investigation of Turbulence Models Applied to Premixed Combustion Using a Level-Set Flamelet Library Approach
Authors: Engdar Ulf , Nilsson Per , Klingmann Jens
Published: November 2004

Most of the common modeling approaches to premixed combustion in engineering applications are either based on the assumption of infinitely fast chemistry or the flamelet assumption with simple chemistry. The level-set flamelet library approach (FLA) has shown great potential in predicting major species and heat release, as well as intermediate and minor species, where more simple models often fail. In this approach, the mean flame surface is tracked by a level-set equation. The flamelet libraries are generated by an external code, which employs a detailed chemical mechanism. However, a model for the turbulent flame speed is required, which, among other considerations, depends on the turbulence intensity, i.e., these models may show sensitivity to turbulence modeling. In this paper, the FLA model was implemented in the commercial CFD program Star-Cd, and applied to a lean premixed flame stabilized by a triangular prism (bluff body). The objective of this paper has been to investigate the impact on the mean flame position, and hence on the temperature and species distribution, using three different turbulent flame speed models in combination with four different turbulence models. The turbulence models investigated are: the standard k-ε model, a cubic nonlinear k-ε model, the standard k-ω model and the shear stress transport (SST) k-ω model. In general, the computed results agree well with experimental data for all computed cases, although the turbulence intensity is strongly underestimated at the downstream position. The use of the nonlinear k-ε model offers no advantage over the standard model, regardless of flame speed model. The k-ω based turbulence models predict the highest turbulence intensity with the shortest flame lengths as a consequence. The Mu¨ller flame speed model shows the least sensitivity to the choice of turbulence model.
Experimental and Computational Study of Hybrid Diffusers for Gas Turbine Combustors
Authors: Walker A. Duncan , Denman Paul A. , McGuirk James J.
Published: November 2004

The increasing radial depth of modern combustors poses a particularly difficult aerodynamic challenge for the pre-diffuser. Conventional diffuser systems have a finite limit to the diffusion that can be achieved in a given length and it is, therefore, necessary for designers to consider more radical and unconventional diffuser configurations. This paper will report on one such unconventional diffuser; the hybrid diffuser which, under the action of bleed, has been shown to achieve high rates of diffusion in relatively short lengths. However, previous studies have not been conducted under representative conditions and have failed to provide a complete description of the relevant flow mechanisms making optimization difficult. Utilizing an isothermal representation of a modern gas turbine combustor an experimental investigation was undertaken to study the performance of a hybrid diffuser compared to that of a conventional, single-passage, dump diffuser system. The hybrid diffuser achieved a 53% increase in area ratio within the same axial length generating a 13% increase in the pre-diffuser static pressure recovery coefficient which, in turn, produced a 25% reduction in the combustor feed annulus total pressure loss coefficient. A computational investigation was also undertaken in order to investigate the governing flow mechanisms. A detailed examination of the flow field, including an analysis of the terms within the momentum equation, demonstrated that the controlling flow mechanisms were not simply a boundary layer bleed but involve a more complex interaction between the accelerating bleed flow and the diffusing mainstream flow. A greater understanding of these mechanisms enabled a more practical design of hybrid diffuser to be developed that not only simplified the geometry but also improved the quality of the bleed air making it more attractive for use in component cooling.
A Study of the Nonlinear Interaction Between an Eccentric Squeeze Film Damper and an Unbalanced Flexible Rotor
Authors: Bonello Philip , Brennan Michael J. , Holmes Roy
Published: November 2004

In this paper, the nonlinear interaction between an eccentric squeeze film damper and an unbalanced flexible rotor is investigated, paying particular attention to the effect of cavitation in the damper. A harmonic balance method that uses the receptance functions of the rotating linear part of the system to determine periodic solutions to the nonlinear problem is used to predict vibration levels in a test rig. By comparing predictions obtained respectively with, and without, lower pressure limits for the squeeze film damper model, it is concluded that cavitation is promoted by increasing static eccentricity and/or unbalance level. This, in turn, is found to have a profound effect on the predictions for the critical vibration levels, which such dampers are designed to attenuate. Experimental results are presented to support the findings.
In Vitro Evaluation of the Effectiveness of the Macrolide Rokitamycin and Chlorpromazine against
Acanthamoeba castellanii
Authors: Mattana A. , Biancu G. , Alberti L. , Accardo A. , Delogu G. , Fiori P. L. , Cappuccinelli P.
Published: November 2004

The present study demonstrates the in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against
Acanthamoeba castellanii
. Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when they were used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal as well as cysticidal. Experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of
A. castellanii
against WKD cells derived from the human cornea. These results may have important significance in the development of new anti-
Acanthamoeba
compounds.
Gln145Met/Leu Changes in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Resistance to Nucleoside and Nonnucleoside Analogs and Impair Virus Replication
Authors: Paolucci Stefania , Baldanti Fausto , Maga Giovanni , Cancio Reynel , Zazzi Maurizio , Zavattoni Maurizio , Chiesa Antonella , Spadari Silvio , Gerna Giuseppe
Published: November 2004

The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.
Functional Cloning of
Bacillus anthracis
Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim
Authors: Barrow Esther W. , Bourne Philip C. , Barrow William W.
Published: November 2004

Bacillus anthracis
is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for
B. anthracis
Sterne is >2,048 but ≤4,096 μg/ml. A putative DHFR sequence was amplified from
B. anthracis
Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into
Escherichia coli
TOP10F′ chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into
E. coli
BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with
E. coli
BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of
B. anthracis
. These results confirm the natural resistance of
B. anthracis
to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.
Molecular Characterization of a β-Lactamase Gene,
bla
GIM-1
, Encoding a New Subclass of Metallo-β-Lactamase
Authors: Castanheira Mariana , Toleman Mark A. , Jones Ronald N. , Schmidt Franz J. , Walsh Timothy R.
Published: November 2004

As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant
Pseudomonas aeruginosa
clinical isolates were detected with metallo-β-lactamase (MβL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the
aacA4
gene cassette. Sequencing revealed a novel MβL gene, designated
bla
GIM-1
. Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MβL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-β-lactamase inhibitors.
bla
GIM-1
was found on a 22-kb nontransferable plasmid. The new MβL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an
aacA4
cassette downstream of the MβL gene that appeared to be truncated with
bla
GIM-1
. The
aacA4
was followed by an
aadA1
gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene,
bla
OXA-2
, before the 3′-CS region. GIM-1 appears to be a unique MβL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MβL enzymes to be characterized.
Antibacterial Activity of a Competence-Stimulating Peptide in Experimental Sepsis Caused by
Streptococcus pneumoniae
Authors: Oggioni Marco R. , Iannelli Francesco , Ricci Susanna , Chiavolini Damiana , Parigi Riccardo , Trappetti Claudia , Claverys Jean-Pierre , Pozzi Gianni
Published: November 2004

Streptococcus pneumoniae
, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood
S. pneumoniae
counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target.
Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells
Authors: Lin Kai , Kwong Ann D. , Lin Chao
Published: November 2004

The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.
Outbreak of
Klebsiella pneumoniae
Producing a New Carbapenem-Hydrolyzing Class A β-Lactamase, KPC-3, in a New York Medical Center
Authors: Woodford Neil , Tierno Philip M. , Young Katherine , Tysall Luke , Palepou Marie-France I. , Ward Elaina , Painter Ronald E. , Suber Deborah F. , Shungu Daniel , Silver Lynn L. , Inglima Kenneth , Kornblum John , Livermore David M.
Published: November 2004

From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant
Klebsiella pneumoniae
. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a
bla
KPC
allele encoding a novel variant, KPC-3, with a His(272)→Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing β-lactamase variant to have been reported in members of the
Enterobacteriaceae
, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.
Two Different Molecular Defects in the Tva Receptor Gene Explain the Resistance of TwotvarLines of Chickens to Infection by Subgroup A Avian Sarcoma and Leukosis Viruses
Authors: Elleder Daniel , Melder Deborah C. , Trejbalova Katerina , Svoboda Jan , Federspiel Mark J.
Published: November 2004

The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. Thetvagenetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility alleletvasencodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chickentvascDNAs had not yet been fully characterized; we cloned the chickentvacDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chickentvascDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line Ctvarcontains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaRfor the ASLV(A) envelope glycoproteins. Line 72tvar2contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.
Nucleolar Localization of Human Hepatitis B Virus Capsid Protein
Authors: Ning Bo , Shih Chiaho
Published: November 2004

Wild-type human hepatitis B virus (HBV) exhibits selective export of virions containing mature genomes. In contrast, changing an isoleucine to a leucine at amino acid 97 (I97L) of the HBV core antigen (HBcAg) causes it to release immature genomes. To elucidate the structure-function relationship of HBcAg at amino acid 97, we systematically replaced the isoleucine residue at this position with 18 other amino acids via mutagenesis. Twelve of the 18 mutants exhibited no significant phenotype, while five new mutants displayed strong phenotypes. The I97D mutant had a near lethal phenotype, the I97P mutant exhibited a significantly reduced level of virion secretion, and the I97G mutant lacked the full-length relaxed circular form of viral DNA. The tip of the spike of the capsid particle is known to contain a predominant B-cell epitope. However, the recognition of this exposed epitope by an anti-HBc antibody appeared to be affected by the I97E mutation or by histidine tagging at the C terminus of mutant HBcAg, which is presumably in the capsid interior. Surprisingly, the nuclear HBcAg of mutants I97E and I97W, produced from either a replicon or an expression vector, was found to be colocalized with nucleolin and B23 at a frequency of nearly 100% by confocal immunofluorescence microscopy. In contrast, this colocalization occurred with wild-type HBcAg only to a limited extent. We also noted that nucleolin-colocalizing cells were often binucleated or apoptotic, suggesting that the presence of HBcAg in the nucleolus may perturb cytokinesis. The mechanism of this phenomenon and its potential involvement in liver pathogenesis are discussed. To our knowledge, this is the first report of nucleolar HBcAg in culture.
Intracellular Viral Processing, Not Single-Stranded DNA Accumulation, Is Crucial for Recombinant Adeno-Associated Virus Transduction
Authors: Hauck Bernd , Zhao Wei , High Katherine , Xiao Weidong
Published: November 2004

Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.
Determinants of High Titer in Recombinant Porcine Endogenous Retroviruses
Authors: Harrison Ian , Takeuchi Yasuhiro , Bartosch Birke , Stoye Jonathan P.
Published: November 2004

Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of
pol
and the middle of the SU region in
env
. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the
env
gene. Using a series of chimeric
env
genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of
env
gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.
Comparison of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses in HIV-1- and HIV-2-Infected Individuals in Senegal
Authors: Zheng N. N. , Kiviat N. B. , Sow P. S. , Hawes S. E. , Wilson A. , Diallo-Agne H. , Critchlow C. W. , Gottlieb G. S. , Musey L. , McElrath M. J.
Published: November 2004

Human immunodeficiency virus type 2 (HIV-2) infection is typically less virulent than HIV-1 infection, which may permit the host to mount more effective, sustained T-cell immunity. We investigated antiviral gamma interferon-secreting T-cell responses by an ex vivo Elispot assay in 68 HIV-1- and 55 HIV-2-infected Senegalese patients to determine if differences relate to more efficient HIV-2 control. Homologous HIV-specific T cells were detected in similar frequencies (79% versus 76%,
P
= 0.7) and magnitude (3.12 versus 3.08 log
10
spot-forming cells/10
6
peripheral blood mononuclear cells) in HIV-1 and HIV-2 infection, respectively. Gag-specific responses predominated in both groups (≥64%), and significantly higher Nef-specific responses occurred in HIV-1-infected (54%) than HIV-2-infected patients (22%) (
P
< 0.001). Heterologous responses were more frequent in HIV-1 than in HIV-2 infection (46% versus 27%,
P
= 0.04), but the mean magnitude was similar. Total frequencies of HIV-specific responses in both groups did not correlate with plasma viral load and CD4
+
T-cell count in multivariate regression analyses. However, the magnitude of HIV-2 Gag-specific responses was significantly associated with lower plasma viremia in HIV-1-infected patients (
P
= 0.04). CD4
+
T-helper responses, primarily recognizing HIV-2 Gag, were detected in 48% of HIV-2-infected compared to only 8% of HIV-1-infected patients. These findings indicate that improved control of HIV-2 infection may relate to the contribution of T-helper cell responses. By contrast, the superior control of HIV-1 replication associated with HIV-2 Gag responses suggests that these may represent cross-reactive, higher-avidity T cells targeting epitopes within Gag regions of functional importance in HIV replication.
A DNAβ Associated with
Tomato Yellow Leaf Curl China Virus
Is Required for Symptom Induction
Authors: Cui Xiaofeng , Tao Xiaorong , Xie Yan , Fauquet Claude M. , Zhou Xueping
Published: November 2004

We report here that all 25 isolates of
Tomato yellow leaf curl China virus
(TYLCCNV) collected from tobacco, tomato, or
Siegesbeckia orientalis
plants in different regions of Yunnan Province, China, were associated with DNAβ molecules. To investigate the biological role of DNAβ, full-length infectious clones of viral DNA and DNAβ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into
Nicotiana benthamiana
,
Nicotiana glutinosa
,
Nicotiana. tabacum
Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAβ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAβ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the
βC1
gene of TYLCCNV-Y10 DNAβ was mutated, the mutants failed to induce leaf curl symptoms in
N. benthamiana
when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAβ molecules were replicated. When
N. benthamiana
and
N. tabacum
plants were transformed with a construct containing the
βC1
gene under the control of the
Cauliflower mosaic virus
35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of
βC1
transcripts, while transgenic plants transformed with the
βC1
gene containing a stop codon after the start codon remained symptomless. Thus, expression of a
βC1
gene is adequate for induction of symptoms of viral infection in the absence of virus.
Attenuating Mutations in Coxsackievirus B3 Map to a Conformational Epitope That Comprises the Puff Region of VP2 and the Knob of VP3
Authors: Stadnick E. , Dan M. , Sadeghi A. , Chantler J. K.
Published: November 2004

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5′ nontranslated region (5′NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5′NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5′NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.