bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino‐acid‐sequence Determination and Biological Activity of Cytin, a Naturally Occurring Specific Chymotrypsin Inhibitor from the Leech Theromyzon tessulatum
Authors: Chopin Vincent , Bilfinger Thomas V. , Stefano George B. , Matias Isabel , Salzet Michel
Published: June 2004

We purified a chymotrypsin inhibitor, designated cytin, from the rhynchobdellid leech Theromyzon tessulatum. 0 This 7.4‐kDa peptide was purified to apparent homogeneity by gel‐permeation and anion‐exchange chromatographies, followed by reverse‐phase HPLC. The structure of cytin was determined by reduction, S‐β‐pyridilethylation, automated Edman degradation, and electrospray mass spectrometry. Cytin is formed by the association of two protein chains, which are linked by a disulfide bridge. Chain A consists of 43 and chain B of 22 amino acid residues. Chain B exhibits 40–63% sequence similarity with the N‐terminal sequences of subtilisin/chymotrypsin inhibitors isolated from barley seeds. Cytin inhibited chymotrypsin (Ki 600 pM) and weakly inhibited trypsin (Ki 350 nM). This chymotrypsin inhibitor, in contrast to others isolated from leeches, does not inhibit elastase or cathepsin G. Furthermore, cytin (10 μM) significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharide (1 U/ml) in a manner similar to that of aprotinin. These data indicate that this chymotrypsin inhibitor may be biomedically significant.
Chemical Characterization of the Predominant Proteins Secreted by Mouse Seminal Vesicles
Authors: Lundwall åke , Peter Anders , Lövgren Janita , Lilja Hans , Malm Johan
Published: June 2004

Mouse seminal vesicles secrete four major protein components with estimated molecular masses of 95, 38, 17, and 16 kDa. Amino acid sequencing revealed that the 95‐kDa component represents a protein with an unknown structure, while the 38‐kDa component was identified as semenoclotin, the 17‐kDa component as seminal‐vesicle‐secreted protein IV, and the 16‐kDa component as seminal‐vesicle‐secreted protein V. Semenoclotin and the 95‐kDa component were readily cross‐linked by transglutaminase, suggesting that the two proteins are involved in the formation of the mouse copulatory plug. Treatment of mouse seminal vesicle fluid with human prostate‐specific antigen rapidly degraded semenoclotin, indicating a structural resemblance of this protein to human semenogelins, despite the vast difference in primary structure. As previously reported for other seminal‐vesicle‐secreted proteins, the semenoclotin transcripts are shown to be under androgen control.
Phorbol‐Ester‐Activated Protein Kinase C‐α Lacking Phosphorylation at Ser657 is Down‐Regulated by a Mechanism Involving Dephosphorylation
Authors: Gysin Stephan , Imber Roland
Published: June 2004

Protein kinase C (PKC) is a key enzyme in the intracellular signaling network. Upon activation by 12‐O‐tetradecanoylphorbol 13‐acetate, the a‐isoform of PKC translocates to the detergent‐soluble and the detergent‐insoluble fractions. Besides cofactors, the activity and stability of this protein is critically regulated by multisite phosphorylations. At least three distinct sites, Thr497, Thr638 and Ser657, are involved. We have previously shown that the replacement of Ser657 by alanine leads to a premature down‐regulation in the detergent soluble compartment of LLC‐PK1 cells [Gysin, S. & Imber, R. (1996) Eur. J. Biochem. 240, 747–750]. More detailed analysis revealed that, in contrast to the wild‐type molecule, the down‐regulation of the mutant protein is in vivo preceded by a rapid dephosphorylation after phorbolester‐induced translocation to both the detergent‐soluble and insoluble compartments. The [Ala657]PKC‐α mutant protein molecule showed in vitro a strongly increased sensitivity towards protein phosphatase 2A whereas its overall proteolytic sensitivity remained unchanged when compared to wild type. The in vitro studies led to the suggestion that further dephosphorylation of the mutant protein is a prerequisite in order to become proteolytically down‐regulated. Therefore phosphorylation of Ser657 controls the duration of activation of this PKC isozyme upon agonist‐induced translocation by preventing premature proteolytic down‐regulation via protecting the protein from dephosphorylation.
In vitro Conversion of the Carbohydrate Moiety of Fungal Glycoproteins to Mammalian‐type Oligosaccharides
Authors: Maras Marleen , Saelens Xavier , Laroy Wouter , Piens Kathleen , Claeyssens Marc , Fiers Walter , Contreras Roland
Published: June 2004

To investigate the potential of filamentous fungi to synthesize N‐glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N ‐acetylglucosaminyltransferase I, human β‐1,4‐galactosyltransferase and rat α‐2,6‐sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High‐mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian‐type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoiα‐1,2‐mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N‐glycan processing in T. reesei apparently involves different steps, including α‐1,2‐mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The α‐1,2‐mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.
Structural and Functional Properties of the 154–171 Wild‐type and Variant Peptides of Human Lecithin‐cholesterol Acyltransferase
Authors: Peelman Frank , Goethals Marc , Vanloo Berlinda , Labeur Christine , Brasseur Robert , Vandekerckhove Joel , Rosseneu Maryvonne
Published: June 2004

The 154–171 segment of the human lecithin‐cholesterol acyltransferase (LCAT) enzyme was identified as the most stable amphipathic helix in the LCAT sequence. Its mean hydrophobicity, hydrophobic moment and its orientation at a lipid/water interface are similar to those of some of the helical repeats of apolipoprotein A‐IV and E. This domain was therefore proposed as a candidate peptide accounting for the association between LCAT and its lipid substrate. To investigate this hypothesis we synthesized the LCAT‐(154–171)‐peptide, two variants containing the natural Y156N and R158C mutations and a variant with increased hydrophobicity through Y156I, L160I, L163I and Y171W substitutions. The structural and lipid‐binding properties of these synthetic peptides were investigated by turbidity, fluorescence, electron microscopy and circular dichroism.

The wild‐type peptide, the R158C variant in its dimeric form, as well as the more hydrophobic peptide, associated with phospholipids, whereas the Y156N and the R158C variant in its monomeric formdid not. However, only the complexes generated with the hydrophobic variant were stable enough to resist dissociation during gel filtration. The wild‐type peptide and hydrophobic variant formed discoidal complexes with dimyristoylglycerophosphocholine (Myr2GraPCho) as shown by negative staining electron microscopy. Comparison of the properties of the wild‐type and hydrophobic variant LCAT‐(154–171)‐peptide stresses the contribution of the hydrophobic face of the amphipathic helix to the formation and stabilization of the peptide/lipid complexes. This is further confirmed by the decreased affinity of the Y156N variant peptide for lipids, as this mutation decreased the mean hydrophobicity of the hydrophobic face of the amphipathic helix. These results support the hypothesis that the 154–171 segment of LCAT might be involved in the interaction of the enzyme with its lipid substrate and suggest that the decreased activity of the Y156N natural LCAT mutant might result from a decreased affinity of this mutant for lipids.
Characterization, Gene Cloning and Expression of Isocitrate Lyase Involved in the Assimilation of One‐Carbon Compounds in Hyphomicrobium methylovorum GM2
Authors: Tanaka Yasuhiro , Yoshida Toyokazu , Watanabe Katsumi , Izumi Yoshikazu , Mitsunaga Toshio
Published: June 2004

Isocitrate lyase of an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2, was purified to homogeneity and characterized. The enzyme is a homotetramer of identical 62‐kDa subunits. After the enzyme had been incubated at temperatures up to 25°C for 30 min, no loss of activity was observed. The enzyme was stable in the pH range of 7.5–9.0. Maximum activity was observed at pH 7.5 and around 45°C. The Kin value for DK‐isocitrate was 0.51 mM. The activity required Mg2+ and was inhibited by oxalate, succinate and glycolate. The gene encoding the isocitrate lyase and its flanking regions were isolated from H. methylovorum GM2. Nucleotide sequencing of recombinant plasmids revealed that the isocitrate lyase gene codes for a 540‐amino‐acid protein. The amino acid sequence of the enzyme is similar to those of the enzymes from Escherichia coll (40% identity) and cucumber (37% identity). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector, pKK223–3, was introduced into E. coli HB101. The transformed E. coli cells expressed isocitrate lyase, which was indistinguishable from the purified H. methylovorum GM2 isocitrate lyase on analysis by SDS/PAGE.
Effects of Dexamethasone Application in Growing Pigs on Hormones, N‐retention and Other Metabolic Parameters
Authors: Lopes S. O. , Claus R. , Lacorn M. , Wagner A. , Mosenthin R.
Published: June 2004

Many single effects of glucocorticoids are known but complex metabolic reactions in pigs in response to a glucocorticoid challenge were not reported. Seven pigs (mean weight 69 kg) with indwelling catheters were kept in metabolic crates. After a 7‐day control period they were fed for 9 days with 0.4 mg dexamethasone (dex) per kg body weight daily, followed by another 9‐day post‐treatment period. Hormones and metabolic parameters were continuously determined in urine or blood plasma. Treatment significantly changed all parameters except non‐esterified fatty acids. Cortisol decreased from 84.5 to 4.9 nmol/l, insulin‐like growth factor I (IGF‐I) from 17.5 to 10.8 nmol/l and aldosterone from 0.36 to 0.13 nmol/l. The N‐retention decreased from 1.07 to 0.53 g/kg0.75 and hydroxyproline from 2.97 to 1.05 mmol/day. An increase was found for urine volume (5.2 versus 13.6 l/day), urea‐N (0.90 versus 1.43 g/kg0.75), allantoin (6.40 versus 8.75 mmol/day), glucose (3.9 versus 4.34 mmol/l) and insulin (6.21 versus 11.16 mU/l). In the post‐treatment period IGF‐I revealed a compensatory pattern (control period versus post‐treatment period: 17.5 versus 22.9 nmol/l) whereas the other parameters were not significantly elevated. Data suggest that dex increased N‐excretion both by inhibiting mitosis and resynthesis of proteins partly via a reduced collagen synthesis. Increased allantoin concentrations additionally pointed to increased apoptosis.
Comparison of the Navicular Region of Newborn Foals and Adult Horses by Magnetic Resonance Imaging
Authors: Hevesi A. , Stanek Ch. , Garamvolgyi R. , Petrasi Zs. , Bogner P. , Repa I.
Published: June 2004

Magnetic resonance imaging (MRI) was tested for evaluation of the soft tissue structures of the equine digit in 16 limbs, derived from three adult warmblood horses and two newborn warmblood foals. The following measuring sequences were used in sagittal, transversal and coronal planes: spin echo, gradient echo, inversion recovery. The images were made with a 1.5 Tesla Siemens scanner in a CP‐Helmholtz circular coil. To compare the visualization of the same tissue structures in adult and in newborn cases the limbs were imaged with the routinly used MRI sequences that are used for mature tissues. In newborn foals the bursa podotrochlearis could not be determined with the used sequences. For both the adult and newborn limbs the most visualization of the same tissue structures including the fluid spaces and growth plates were taken by the inversion recovery sequence. T2 sequence was very informative in adult cases but moderately in newborn foals.
Constructing a Quality‐Adjusted Price Index for a Heterogeneous Oligopoly
Authors: Prentice David , Yin Xiangkang
Published: June 2004

Both the theory and practice of using hedonic regressions to remove quality effects in price indexes are implicitly developed for monopolistic competitive markets. In this paper, we theoretically and practically analyse the application of a standard hedonic regression for an oligopoly. In the theoretical work, we recast how for an oligopoly the standard hedonic regression may be unstable. Then in the empirical work, we recommend using the weighted imputation method for constructing an index and estimating separate hedonic regressions for market segments. We apply these recommendations to estimating a quality‐adjusted price index for the Australian passenger vehicle market and find they make a substantial difference.
Tradeable Emissions Permits, Emissions Taxes and Growth
Authors: Crettez Bertrand
Published: June 2004

This paper uses a dynamic general equilibrium model with overlapping generations in order to analyse and to compare emissions taxes and tradeable emissions permits. Even in the context of a perfect environment, i.e. with perfect information, perfect competition…, it is shown that privately owned emissions permits have some disadvantages. An equilibrium with emissions permits would certainly be better than a laissez‐faire equilibrium since it would entail a lower pollution level. However, it is far from clear that an economy with pollution permits would be preferable over an economy with emissions taxes. While in both cases pollution would be lower, growth would be higher in an economy with emissions taxes. This is because emissions permits divert saving from ‘productive’ resources and have a negative impact on capital accumulation. This happens whatever the way emissions taxes are redistributed.
Quantile Regression Estimates of the Union Wage Effect for Great Britain
Authors: O'Leary N. C. , Murphy P. D. , Blackaby D. H.
Published: June 2004

This paper assesses the impact that trade unions have on the wage rates paid to workers in Great Britain using data from the Labour Force Survey. By employing a quantile regression model, this analysis is conducted over the entire range of the earnings distribution, where it is found that unions have more scope for increasing the earnings of workers on the very lowest of wage rates. Asymmetries in the effects of union membership and union coverage are also found to exist for employees across all earnings levels.
Wage Bargaining and Employer Objectives
Authors: Falch Torberg
Published: June 2004

This paper compares union wage bargaining outcomes across different types of employers. Five different employer objectives are discussed: profit, welfare and output maximization, and two specifications of a Leviathan. The model shows that the ordering of the union wage level across employer types depends on the functional form of product demand. With constant elasticity of product demand wage tends to be lowest in the output maximization case, while with a linear product demand wage tends to be lowest under welfare maximization.
The impact of organisational change on the nature and extent of training in Australian enterprises
Authors: Smith Andrew , Oczkowski Edward , Noble Charles , Macklin Robert
Published: June 2004

This article reports on a study investigating the relationship between the introduction of new management practices and the training provided by Australian enterprises for their employees. The new management practices investigated include teamworking, total quality management, lean production, business process re‐engineering and the learning organisation. The study involved a survey of human resource managers in medium to large size Australian enterprises. The study models the impact of the introduction of new management practices and the extent of training provision in the enterprises and on the form and nature of the training. The article concludes that whereas new management practices have a significant impact on training provision in enterprises, the effect is highly differentiated and that a critical relationship is the integration of training with the business strategy of the enterprise.
Book reviews
Authors: Stewart Jim
Published: June 2004

Assessing the Value of Your Training: The Evaluation Process from Training Needs to the Report to the Board  Leslie Rae, 2002. Published by Gower Publishing Ltd, Aldershot, Hants, UK. ISBN 0 566 68535 6. £49.50, 240 pages.
Pulmonary metastatectomy in the management of four dogs with hypertrophic osteopathy
Authors: Liptak J. M. , Monnet E. , Dernell W. S. , Withrow S. J.
Published: June 2004

The efficacy and outcome of pulmonary metastatectomy in the management of hypertrophic osteopathy (HO) secondary to metastatic osteosarcoma was retrospectively evaluated in four dogs. Metastatectomy was performed by subpleural enucleation, partial lung lobectomy or complete lung lobectomy through either a median sternotomy or thoracoscopically. Perioperative morbidity was minimal. Clinical signs associated with HO resolved within 24 h of pulmonary metastatectomy in all dogs. Durable remission of symptomatic HO was achieved in all dogs (range, 50–294 days), although recurrence of HO was noted in one dog, 246 days postmetastatectomy due to metastasis to the lungs and chest wall. Pulmonary metastatectomy resulted in a rapid and prolonged resolution of HO, and the clinical benefits of metastatectomy potentially exceed the morbidity associated with the surgical procedure.
Saint-Venant Decay Rates for the Rectangular Cross Section Rod
Authors: Stephen N. G. , Wang P. J.
Published: June 2004

A finite element-transfer matrix procedure developed for determination of Saint-Venant decay rates of self-equilibrated loading at one end of a semi-infinite prismatic elastic rod of general cross section, which are the eigenvalues of a single repeating cell transfer matrix, is applied to the case of a rectangular cross section. First, a characteristic length of the rod is modelled within a finite element code; a superelement stiffness matrix relating force and displacement components at the master nodes at the ends of the length is then constructed, and its manipulation provides the transfer matrix, from which the eigenvalues and eigenvectors are determined. Over the range from plane stress to plane strain, which are the extremes of aspect ratio, there are always eigenmodes which decay slower than the generalized Papkovitch-Fadle modes, the latter being largely insensitive to aspect ratio. For compact cross sections, close to square, the slowest decay is for a mode having a distribution of axial displacement reminiscent of that associated with warping during torsion; for less compact cross sections, slowest decay is for a mode characterized by cross-sectional bending, caused by self-equilibrated twisting moment.
PCB Migration and Cleanup Scenarios in Natural Gas Pipelines
Authors: Eltohami E. S. , Adewumi M. A. , Erdogmus M.
Published: June 2004

A model for tracking PCB migration under a variety of solvent injection scenarios is presented. The model serves as a tool for evaluating the efficacy and efficiency of solvents as a means of decontaminating PCB-contaminated natural gas pipelines. The model accounts for the continuous mass transfer between the pipe wall and the multiphase fluids flowing in the pipeline. Consideration is given to the processes of PCB desorption from the pipe wall, and the possibility of subsequent re-adsorption to the pipe wall. To capture the multiple mechanisms involved adequate coupling of hydrodynamic, thermodynamic, and mass transfer modeling is called for and used in this study. The study shows that PCBs concentrate in the liquid phase which is the primary transport phase. It also demonstrates that the model could be used to design an optimal pipeline decontamination scenario.
Analysis of Variable Porosity, Thermal Dispersion, and Local Thermal Nonequilibrium on Free Surface Flows Through Porous Media
Authors: Alazmi Bader , Vafai Kambiz
Published: June 2004

Characteristics of momentum and energy transport for free surface flows through porous media are explored in this study. Effects of variable porosity and an impermeable boundary on the free surface front are analyzed. In addition, effects of thermal dispersion and local thermal nonequilibrium (LTNE) are also analyzed. Pertinent parameters such as porosity, Darcy number, inertia parameter, Reynolds number, particle diameter, and solid-to-fluid conductivity ratio are used to investigate the significance of the above mentioned effects. Results show that considering the effect of variable porosity is significant only in the neighborhood of the solid boundary. The range of parameters which enhance the dispersion and LTNE effects are prescribed. Finally, it is shown that adding the effect of thermal dispersion to LTNE increases the sensitivity of LTNE between the two phases.
Thermal and Thermomechanical Phenomena in Picosecond Laser Copper Interaction
Authors: Wang Xinwei
Published: June 2004

Thermal and thermomechanical phenomena in laser metal interaction are of great importance in terms of understanding the underlying mechanisms in laser materials processing, optimizing the efficiency of laser micro-machining, and minimizing laser induced damage. In this work, Molecular Dynamics (MD) simulation is carried out to investigate picosecond laser copper interaction. A method has been developed to account for the laser beam absorption in, and the thermal transport sustained by, free electrons. Superheating is observed, and an evident temperature drop is revealed at the solid-liquid interface, which moves at a speed of 4400 m/s. However, the later phase change from solid to liquid happens in the target simultaneously and no visible movement of solid-liquid interface is observed. The results show that the laser induced stress wave consists of a strong compressive stress and a weak tensile stress. After reflection at the back side of the MD domain, the strong compressive stress becomes a strong tensile stress, which results in a visible drop of the number density of atoms. In the presence of this strong tensile stress, voids have formed in the region near the back side of the MD domain, indicating that the strong tensile stress in laser materials interaction plays an important role in terms of inducing structural damage.
Critical Heat Flux of Multi-Nozzle Spray Cooling
Authors: Lin Lanchao , Ponnappan Rengasamy
Published: June 2004

Tiny nozzles are developed that are capable of creating the swirling flow necessary to generate a full cone spray. Eight miniature nozzles are embedded in a multi-nozzle plate used to generate a spray array for the cooling of high heat flux laser diodes. The target spray cooling area is a 1×2 cm2 flat surface of a copper heater plate. A closed loop spray cooling test setup is established. FC-87, FC-72 and methanol are used as the working fluids. Critical heat flux (CHF) is experimentally investigated at various spray saturation temperatures and nozzle pressure drops (from 0.690 bar to 3.10 bar). It is demonstrated that the spray cooler can reach the CHF levels up to 91.5 W/cm2 with FC-87 and 490 W/cm2 with methanol.
Comparison of Two Procedures for the Estimation of Surface Temperature History Using Function Specification Method
Authors: Corz Alfonso , Gutie´rrez Jose´ M. , Martı´n Juan A.
Published: June 2004

This work presents a comparative study of two alternative procedures for the estimation of surface temperature of a heated body from transient interior temperature measurements. This Inverse Heat Conduction Problem (IHCP) is solved (in both procedures) by using the Function Specification Method. A numerical test was used in order to compare the best estimation achieved in each procedure. The influences of the time step size, the total number of measurements and the noise level in the measurement have been considered in the estimation. Two criteria (minimization of total error and residual principle [3] are used to choose the best hyper-parameter (r). The comparisons confirm that the procedures and criteria used provide similar results, nevertheless this study reveals slight differences with respect to the accurate and the CPU time.
Polymer Electrolyte Fuel Cells With Porous Materials as Fluid Distributors and Comparisons With Traditional Channeled Systems
Authors: Senn S. M. , Poulikakos D.
Published: June 2004

In this study, a novel concept is investigated, according to which the traditional ribbed flow delivery systems are replaced with permeable porous fluid distributors, which circumvent a number of known performance hindering drawbacks. A thorough single-phase model, including the conservation of mass, momentum, energy, species, and electric current, using Butler-Volmer kinetics, is numerically solved in three dimensions, to investigate the impact of different flow configurations on the performance of hydrogen fuel cells. It is found that cells with porous gas distributors generate substantially higher current densities and therefore are more advantageous with respect to mass transfer. Another advantage of porous flow distributors is the potential for higher power densities and reduced stack weight.
Generalized Model of Prediction of Natural Gas Consumption
Authors: Gil S. , Deferrari J.
Published: June 2004

We present a model intended to predict mainly the residential and commercial natural gas consumption in urban areas, for the short and intermediate ranges of time. In the short range, the model has been successfully used to forecast the daily gas consumption of major cities of Argentina. It is able to predict the consumption 1 to 5 days in advance with 10% of uncertainty. In the intermediate range (1 to 5 years), the model allows us to estimate the annual peak consumption, load factors and the optimal transportation capacity for a given region of interest. We also present a novel procedure to obtain the distribution of daily consumption from the monthly consumption obtained from the monthly billing.
On Source-Limited Dislocations in Nanoindentation
Authors: Shi M. X. , Huang Y. , Li M. , Hwang K. C.
Published: June 2004

The discrete dislocation model is used in this note to investigate the source-limited dislocation generation and glide in nanoindentation. It is shown that once there are enough sources for dislocation generation, the material behavior becomes independent of the dislocation source distribution.
On Timoshenko Beams of Rectangular Cross-Section
Authors: Hutchinson James R.
Published: June 2004

It was recently shown that the shear coefficient for a rectangular Timoshenko beam is highly dependent on the aspect ratio of the beam. This research investigates the reasons for that behavior by comparison of the Timoshenko beam solution with a new three-dimensional solution for a simply supported beam. The new solution is a series solution that converges to any desired accuracy. Comparisons are also made to both elementary and Mindlin plates. The Mindlin plate solution is in excellent agreement with the three-dimensional solution for the simply supported case, and is used as a basis of comparison for a free-free beam. It is found that a shear coefficient which would cause matching of the three-dimensional and Mindlin solutions would have to be a function of the wave length as well as the aspect ratio. Physical explanations are given for the high dependence on aspect ratio and for the dependence on wave length.
A Mode III Crack in a Functionally Graded Piezoelectric Material Strip
Authors: Wang B. L. , Zhang X. H.
Published: June 2004

This paper considers a mode III crack problem for a functionally graded piezoelectric material strip. The mechanical and electrical properties of the strip are considered for a class of functional forms for which the equilibrium equation has an analytical solution. The problem is solved by means of singular integral equation technique. Both a single crack and two collinear cracks are investigated. The results are tabulated and plotted to show the effect of the material nonhomogeneity and crack location on the stress and electric displacement intensity factors.
Defined Anaerobic Growth Medium for Studying
Candida albicans
Basic Biology and Resistance to Eight Antifungal Drugs
Authors: Dumitru Raluca , Hornby Jacob M. , Nickerson Kenneth W.
Published: June 2004

The polymorphic fungus
Candida albicans
is one of the most versatile opportunistic pathogens in humans. Many organs of the human body are potential targets for infection by this pathogen, but infection is commonly localized in the gastrointestinal tract, an environment providing anaerobic growth conditions. We describe a chemically defined anaerobic growth medium for four strains of
Candida albicans
(A72, SC5314, MEN, and 10261). It is a defined liquid glucose-phosphate-proline growth medium supplemented with oleic acid, nicotinic acid, and ammonium chloride. The cells did not require or respond to added ergosterol. Oleic acid and nicotinic acid are growth factors which are required only for the anaerobic growth of
C. albicans
. An important technical feature of this study was the use of anaerobically grown inocula to study anaerobic growth. Anaerobically, the cells grew exclusively as mycelia at 25, 30, and 37°C. The doubling time at 30°C was ca. 20 h. The cells did not produce farnesol and did not respond to exogenous farnesol, and they were resistant to the highest tested levels of amphotericin B and four of the azole antifungals. We suggest that the anaerobic growth of
C. albicans
may contribute to the trailing end point phenomenon and the resistance of
C. albicans
biofilms to antifungal drugs.
Factors Associated with Relative Rates of Antibiotic Resistance in
Pseudomonas aeruginosa
Isolates Tested in Clinical Laboratories in the United States from 1999 to 2002
Authors: Flamm Robert K. , Weaver Mellany K. , Thornsberry Clyde , Jones Mark E. , Karlowsky James A. , Sahm Daniel F.
Published: June 2004

For the period from 1999 to 2002 in the United States, the in vitro susceptibilities of 52,637
Pseudomonas aeruginosa
isolates to 10 antimicrobial agents were evaluated. The isolates were from 29 laboratories, 11 of which participated in The Surveillance Network for four consecutive years. Isolates were collected from adult patients (≥18 years of age) in intensive care units (ICU), non-ICU inpatients, nursing home patients, and outpatients; data were analyzed to evaluate factors, such as year of isolation, patient age group, isolate specimen source, and patient type (hospitalized patients [ICU, non-ICU, or nursing home] or outpatients). Rates of resistance for the 4-year period were highest for isolates from patients in ICU and 18- to 39-year-old patients and for isolates from the lower respiratory tract. Resistance decreased with age. Resistance was lowest in isolates from outpatients, in isolates from ≥70-year-old patients, and from specimens from the upper respiratory tract. Multidrug resistance (MDR) (resistance to three or more antimicrobial agents) accounted for 24.9% of all isolates. The MDR rate was highest in isolates from patients in nursing homes (29.9%) and ICU (29.5%).
Optimizing Pharmacodynamic Target Attainment Using the MYSTIC Antibiogram: Data Collected in North America in 2002
Authors: Kuti Joseph L. , Nightingale Charles H. , Nicolau David P.
Published: June 2004

The OPTAMA Program is intended to examine typical antimicrobial regimens used in the treatment of common nosocomial pathogens and the likelihood of these regimens attaining appropriate pharmacodynamic exposure in different parts of the world. A 5,000-subject Monte Carlo simulation was used to estimate pharmacodynamic target attainment for meropenem, imipenem, ceftazidime, cefepime, piperacillin-tazobactam, and ciprofloxacin against
Escherichia coli
,
Klebsiella pneumoniae
,
Acinetobacter baumannii
, and
Pseudomonas aeruginosa
. Standard dosing regimens from North America were used. Pharmacokinetic parameter variability was derived from existing healthy volunteer data, and MIC data came from the 2002 MYSTIC Program. Ciprofloxacin displayed the lowest target attainment against all bacterial species (41 to 46% for
A. baumannii
, 53 to 59% for
P. aeruginosa
, and 80 to 85% for the
Enterobacteriaceae
). Increasing the dose to 400 mg every 8 h did not significantly increase target attainment against nonfermenters. Piperacillin-tazobactam target attainments were similar to that of ceftazidime against all pathogens. Higher doses of both compounds were needed to achieve better target attainments against
P. aeruginosa
. Overall, meropenem, imipenem, and cefepime attained the highest probabilities of attainment against the
Enterobacteriaceae
(99 to 100%). The carbapenems appear to be the most useful agents against
A. baumannii
(88 to 92%), and these agents, along with higher doses of any of the β-lactams, would be the most appropriate choices for empirical therapy for
P. aeruginosa
infection. Given the lack of agreement between percent susceptibility and probability of target attainment for certain antimicrobial regimens, a methodology employing stochastic pharmacodynamic analyses may be a more useful tool for differentiating the most-optimal compounds and dosing regimens in the clinical setting of initial empirical therapy.
Patterns of Amphotericin B Killing Kinetics against Seven
Candida
Species
Authors: Cantón Emilia , Pemán Javier , Gobernado Miguel , Viudes Angel , Espinel-Ingroff Ana
Published: June 2004

In a previous study tolerance to amphotericin B (AMB) was found among
Candida parapsilosis
and
C. dubliniensis
strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non-
C. albicans Candida
strains were demonstrated to be above the levels safely achievable in serum. As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6
C. albicans
, 5
C. parapsilosis
, 5
C. krusei
, 4 C.
glabrata
, 3
C. lusitaniae
, and 3
C. tropicalis
isolates), 3 oropharyngeal
C. dubliniensis
isolates, 3 AMB-susceptible isolates (ATCC 90028, ATCC 22019, ATCC 6254), and 6 AMB-resistant isolates (ATCC 200955, ATCC 200956, ATCC 200950, ATCC 200951, ATCC 200952, ATCC 200953) using RPMI 1640 medium and 0.12 to 32 μg of AMB per ml and determined the numbers of CFU per milliliter at 0, 2, 4, 8, 12, 24, and 48 h. MFCs and time-kill patterns were species specific (MFCs, ≤1 μg/ml for all
C. dubliniensis
and
C. albicans
isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 μg/ml for the other isolates). The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for
C. albicans
and
C. dubliniensis
, 16 h for
C. glabrata
, 24 h for
C. parapsilosis
and
C. lusitaniae
, and ≥40 h for
C. tropicalis
and
C. krusei
. The killing rate increased as the AMB concentration was increased up to 2 μg/ml. The highest killing rates were achieved for
C. albicans
,
C. dubliniensis
, and
C. lusitaniae
, while viable
C. tropicalis, C. krusei
, and
C. parapsilosis
cells were present after 48 h (MICs, ≤2 μg/ml) when AMB was used at 2 μg/ml. Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at ≥2 μg/ml. The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum.
Terpenes Arrest Parasite Development and Inhibit Biosynthesis of Isoprenoids inPlasmodium falciparum
Authors: Rodrigues Goulart Herbert , Kimura Emília A. , Peres Valnice J. , Couto Alicia S. , Aquino Duarte Fulgencio A. , Katzin Alejandro M.
Published: June 2004

Development of new drugs is one of the strategies for malaria control. The biosynthesis of several isoprenoids inPlasmodium falciparumwas recently described. Interestingly, some intermediates and final products biosynthesized by this pathway in mammals differ from those biosynthesized inP. falciparum. These facts prompted us to evaluate various terpenes, molecules with a similar chemical structure to the intermediates of the isoprenoids pathway, as potential antimalarial drugs. Different terpenes andS-farnesylthiosalicylic acid were tested on cultures of the intraerythrocytic stages ofP. falciparum, and the 50% inhibitory concentrations for each one were found: farnesol, 64 μM; nerolidol, 760 nM; limonene, 1.22 mM; linalool, 0.28 mM; andS-farnesylthiosalicylic acid, 14 μM. All the terpenes tested inhibited dolichol biosynthesis in the trophozoite and schizont stages when [1-(n)-3H]farnesyl pyrophosphate triammonium salt ([3H]FPP) was used as precursor. Farnesol, nerolidol, and linalool showed stronger inhibitory activity on the biosynthesis of the isoprenic side chain of the benzoquinone ring of ubiquinones in the schizont stage. Treatment of schizont stages withS-farnesylthiosalicylic acid led to a decrease in intensity of the band corresponding a p21rasprotein. The inhibitory effect of terpenes andS-farnesylthiosalicylic acid on the biosynthesis of both dolichol and the isoprenic side chain of ubiquinones and the isoprenylation of proteins in the intraerythrocytic stages ofP. falciparumappears to be specific, because overall protein biosynthesis was not affected. Combinations of some terpenes orS-farnesylthiosalicylic acid tested in this work with other antimalarial drugs, like fosmidomycin, could be a new strategy for the treatment of malaria.
Saccharomyces cerevisiae
Multidrug Transporter Qdr2p (Yil121wp): Localization and Function as a Quinidine Resistance Determinant
Authors: Vargas Rita C. , Tenreiro Sandra , Teixeira Miguel C. , Fernandes Alexandra R. , Sá-Correia Isabel
Published: June 2004

This work reports the functional analysis of
Saccharomyces cerevisiae
open reading frame
YIL121w
, encoding a member of a family of drug:H
+
antiporters with 12 predicted membrane-spanning segments (DHA12 family). Like its close homologue Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression also led to increased tolerance to the antiarrhythmia and antimalarial drug quinidine. The quinidine resistance phenotype was confirmed for different yeast strains and growth media, including a prototrophic strain, and
YIL121w
was named the
QDR2
gene. Both
QDR1
and
QDR2
were also implicated in yeast resistance to the herbicide barban (4-chloro-2-butynyl [3-chlorophenyl] carbamate), and the genes are functionally interchangeable with respect to both resistance phenotypes. The average intracellular pH of a yeast population challenged with quinidine added to the acidic growth medium was significantly below the intracellular pH of the unstressed population, suggesting plasma membrane permeabilization by quinidine with consequent increase of the H
+
influx rate. For the same extracellular quinidine concentration, internal acidification was more intense for the Δ
qdr2
deletant compared with the parental strain. Although
QDR2
transcription was not enhanced in response to quinidine, the results confirmed that Qdr2p is involved in the active export of quinidine out of the cell, thus conferring resistance to the drug.
Multiple-Dose Pharmacokinetics and Safety of a Novel Broad-Spectrum Cephalosporin (BAL5788) in Healthy Volunteers
Authors: Schmitt-Hoffmann Anne , Nyman Lars , Roos Brigitte , Schleimer Michael , Sauer Jill , Nashed Norman , Brown Thomas , Man Anthony , Weidekamm Erhard
Published: June 2004

BAL5788 is the water-soluble prodrug of BAL9141, a novel broad-spectrum cephalosporin with potent bactericidal activity against methicillin-resistant
Staphylococcus aureus
(MRSA) and penicillin-resistant
Streptococcus pneumoniae
. Safety and pharmacokinetic data from a multiple-dose study with 16 healthy male volunteers are reported. Subjects were randomized to receive BAL5788 at 500 or 750 mg (as BAL9141 equivalents;
n
= 6 subjects per dose) or placebo (
n
= 2 subjects per dose). The doses were given as 200-ml infusions over 30 min once daily on days 1 and 8 and twice daily on days 2 to 7. BAL5788 was well tolerated, with no severe or serious adverse events (AEs) or dosing-related changes in laboratory parameters, electrocardiographic findings, or vital signs. Drug accumulation in plasma was negligible during the dosing period. The results of pharmacokinetic analyses agreed well with data reported from a previous single-ascending-dose study. The elimination half-life of BAL9141 was about 3 h. The volume of distribution at steady state was equal to the volume of the adult extracellular water compartment. BAL9141 was predominantly eliminated in urine, and renal clearance of the free drug corresponded to the normal glomerular filtration rate in adults. After multiple infusions of 750 mg, the mean concentrations of BAL9141 in plasma exceeded the MIC at which 100% of MRSA isolates are inhibited (4 μg/ml) for approximately 7 to 9 h, corresponding to 58 to 75% of a 12-h dosing interval.
Absorption of Ester Prodrugs in Caco-2 and Rat Intestine Models
Authors: He Xin , Sugawara Mitsuru , Takekuma Yoh , Miyazaki Katsumi
Published: June 2004

The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs. Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs. The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when
p
-nitrophenyl acetate was used as a substrate. Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer. When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation. On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed. The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract. The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model. It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium. Broader evaluation of such a system is warranted.
Pneumocystis jiroveci
Dihydropteroate Synthase Polymorphisms Confer Resistance to Sulfadoxine and Sulfanilamide in
Saccharomyces cerevisiae
Authors: Meneau I. , Sanglard D. , Bille J. , Hauser P. M.
Published: June 2004

Failure of anti-
Pneumocystis jiroveci
prophylaxis with sulfa drugs is associated with mutations within the putative active site of the fungal dihydropteroate synthase (DHPS), an enzyme encoded by the multidomain
FAS
gene. This enzyme is involved in the essential biosynthesis of folic acid. The most frequent polymorphisms are two mutations leading to two amino acid changes (
55
Trp-Arg-
57
Pro to
55
Ala-Arg-
57
Ser), observed as a single or double mutation in the same
P. jiroveci
isolate. In the absence of a culture method for
P. jiroveci
, we studied potential resistance to sulfa drugs conferred by these polymorphisms by using
Saccharomyces cerevisiae
as a model. Single or double mutations identical to those observed in the DHPS domain of the
P. jiroveci FAS
gene were introduced by in vitro site-directed mutagenesis into alleles of the
S. cerevisiae FOL1
gene, which is the orthologue of the
P. jiroveci FAS
gene. The mutated alleles were integrated at the genomic locus in
S. cerevisiae
and expressed by functional complementation in a strain with a disrupted
FOL1
allele. The single mutation
55
Trp to
55
Ala conferred resistance to sulfanilamide, whereas the single mutation
57
Pro to
57
Ser conferred resistance to both sulfanilamide and sulfadoxine. Both single mutations also separately conferred hypersensitivity to sulfamethoxazole and dapsone. The resistance to sulfadoxine is consistent with epidemiological data on
P. jiroveci
. The double mutation
55
Trp-Arg-
57
Pro to
55
Ala-Arg-
57
Ser conferred on
S. cerevisiae
a requirement for
p
-aminobenzoate, suggesting reduced affinity of DHPS for this substrate. This characteristic is commonly observed in mutated DHPS enzymes conferring sulfa drug resistance from other organisms. However, the double mutation conferred hypersensitivity to sulfamethoxazole, which is not in agreement with epidemiological data on
P. jiroveci
. Taken together, our results suggest that the DHPS polymorphisms observed in
P. jiroveci
confer sulfa drug resistance on this pathogen.
Dihydropteroate Synthase Mutations in
Pneumocystis jiroveci
Can Affect Sulfamethoxazole Resistance in a
Saccharomyces cerevisiae
Model
Authors: Iliades Peter , Meshnick Steven R. , Macreadie Ian G.
Published: June 2004

Dihydropteroate synthase (DHPS) mutations in
Pneumocystis jiroveci
have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since
P. jiroveci
cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism,
Saccharomyces cerevisiae
. Based on the sequence conservation between the DHPS enzymes of
P. jiroveci
and
S. cerevisiae
, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in
P. jiroveci
were reverse engineered into the
S. cerevisiae
DHPS. Those mutations, T
597
A and P
599
S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the
S. cerevisiae dhps
were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with
dhps
deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T
597
and P
599
substitutions had a requirement for
p
-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.