bluuebunny/crossref_metadata_2025 huggingface dataset:
The Mechanism of Tubulin‐Colchicine Recognition
Authors: Dumortier Chantal , Potenziano James L. , Bane Susan , Engelborghs Yves
Published: June 2004

2‐Methoxy‐5‐(2′,3′,4′‐trimethoxy)‐2,4,6‐cycloheptatrien‐1‐one (MTC) is a colchicine analogue that lacks the B ring. 2‐Methoxy‐5‐(2′,4′‐dimethoxyphenyl)‐2,4,6‐cycloheptatrien‐1‐one (MD) is an A‐ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A‐ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped‐flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo‐first‐order rate constant of the fluorescence increase upon binding increases in a non‐linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25°C were 540 ± 65 M−1 and 70 ± 6 s−1, respectively. From the temperature dependence, a reaction enthalpy change (ΔHo1) of the initial binding of 49 ± 11 kJ/mol−1 and an activation energy for the second step of 28 ± 9kJ/mol−1 were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 ± 0.07 s−1 at 25°C and the activation energy of 81 ± 6 kJ/mol−1. The overall binding constant was (6.3 ± 0.2) ± 104M−1 at 25°C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A‐ring colchicine analogues (podophyllotoxin, 3′,4′,5′‐trimethoxy‐acetophenone and N‐acetylmescaline) and a C‐ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.
The Action of α‐Mannosidase from Oerskovia sp. on the Mannose‐Rich O‐Linked Sugar Chains of Glycoproteins
Authors: Bagiyan Foma G. , Eneyskaya Elena V. , Kulminskaya Anna A. , Savel'ev Andrew N. , Shabalin Konstantin A. , Neustroev Kirill N.
Published: June 2004

α‐Mannosidase was isolated from the culture liquid of Oerskovia sp. The purified enzyme had a molecular mass of 480 kDa and comprises four identical subunits. The enzyme cleaves bonds in side chains of yeast mannan (Km= 0.08 mM, kcat= 1.02 μmol · min−1· mg−1) and reveals a low activity towards p‐nitrophenyl α‐D‐mannopyranoside. The α‐mannosidase is a Ca2+‐dependent enzyme and is inhibited by EDTA. The enzyme possess no endo‐mannosidase activity releasing only mannose in the reaction with the inversion of anomeric configuration and could be classified as exo‐α‐mannanase. The enzyme revealed a high deglycosylating activity towards the short mannose‐rich O‐linked carbohydrate chains of glycoproteins.
The 11β‐Hydroxysteroid Dehydrogenase System, A Determinant of Glucocorticoid and Mineralocorticoid Action
Authors: Oppermann Udo C. T. , Persson Bengt , Jörnvall Hans
Published: June 2004

Enzymatic interconversion of active and inactive glucocorticoid hormone is important, and is carried out physiologically by 11β‐hydroxysteroid dehydrogenase (11β‐HSD) isoforms, explaining their role in cellular and toxicological processes. Two forms of the enzyme, 11β‐HSD‐1 and 11β‐HSD‐2, belonging to the protein superfamily of short‐chain dehydrogenases/reductases, have been structurally and functionally characterised. Although displaying dehydrogenase and reductase activities in vitro, the dominant in vivo function of the type‐1 enzyme might be to work as a reductase, thus generating active cortisol from inactive cortisone precursors. On the other hand, for adrenal glucocorticoids the type‐2 enzyme seems to be exclusively a dehydrogenase and, by inactivating glucocorticoids, confers specificity to peripheral mineralocorticoid receptors.
Transport of CtpA Protein from the Cyanobacterium Synechocystis 6803 Across the Thylakoid Membrane in Chloroplasts
Authors: Karnauchov Ivan , Herrmann Reinhold G. , Pakrasi Himadri B. , Klösgen Ralf Bernd
Published: June 2004

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C‐terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast‐targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec‐dependent thylakoid transport route by high concentrations of the precursor of the 33‐kDa subunit of the oxygen‐evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23‐kDa subunit of the oxygen‐evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the pH‐dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.
Molecular Characterisation of the pifC Gene Encoding Translation Initiation Factor 3, which is Required for Normal Photosynthetic Complex Formation in Rhodobacter Sphaeroides NCIB 8253
Authors: Babic Slobodan , Hunter C. Neil , Rakhlin Nina J. , Simons Robert W. , Phillips‐Jones Mary K.
Published: June 2004

In order to determine whether translation initiation events play a selective role in regulating the expression of photosynthetic complexes in the photosynthetic bacterium Rhodobacter sphaeroides, we have undertaken an initial study to investigate the potential role of translation initiation factor IF3, which also behaves as a pleiotropic regulatory factor in some bacteria. Following the isolation and purification of a 24‐kDa IF3‐like protein (PifC) from R. sphaeroides, we used nested PCR to clone and characterize the encoding gene, pifC (photosynthesis‐affecting initiation factor). The 545‐bp pifC encodes a protein exhibiting 60% identity (78.6% similarity) with the Escherichia coli IF3 (infC) protein and, in common with all other IF3 genes identified to date, pifC possesses a rare initiation codon (AUA). Furthermore, in common with IF3, PifC was shown here to perform a discriminatory function towards CUG start codons, confirming its role and function as an IF3 in R. sphaeroides. Insertion of a kanamycin resistance cassette into the 5' end of pifC resulted in a viable phenotype which exhibits growth rates similar to wild type but which possesses reduced bacteriochlorophyll and photosynthetic complexes in semi‐aerobic cultures. It is shown here that the mutant is still able to produce a PifC protein but that it possesses reduced IF3 activity. This may account for the viable nature of the mutant strain, and may indicate that the effect of the mutation on photosynthesis can be more severe than shown in the present study. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.
5′ Sequences Direct Developmental Expression and Hormone Responsiveness of Tyrosine Aminotransferase in Primary Cultures of Fetal Rat Hepatocytes
Authors: Pech Cheryl M. , Tay Tisha S. , Yeoh George C. T.
Published: June 2004

Tyrosine aminotransferase (TyrAT) is one of several gluconeogenic enzymes which appear postnatally in humans and rodents in response to increased glucocorticoid and glucagon levels and decreased insulin. Primary cultured fetal rat hepatocytes older than day 15 of gestation (>E15) transcribe the TyrAT gene in response to the synergistic effect of dexamethasone and N6,2′‐O ‐dibutyryl‐adenosine 3′,5′‐monophosphate (Bt2cAMP), whereas less mature hepatocytes (E15 hepatocytes, and not <E15 hepatocytes, to be determined. This study reports that 11.1 kb of sequences upstream of the TyrAT transcription start site, which include a cAMP‐responsive element (CRE) and a glucocorticoid‐responsive element (GRE), are required for correct developmental regulation of gene expression in determined fetal hepatocytes. In contrast, the TyrAT CRE alone does not have this capability. Dexamethasone augments basal and Bt2cAMP‐stimulated activity of the TyrAT CRE alone, suggesting that synergism may be due to interaction between the glucocorticoid and cAMP‐signaling pathways. However, Bt2cAMP does not further increase dexamethasone‐induced activity of the 11.1 kb 5′ sequences when the TyrAT CRE is removed, thus excluding interaction of Bt2cAMP with the glucocorticoid pathway. Finally, insulin inhibition of dexamethasone‐induced gene transcription is shown to be conferred by TyrAT 5′ sequences. This study shows that cellular components, other than those which mediate hormonal regulation of genes, are required for determination of hepatocytes with respect to TyrAT. Since this phenomenon is observed with transient transfections, it is unlikely to involve higher‐order chromatin structure.
Change in the Mode of Gene Expression of the Hypopharyngeal Gland Cells with an Age‐dependent Role Change of the Worker Honeybee Apis mellifera L.
Authors: Ohashi Kazuaki , Natori Shunji , Kubo Takeo
Published: June 2004

Major proteins synthesized in the hypopharyngeal gland of the worker honeybee change from bee‐milk proteins to α‐glucosidase in accordance with the age‐dependent role change of the worker bee. Previously, we showed that the gene for α‐glucosidase is expressed specifically in the forager‐bee gland [Ohashi, K., Sawata, M., Takeuchi, H., Natori, S. & Kubo, T. (1996) Biochem. Biophys. Res. Commun. 221, 380–385]. Here, we describe the isolation and analysis of cDNAs for two bee‐milk 56‐kDa and 64‐kDa proteins. The 56‐kDa protein was a glycoprotein which shared 63.2% and 56.9% amino acid sequence identities with proteins encoded by cDNA for royal‐jelly‐related protein 57–1 (pRJP57‐l) and pRJP57–2. The 64‐kDa protein cDNA was identical to pRJP57‐l. Thus, these bee‐milk proteins seem to form a structurally related protein family. The gene for the 64‐kDa protein/RJP57‐1 was expressed specifically in the nurse‐bee gland, whereas that for the 56‐kDa protein was expressed in both the nurse‐bee and forager‐bee glands. mRNAs for the 56‐kDa and 64‐kDa proteins were detected by in situ hybridization in a whole acinus of the nurse‐bee gland, whereas mRNAs for the 56‐kDa protein and α‐glucosidase were detected in that of the forager‐bee gland. Therefore, the individual secretory cells of the acinus of the hypopharyngeal gland were shown to express these genes differently with the age‐dependent role change of the worker bee.
Differential Stimulation by CCAAT/Enhancer‐Binding Protein α Isoforms of the Estrogen‐Activated Promoter of the Very‐Low‐Density Apolipoprotein II Gene
Authors: Calkhoven Cor F. , Snippe Lenie , Ab Geert
Published: June 2004

The transcription factors CCAAT/enhancer‐binding proteins α and β (C/EBPα and C/EBPβ) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes. C/EBPα appears to be a critical regulator of genes involved in metabolic processes. We are interested in the roles of C/EBP in the expression of the very‐low‐density apolipoprotein II (apoVLDL II) gene. This gene encodes an avian yolk protein, is induced by estrogens and is only expressed in liver. To examine the role of C/EBP in apoVLDL II expression, footprinting and electromobility‐shift analysis were performed. For three of the protein‐binding sites in the apoVLDL II promoter region, C/EBPα and C/EBPβ were identified as the major DNA‐binding activities. For one of the C/EBP genes, C/EBPα, the effect of the gene products on apoVLDL II transcription was examined. From transfection experiments we conclude that maximal estrogen‐dependent activity of the apoVLDL II promoter requires the dual action of the estrogen receptor and C/EBP. The level of activity is different depending on the nature of the C/EBPα translational isoform transfected, the full‐length C/EBPα polypeptide being the most active isoform and the N‐terminally truncated isoform being moderately active. The present results suggest a role of C/EBPα translational isoform ratio in the modulation of expression of C/EBP target genes, such as those involved in metabolic processes.
Fluoresceinyl‐Ethylenediamine‐Ouabain Detects an Acidic Environment in the Cardiac Glycoside binding Site of Na+/K+‐ATPase
Authors: Brinkmann Klaus , Linnertz Holger , Amler Evzen , Lanz Edvard , Herman Petr , Schoner Wilhelm
Published: June 2004

To probe the pH value in the microenvironment of the cardiac glycoside‐binding site of Na+/K+‐ATPase, pH‐sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino‐ouabain (FEDO) had a pKs of 6.0 and showed a H+‐dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+‐ATPase at 37°C and pH 7.25 in a slow time‐dependent process under the conditions of backdoor phosphorylation with kon of 891 s−1 M−1. The complex dissociated with koff of 0.35°10−3 s−1 resulting in a Kd value of 0.4 μM for the FEDO · enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 · 0.2 was calculated to exist in the microenvironment of the FEDO · enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO · enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO · enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern‐Volmer constant from 6.2 mM−1 (free FEDO) to 1.5 mM−1 (FEDO · enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO · enzyme complex by measurements of the anisotropy and the fluorescence half‐life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1 ± 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain · receptor complex has been formed.
Reactivity of the Tyrosyl Radical of Escherichia Coli Ribonucleotide Reductase
Authors: Gerez Catherine , Elleingand Eric , Kauppi Björn , Eklund Hans , Fontecave Marc
Published: June 2004

Ribonucleotide reductase is a key enzyme for DNA synthesis. Its small component, named protein R2, contains a tyrosyl radical essential for activity. Consequently, radical scavengers are potential antiproliferative agents. In this study, we show that the reactivity of the tyrosyl radical towards phenols, hydrazines, hydroxyurea, dithionite and ascorbate can be finely tuned by relatively small modifications of its hydrophobic close environment. For example, in this hydrophobic pocket, Leu77→Phe mutation resulted in a protein with a much higher susceptibility to radical scavenging by hydrophobic agents. This might suggest that the protein is flexible enough to allow small molecules to penetrate in the radical site. When mutations keeping the hydrophobic character are brought further from the radical (for example IIe74→Phe) the reactivity of the radical is instead very little affected. When a positive charge was introduced (for example IIe74→Arg or Lys) the protein was more sensitive to negatively charged electron donors such as dithionite. These results allow us to understand how tyrosyl radical sites have been optimized to provide a good stability for the free radical.
Interleukin‐3 Activates Syk in a Human Myeloblastic Leukemia Cell Line, AML193
Authors: Tsubokawa Misao , Tohyama Yumi , Tohyama Kaoru , Asahi Momoyo , Inazu Tetsuya , Nakamura Hideo , Saito Hitoshi , Yamamura Hirohei
Published: June 2004

Protein‐tyrosine kinases and phosphatases play an important role in cytokine‐mediated cell growth. The proliferation of a human myeloid leukemia cell line, AML193, is dependent on interleukin‐3 (IL‐3) or granulocyte colony‐stimulating factor. In the current study, we demonstrated that a non‐receptor‐type protein‐tyrosine kinase, Syk, was immediately activated by the stimulation with IL‐3 or granulocyte colony‐stimulating factor in AML193 cells. We further investigated the relation of Syk with IL‐3‐mediated signaling and found that the IL‐3 receptor β subunit was immunoprecipitated with Syk. Since the IL‐3 receptor β subunit is known to mediate growth signaling, our results indicate that Syk may be involved in the proliferation of myeloid cells.
The Role of Subunit VIII in the Structural Stability of the bc1 Complex from Saccharomyces cerevisiae Studied Using Hybrid Complexes
Authors: Boumans Hans , Berden Jan A. , Grivell Leslie A.
Published: June 2004

The QCR8 genes encoding subunit VIII of the be, complex from Kluyveromyces lactis and Schizosaccharomyces pombe partially complement the respiratory‐deficient phenotype of a S. cerevisiaeQCR8 ‐null mutant. This implies that the heterologous Qcr8 subunits can be imported by S. cerevisiae mitochondria and that they assemble to form a hybrid be, complex that is sufficiently active to support growth. In contrast, the QCR8 gene from bovine heart, encoding the 9.5‐kDa subunit, is not able to restore respiratory function to the S. cerevisiae null mutant. This lack of functional complementation is directly attributable to the inability of S. cerevisiae mitochondria to import this protein as shown by in vitro assays. However, a hybrid gene encoding the N‐terminal 26 residues of S. cerevisiae subunit VIII and the rest of the 9.5‐kDa bovine heart homologue, was able to functionally complement the QCR8‐null mutant, albeit to a very low extent. Successful import into S. cerevisiae mitochondria was confirmed by in vitro import experiments. Surprisingly, although assembly of these hybrid complexes is reduced to an extent that is proportional to the evolutionary distance of the homologue to S. cerevisiae, the specific activities of the assembled complexes are the same as for the wild‐type be, complex. After solubilisation of the mitochondrial membranes with the mild detergent dodecyl maltoside, the wild‐type enzyme can be inactivated by incubation at increased temperature, independent of protease activity. The rate of inactivation can be significantly increased by the addition of o‐phenanthroline [Boumans, H., Grivell, L. A. & Berden, J. A. (1997) J. Biol. Chem. 272, 16753–16760]. The hybrid complexes are much more sensitive to both types of treatment. We conclude that substitution of subunit VIII by a homologous counterpart results in a loosening of the structure of the bc1, complex on the intermembrane space side, resulting in a less stable insertion of the Rieske Fe‐S protein in vivo and therefore a lower stability of the assembled enzyme under certain in vitro conditions, but without an effect on catalytic activity.
Asthma prevention: Breast is best?
Authors: Kemp A , Kakakios A
Published: June 2004

Abstract:  Whilst breastfeeding has been considered to exert a preventative effect on the development of allergic disease, several recent publications have challenged this view, particularly with respect to the long‐term outcomes for asthma. There are many other beneficial effects of breastfeeding apart from the possibility of allergy prevention. The suggestion that breastfeeding may increase the development of allergic disease raises concerns about the appropriate steps to take for primary prevention of allergy. It is concluded that breastfeeding can still be recommended for the beneficial effects in reducing atopic disease in childhood in addition to the other demonstrated benefits, and that there are unresolved questions concerning the few studies that suggest the possibility of increased allergic disease in later life.
Over‐Education and Ethnic Minorities in Britain
Authors: Battu H. , Sloane P. J.
Published: June 2004

This study examines the utilization of education across ethnic minorities in the UK. In particular, we examine the incidence of mismatch between educational qualifications and occupational attainment, the determinants of any mismatch and the consequences for earnings and other labour market outcomes. Using a modal measure of required education across 60 occupations we find that different ethnic groups have varying levels of over‐education with the highest incidence being amongst the Indian and African‐Asian groups. When we introduce controls we find that the African‐Asians are more likely to be both over‐ and under‐ educated relative to Indians (the omitted group), but this is reversed for those born in the UK. Foreign qualifications increase the likelihood of over‐education for Pakistanis and Bangladeshis but reduce it for African‐Asians. Language fluency increases the likelihood of mismatch. The results from our earnings regressions support previous work in relation to the returns to over‐education, required education and under‐education, though with differences between native and immigrant non‐whites.
Skill upgrading within informal training: lessons from the Indian auto mechanic
Authors: Barber Jamie
Published: June 2004

Informal training is known to be the dominant skill acquisition strategy for the majority of workers in India and many other economically developing countries and there is much benefit in understanding the strengths and weaknesses of this form of training. A participant observation case study was undertaken in northern India to investigate these strengths and weaknesses as well as to search out causes and influences that may be of benefit to those that seek to understand the process of informal learning.
HRD and the challenges of assessing learning in the workplace
Authors: Clarke Nicholas
Published: June 2004

Increasingly the different ways in which learning can be conceptualised alongside debates within the field of human resource development (HRD) regarding its nature and purpose, potentially lead to confusion regarding how learning is to be assessed in the workplace. This article identifies some of the complexities associated with assessing learning in today's workplace and presents empirical findings suggesting that different aspects of an organisation's training and development system are likely to differentially determine the extent to which either formal or informal learning is assessed. Explanations as to why this may be the case are put forward.
On the Nearly Viscometric Torsional Motion of Viscoelastic Liquids Between Shrouded Rotating Disks
Authors: Siginer Dennis A.
Published: June 2004

Flow of a viscoelastic liquid in a cylindrical cavity, driven by rotating finite disks is investigated. The cylindrical sidewall is fixed and the covers rotate with different angular velocities either in the same or in opposite directions. A regular perturbation in terms of the angular velocity of the caps is used. The flow field is resolved into a primary azimuthal stratified viscometric field and a weaker secondary meridional field. Results are presented for a range of cylinder aspect and cap rotation ratios and viscoelastic parameters. Interesting instabilities of the fluid of second grade are discussed. The controversy surrounding the sign of the first Rivlin-Ericksen constant is completely irrelevant to the discussion. It is shown qualitatively that loss of stability occurs repeatedly and bifurcating flows exist for critical values of an elasticity parameter at fixed aspect and cap rotation ratio. Branching flows also occur at a fixed value of the elasticity parameter for critical values of the cap rotation ratio, when the aspect ratio is fixed.
Critical Heat Fluxes of Subcooled Water Flow Boiling Against Outlet Subcooling in Short Vertical Tube
Authors: Hata Koichi , Shiotsu Masahiro , Noda Nobuaki
Published: June 2004

The critical heat fluxes (CHFs) of subcooled water flow boiling are systematically measured for the flow velocities (u=4.0 to 13.3 m/s), the outlet subcoolings (ΔTsub,out=3 to 129 K) and the outlet pressure Pout=800kPa. The SUS304 test tubes of 3, 6, 9 and 12 mm in inner-diameter, d, and 33, 66, 99 and 133 mm in length, L, respectively for L/d=11 are used. The CHFs first become lower and then become higher with the increase in subcooling. The CHFs for four different inner-diameters with L/d=11 measured here become higher with the decrease in the diameter. CHF correlation for the latter increasing regime was given in non-dimensional form against average outlet subcoolings based on the experimental data. The correlation can describe not only the CHFs obtained in this work at the outlet pressure of 800 kPa but also the authors’ published CHFs (1284 points) for the wide range of Pout=159kPa to 1 MPa, d=6, 9 and 12 mm, L=49, 99 and 149 mm, ΔTsub,out=−4 to 140 K and u=4.0 to 13.3 m/s within 15% difference for 50K⩽ΔTsub,out⩽140K and within −10 to +30% for 30K<ΔTsub,out<50K.
Two-Phase Flow Modeling of Inducers
Authors: Harun Amrin F. , Prado Mauricio G. , Shirazi Siamack A. , Doty Dale R.
Published: June 2004

Inducers, which are classified as axial flow pumps with helical path blades, are used within rotary gas separators commonly used in electrical submersible pump installations. A two-phase flow model has been developed to study the inducer performance, focusing on head generation. The proposed model is based on a meridional flow solution technique and utilizes a two-fluid approach. The model indicates that head degradation due to gas presence is a function of flow pattern. The effect of flow pattern diminishes when the void fraction is greater than 15 percent since the centrifugal force dominates the interfacial drag force. In this case, the two-phase flow can be approximated as a homogeneous mixture. The model also suggests that a liquid displacement correction is needed when phase segregation occurs inside the inducer. The new model significantly improves the ability to predict separation efficiency of a rotary gas separator over existing models. Hydrocarbon-air and water-air experimental data were gathered to validate the new model.
Hysteresis in Liquid Crystal Thermography
Authors: Anderson M. R. , Baughn J. W.
Published: June 2004

Hysteresis in five different Thermochromic Liquid Crystals (TLCs), both narrow-band and broad-band, has been investigated. All were found to exhibit a similar hysteresis behavior during cooling relative to heating. This hysteresis is characterized by a decrease in reflectivity and a shift in the temperature associated with the peak reflected intensity for each of the R, G, and B components during cooling. This causes a shift in the hue-temperature calibration of the TLC causing temperature biases, when cooled rather than heated, of 20–60% of the useful calibration range. The hysteresis effect increases as the peak temperature during a heating and cooling cycle is increased. Repeatable heating calibrations were obtained when the TLC was cooled below the red start temperature to an apparent low reset temperature. Somewhat repeatable cooling calibrations, different from the heating calibrations, were obtained when the TLC was heated somewhat above the blue stop temperature. A possible explanation of the hysteresis based on the texture of the liquid crystal helices is provided. In addition to hysteresis, a permanent decrease in reflectivity and a shift in the temperature associated with the peak reflected intensity was observed when the TLCs were exposed to extended higher temperatures (60°C–80°C).
Lateral Deflections of Webs in Air-Flotation Ovens
Authors: Moretti Peter M.
Published: June 2004

A long web span supported by many, regularly spaced, alternating air-bars is studied. The focus is on the lateral forces on the web due to the interaction of lateral curvature with out-of-plane deflections. The effect of stretching of the elastic web material is included, and the effect of high web speed is handled by distinguishing between the tension in the material Tmat and the apparent tension Tapp=Tmat−μv2. The governing partial differential equations for a continuous representation of the web’s lateral deflection, stability, and control is developed for both straight and cambered webs. The dimensionless parameters for web-tension effect, web-camber effect, and stretching effect are identified. The influence of tilted air-bars is studied, towards developing a control mechanism to compensate for camber.
Virtual Circular Dislocation-Disclination Loop Technique in Boundary Value Problems in the Theory of Defects
Authors: Kolesnikova A. I. , Romanov A. E.
Published: June 2004

A technique for elastic boundary value problem solutions for defects in solids is developed. The method is based on the introduction of virtual circular dislocation-disclination loops distributed continuously for satisfying the prescribed boundary conditions at free surfaces and interfaces. The set of dislocation-disclination loops which may be used as virtual ones is considered. The elastic fields and energies of the selected dislocation and disclination loops are presented. The method of the virtual circular dislocation-disclination loops is then applied to obtain the elastic fields and energies of a spherical dilatating inclusion in a plate and a half-space, of a prismatic dislocation loop parallel to free surfaces of a plate and a half-space, and the elastic fields of a twist disclination loop coaxial to a cylinder.
Single-Nucleotide Polymorphism Mutation Spectra and Resistance to Quinolones in
Salmonella enterica
Serovar Enteritidis with a Mutator Phenotype
Authors: Levy Dan D. , Sharma Bhavana , Cebula Thomas A.
Published: June 2004

Resistance to quinolone antibiotics has been associated with single-nucleotide polymorphisms (SNPs) in the quinolone resistance-determining region (QRDR) of
gyrA
. Mutations in the
gyrA
gene were compared by using mutant populations derived from wild-type
Salmonella enterica
serovar Enteritidis and its isogenic
mutS
::Tn
10
mutator counterpart. Spontaneous mutants arising during nonselective growth were isolated by selection with either nalidixic acid, enrofloxacin, or ciprofloxacin. QRDR SNPs were identified in approximately 70% (512 of 695) of the isolates via colony hybridization with radiolabeled oligonucleotide probes. Notably, transition base substitution SNPs in the QRDR were dramatically increased in mutants derived from the
mutS
strain. Some, but not all, antibiotic-resistant mutants lacking QRDR SNPs were resistant to tetracycline and chloramphenicol, consistent with alterations in nonspecific efflux pumps or other membrane transport mechanisms. Changing the selection conditions shifted the mutation spectrum. Selection with ciprofloxacin was least likely to yield a mutant harboring either a QRDR SNP or chloramphenicol resistance. Selection with enrofloxacin was more likely to yield mutants containing Ser83→Phe mutations, whereas selection with ciprofloxacin or nalidixic acid favored recovery of Asp87→Gly mutants. Fluoroquinolone-resistant
Salmonella
strains isolated from veterinary or clinical settings frequently display a mutational spectrum with a preponderance of transition SNPs in the QRDR, the pattern found in vitro among
mutS
mutator mutants reported here. Both the preponderance of transition mutations and the varied mutation spectra reported for veterinary and clinical isolates suggest that bacterial mutators defective in methyl-directed mismatch repair may play a role in the emergence of quinolone and fluoroquinolone resistance in feral settings.
Cloning of
S
-Adenosyl-
l
-Methionine:C-24-Δ-Sterol-Methyltransferase (ERG6) from
Leishmania donovani
and Characterization of mRNAs in Wild-Type and Amphotericin B-Resistant Promastigotes
Authors: Pourshafie Mohammad , Morand Stanislas , Virion Alain , Rakotomanga Michaelle , Dupuy Corinne , Loiseau Philippe M.
Published: June 2004

The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant
Leishmania donovani
promastigotes, suggesting that the
S
- adenosyl-
l
-methionine:C-24-Δ-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites. From an
L. donovani
wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position. The two cDNAs differed by their 3′-untranslated region (3′-UTR) and 5′-UTR sequences. One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells. The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells. As a functional test, ERG6 null mutant
Saccharomyces cerevisiae
yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate
SCMT1/ERG6
coding sequence cloned from
L. donovani
. The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation. In AmB-R
L. donovani
promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells. Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in
Leishmania
.
RamA Is an Alternate Activator of the Multidrug Resistance Cascade in
Enterobacter aerogenes
Authors: Chollet Renaud , Chevalier Jacqueline , Bollet Claude , Pages Jean-Marie , Davin-Regli Anne
Published: June 2004

Multidrug resistance (MDR) in
Enterobacter aerogenes
can be mediated by induction of MarA, which is triggered by certain antibiotics and phenolic compounds. In this study, we identified the gene encoding RamA, a 113-amino-acid regulatory protein belonging to the AraC-XylS transcriptional activator family, in the
Enterobacter aerogenes
ATCC 13048 type strain and in a clinical multiresistant isolate. Overexpression of RamA induced an MDR phenotype in drug-susceptible
Escherichia coli
JM109 and
E. aerogenes
ATCC 13048, as demonstrated by 2- to 16-fold-increased resistance to β-lactams, tetracycline, chloramphenicol, and quinolones, a decrease in porin production, and increased production of AcrA, a component of the AcrAB-TolC drug efflux pump. We show that RamA enhances the transcription of the
marRAB
operon but is also able to induce an MDR phenotype in a
mar
-deleted strain. We demonstrate here that RamA is a transcriptional activator of the Mar regulon and is also a self-governing activator of the MDR cascade.
Efficacy of Organic Acids in Hand Cleansers for Prevention of Rhinovirus Infections
Authors: Turner Ronald B. , Biedermann Kim A. , Morgan Jeffery M. , Keswick Bruce , Ertel Keith D. , Barker Mark F.
Published: June 2004

Direct hand-to-hand contact is an important mechanism of transmission of rhinovirus infection. The rhinoviruses are inactivated at a low pH. A survey of organic acids in vitro revealed that these compounds have antirhinoviral activity that persists for at least 3 h after application to the skin. In additional studies of salicylic acid (SA) and pyroglutamic acid (PGA), the hands of volunteers were contaminated with rhinovirus at defined times after application of the acid, and then volunteers attempted to inoculate the nasal mucosa with one hand and quantitative viral cultures were done on the other hand. In one study, 3.5% SA or 1% SA with 3.5% PGA was compared with controls 15 min after application to assess the efficacy of the inactivation of virus and prevention of infection. Virus was recovered from the hands of 28 out of 31 (90%) of the volunteers in the control group compared to 4 out of 27 (15%) and 0 out of 27 in the groups administered 3.5 and 1% SA, respectively (
P
< 0.05). Rhinovirus infection occurred in 10 out of 31 (32%) of the controls and 2 out of 27 (7%) of volunteers in both treatment groups (
P
< 0.05 compared with control). In a second study, the efficacy of 4% PGA was evaluated 15 min, 1 h, and 3 h after application. Significantly fewer volunteers had positive hand cultures at all time points compared with the control group, but the proportion that developed rhinovirus infection was not significantly reduced. These results suggest the feasibility of the prevention of rhinovirus transmission by hand treatments that are virucidal on contact and have activity that persists after application.
Lysostaphin-Coated Catheters Eradicate
Staphylococccus aureus
Challenge and Block Surface Colonization
Authors: Shah Anjali , Mond James , Walsh Scott
Published: June 2004

Lysostaphin is an endopeptidase that kills
Staphylococcus aureus
, a predominant organism in catheter-related infections. Lysostaphin-coated catheters prevented catheter colonization by several strains of
S. aureus
, and activity was maintained for at least 4 days. Prophylactic use of lysostaphin in catheters may help prevent the occurrence of catheter-related staphylococcal infections.
In Vitro Interactions of Approved and Novel Drugs against
Paecilomyces
spp
Authors: Ortoneda Montserrat , Capilla Javier , Pastor F. Javier , Pujol Isabel , Yustes Clara , Serena Carolina , Guarro Josep
Published: June 2004

We have evaluated the in vitro activity of 15 combinations of antifungal drugs (amphotericin B, itraconazole, voriconazole, albaconazole, ravuconazole, terbinafine, and micafungin) against four isolates of
Paecilomyces variotii
and three of
P. lilacinus
. The interaction of terbinafine with the four azoles was synergistic for 53% of the combinations, while the interactions of both amphotericin B and micafungin with the rest of antifungal agents were mainly indifferent.
Dihydroethanoanthracene Derivatives as In Vitro Malarial Chloroquine Resistance Reversal Agents
Authors: Millet Julie , Torrentino-Madamet Marylin , Alibert Sandrine , Rogier Christophe , Santelli-Rouvier Christiane , Mosnier Joel , Baret Eric , Barbe Jacques , Parzy Daniel , Pradines Bruno
Published: June 2004

The ability of four 9,10-dihydroethanoanthracene derivatives (BG920, BG932, BG958, and BG996), as well as verapamil and promethazine, to reverse chloroquine resistance was assessed against 24 chloroquine-resistant and 10 chloroquine-susceptible strains of
Plasmodium falciparum
from different countries. The 9,10-dihydroethanoanthracene derivatives clearly increase chloroquine susceptibility only in chloroquine-resistant isolates.
Rhesus Cytomegalovirus Is Similar to Human Cytomegalovirus in Susceptibility to Βenzimidazole Nucleosides
Authors: North Thomas W. , Sequar Getachew , Townsend Leroy B. , Drach John C. , Barry Peter A.
Published: June 2004

Rhesus and human cytomegalovirus (RhCMV and HCMV, respectively) exhibit comparable inhibition by benzimidazole nucleosides, including 2,5,6-trichloro-(1-β-
d
-ribofuranosyl)benzimidazole (TCRB), and pyrrolo[2,3-
d
]pyrimidines. The two HCMV protein targets of TCRB, UL89 and UL56, are highly conserved with their RhCMV homologues. These data indicate that infection of rhesus macaques with RhCMV represents a useful model to test novel anti-HCMV drugs.
Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH
2
-Terminal Sequence
Authors: Saavedra Lucila , Minahk Carlos , de Ruiz Holgado Aída P. , Sesma Fernando
Published: June 2004

The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol.
68:
3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system.
Identification of Murine H2-Dd- and H2-Ab-Restricted T-Cell Epitopes on a Novel Protective Antigen, MPT51, ofMycobacterium tuberculosis
Authors: Suzuki Mina , Aoshi Taiki , Nagata Toshi , Koide Yukio
Published: June 2004

Both CD4+type 1 helper T (Th1) cells and CD8+cytotoxic T lymphocytes (CTL) play pivotal roles in protection againstMycobacterium tuberculosisinfection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-γ) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-γ. Flow cytometric analysis with intracellular IFN-γ and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8+T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with Kd, Dd, or Ldindicated that the epitope is presented by Dd. Finally, we proved that the p24-32/Ddcomplex is recognized by IFN-γ-producing CTL. In C57BL/6 mice, we observed H2-Ab-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine againstM. tuberculosisinfection.
Cleavage of Host Keratin 8 by a Chlamydia-Secreted Protease
Authors: Dong Feng , Su Heng , Huang Yanqing , Zhong Youmin , Zhong Guangming
Published: June 2004

Chlamydiae have to replicate within a cytoplasmic vacuole in eukaryotic cells. Expansion of the chlamydia-laden vacuole is essential for chlamydial intravacuolar replication, which inevitably causes host cell cytoskeleton rearrangements. A cleavage fragment of keratin 8 corresponding to the central rod region was detected in the soluble fraction of chlamydia-infected cells. Since keratin 8 is a major component of the intermediate filaments in simple epithelial cells, cleavage of keratin 8 may increase the solubility of the host cell cytoskeleton and thus permit vacuole expansion in chlamydia-infected cells. A chlamydia-secreted protease designated CPAF (chlamydial protease/proteasome-like activity factor) was both necessary and sufficient for keratin 8 cleavage in chlamydia-infected cells, suggesting that chlamydiae have evolved specific mechanisms for modifying the host cell cytoskeleton.
The Ami-AliA/AliB Permease of
Streptococcus pneumoniae
Is Involved in Nasopharyngeal Colonization but Not in Invasive Disease
Authors: Kerr A. R. , Adrian P. V. , Estevão S. , de Groot R. , Alloing G. , Claverys J.-P. , Mitchell T. J. , Hermans P. W. M.
Published: June 2004

The Ami-AliA/AliB oligopeptide permease is an ATP-binding cassette transporter which is found in
Streptococcus pneumoniae
and which is involved in nutrient uptake. We investigated the role of the three paralogous oligopeptide-binding lipoproteins AmiA, AliA, and AliB by using murine models of pneumococcal colonization and invasive disease. A series of mutants lacking
aliA
,
aliB
, and
amiA
either alone or in combination as double or triple mutations were used. Inoculation of the nasopharynx with a mixture of the
obl
(oligopeptide-binding lipoprotein-negative) triple-mutant and wild-type (D39) bacteria resulted in significantly smaller numbers of
obl
bacteria colonizing the nasopharynx. The use of a mixture of individual mutants and wild-type pneumococci revealed that AmiA, AliA, and AliB were all required for successful colonization of the nasopharynx. The
obl
mutant was more attenuated than the
aliB
mutant but not the
aliA
or
amiA
mutant. Therefore, there is some redundancy in the Ami-AliA/AliB complex in terms of nasopharyngeal colonization, with AliA and AmiA being able to compensate for the removal of AliB. Animals with invasive disease caused by these mutants had survival times, bacterial loads, and inflammatory cytokine production levels similar to those of animals infected with wild-type pneumococci. Our results show that although the Ami-AliA/AliB complex is not required for virulence during pneumococcal pneumonia, it does play a role in colonization of the nasopharynx.
Dual Role of Interleukin-4 (IL-4) in Pulmonary Paracoccidioidomycosis: Endogenous IL-4 Can Induce Protection or Exacerbation of Disease Depending on the Host Genetic Pattern
Authors: Arruda Celina , Valente-Ferreira Rita C. , Pina Adriana , Kashino Suely S. , Fazioli Raquel A. , Vaz Celidéia A. C. , Franco Marcello F. , Keller Alexandre C. , Calich Vera L. G.
Published: June 2004

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 106Paracoccidioides brasiliensisyeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant toP. brasiliensisinfection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.