bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect
Authors: Ramos Carlos H.I. , Baldwin Robert L. 
Published: July 2002

AbstractData are reported for Tm, the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2–9) and salt concentration (range 0–1 M) for two salts, Na2SO4 and NaCl. The results show stabilization by sulfate via anion‐specific binding in the concentration range 0–0.1 M and via the Hofmeister effect in the concentration range 0.1–1.0 M. The increase in Tm caused by anion binding at 0.1 M sulfate is 20° at pH 2 but only 1° at pH 9, where the net proton charge on the protein is near 0. The 10° increase in Tm between 0.1 and 1.0 M Na2SO4, caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH‐dependent stabilization is produced by 1 M NaCl: the increase in Tm between 0 and 1.0 M is 14° at pH 2 but only 1° at pH 9. The 14° increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na2SO4 and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.
Interactions between PEG and type I soluble tumor necrosis factor receptor: Modulation by pH and by PEGylation at the N terminus
Authors: Kerwin Bruce A. , Chang Byeong S. , Gegg Colin V. , Gonnelli Margherita , Li Tiansheng , Strambini Giovanni B. 
Published: July 2002

AbstractThe effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF‐RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF‐RI bound free PEG in a pH‐dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp‐107, at the C terminus, with the pH‐dependent and PEGylation‐dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF‐RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp‐107. Deprotonation of His‐105, which is perpendicular to Trp‐107, was integral to the binding mechanism producing a pH‐dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.
Key interactions in the immunoglobulin‐like structure of apo‐neocarzinostatin: Evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations
Authors: Izadi‐Pruneyre Nadia , Quiniou Éric , Blouquit Yves , Perez Javier , Minard Philippe , Desmadril Michel , Mispelter Joël , Adjadj Élisabeth 
Published: July 2002

AbstractThe three‐dimensional structure of apo‐neocarzinostatin (apo‐NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven‐stranded antiparallel β‐sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo‐NCS by 13C‐NMR relaxation measurements and molecular dynamics simulation. Model‐free parameters determined from the experimental data are compared with a 1.5‐nsec molecular simulation of apo‐NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this β‐protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.
Prediction of the membrane‐spanning β‐strands of the major outer membrane protein of Chlamydia
Authors: Rodríguez‐Marañón María José , Bush Robin M. , Peterson Ellena M. , Schirmer Tilman , de la Maza Luis M. 
Published: July 2002

AbstractThere is preliminary experimental evidence indicating that the major outer‐membrane protein (MOMP) of Chlamydia is a porin. We tested this hypothesis for the MOMP of the mouse pneumonitis serovar of Chlamydia trachomatis using two secondary structure prediction methods. First, an algorithm that calculates the mean hydrophobicity of one side of putative β‐strands predicted the positions of 16 transmembrane segments, a structure common to known porins. Second, outer loops typical of porins were assigned using an artificial neural network trained to predict the topology of bacterial outer‐membrane proteins with a predominance of β‐strands. A topology model based on these results locates the four variable domains (VDs) of the MOMP on the outer loops and the five constant domains on β‐strands and the periplasmic turns. This model is consistent with genetic analysis and immunological and biochemical data that indicate the VDs are surface exposed. Furthermore, it shows significant homology with the consensus porin model of the program FORESST, which contrasts a proposed secondary structure against a data set of 349 proteins of known structure. Analysis of the MOMP of other chlamydial species corroborated our predicted model.
Ca2+ and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III
Authors: Sopkova Jana , Raguenes‐Nicol Céline , Vincent Michel , Chevalier Anne , Lewit‐Bentley Anita , Russo‐Marie Françoise , Gallay Jacques 
Published: July 2002

AbstractAnnexin 3 (ANX A3) represents ∼1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca2+‐dependent manner, via Ca2+‐binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino‐acid, N‐terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N‐terminal segment) in the wild type and in single‐tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca2+, domain III of ANX A3 exhibits an open conformation and a widely solvent‐accessible W190 residue in the same conditions. This is in agreement with the three‐dimensional structure of the ANX A3‐E231A mutant lacking the bidentate Ca2+ ligand in domain III. Ca2+ in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N‐terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca2+ and returns to its original value when interacting with membranes. Ca2+ concentration for half binding of the W5A mutant to negatively charged membranes is ∼0.5 mM while it increases to ∼1 mM for the ANX A3 wild type and to ∼3 mM for the W190 ANX A3 mutant. In addition to the expected perturbation of the W190 environment at the contact surface between the protein and the membrane bilayer, binding of the protein to Ca2+ and to membranes modulates the flexibility of the ANX A3 hinge region at the opposite of this interface and might affect its membrane permeabilizing properties.
Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the
mec
Element in Methicillin-Resistant
Staphylococcus aureus
Authors: Oliveira Duarte C. , Lencastre Hermínia de 
Published: July 2002

ABSTRACT

Full characterization of methicillin-resistant
Staphylococcus aureus
(MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous
mec
element these bacteria carry, which is associated with drug resistance determinant
mecA
. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the
mec
element types based on the structural features that were shown to be typical of
mec
elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated
mec
elements was previously determined by hybridization and PCR screenings and also by DNA sequencing. The method was tested together with multilocus sequence typing and other typing methods for the characterization of 18 isolates representative of the MRSA clones recovered during a hospital outbreak in Barcelona, Spain. The multiplex PCR was shown to be rapid, robust, and capable in a single assay of identifying five structural types of the
mec
element among these strains, three major and two minor variants, each one of which has been already been seen among MRSA characterized earlier. This technique should be a useful addition to the armamentarium of molecular typing tools for the characterization of MRSA clonal types and for the rapid tentative identification of structural variants of the
mec
element.
ATP-Dependent Removal of Nucleoside Reverse Transcriptase Inhibitors by Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Naeger Lisa K. , Margot Nicolas A. , Miller Michael D. 
Published: July 2002

ABSTRACTRemoval of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) ≫ zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.
Physiologically Based Pharmacokinetic Model for Terbinafine in Rats and Humans
Authors: Hosseini-Yeganeh Mahboubeh , McLachlan Andrew J. 
Published: July 2002

ABSTRACTThe aim of this study was to develop a physiologically based pharmacokinetic (PB-PK) model capable of describing and predicting terbinafine concentrations in plasma and tissues in rats and humans. A PB-PK model consisting of 12 tissue and 2 blood compartments was developed using concentration-time data for tissues from rats (n= 33) after intravenous bolus administration of terbinafine (6 mg/kg of body weight). It was assumed that all tissues except skin and testis tissues were well-stirred compartments with perfusion rate limitations. The uptake of terbinafine into skin and testis tissues was described by a PB-PK model which incorporates a membrane permeability rate limitation. The concentration-time data for terbinafine in human plasma and tissues were predicted by use of a scaled-up PB-PK model, which took oral absorption into consideration. The predictions obtained from the global PB-PK model for the concentration-time profile of terbinafine in human plasma and tissues were in close agreement with the observed concentration data for rats. The scaled-up PB-PK model provided an excellent prediction of published terbinafine concentration-time data obtained after the administration of single and multiple oral doses in humans. The estimated volume of distribution at steady state (Vss) obtained from the PB-PK model agreed with the reported value of 11 liters/kg. The apparent volume of distribution of terbinafine in skin and adipose tissues accounted for 41 and 52%, respectively, of theVssfor humans, indicating that uptake into and redistribution from these tissues dominate the pharmacokinetic profile of terbinafine. The PB-PK model developed in this study was capable of accurately predicting the plasma and tissue terbinafine concentrations in both rats and humans and provides insight into the physiological factors that determine terbinafine disposition.
Pharmacodynamic Effects of Nitroimidazoles Alone and in Combination with Clarithromycin on
Helicobacter pylori
Authors: Svensson Margareta , Ström Magnus , Nilsson Maud , Sörberg Mikael , Nilsson Lennart E. 
Published: July 2002

ABSTRACT

Pharmacodynamic studies of
Helicobacter pylori
exposed to metronidazole and tinidazole alone and in combination with clarithromycin were performed by bioluminescence assay of intracellular ATP. The pharmacodynamic parameter control-related effective regrowth time (CERT) was used. CERT is defined as the time required for the resumption of logarithmic growth and a return of the level of growth to the preexposure inoculum in the test culture minus the corresponding time in the control culture. CERT measures the combined effects of the initial level of killing and postantibiotic effect. The incubation times and drug concentrations were chosen according to their half-lives and their clinically achievable concentrations. The study shows that the parameter CERT is useful for the testing of antibiotic combinations. The CERTs induced by clarithromycin, metronidazole, and tinidazole alone and in the combinations tested were concentration dependent, with no maximum response, indicating that the use of high doses may be preferable. The combinations with the highest concentrations induced synergistic effects and prevented regrowth. The use of tinidazole in combination with clarithromycin proved to give the longest CERTs, indicating that this is the most effective combination.
Novel Plasmid-Encoded Class C β-Lactamase (MOX-2) in
Klebsiella pneumoniae
from Greece
Authors: Raskine Laurent , Borrel Isabelle , Barnaud Guilène , Boyer Sophie , Hanau-Berçot Béatrice , Gravisse Jérome , Labia Roger , Arlet Guillaume , Sanson-Le-Pors Marie-José 
Published: July 2002

ABSTRACT

Klebsiella pneumoniae
KOL, a clinical strain resistant to various β-lactams, was isolated from the stools of a patient from Greece. This strain harbored a new pI 9.1 plasmid-mediated AmpC β-lactamase with unusually high levels of hydrolytic activity for cefoxitin and cefotetan that we named MOX-2. Sequencing of
bla
MOX-2
revealed 93.2, 92.9, 92.7, and 73.1% identities with the deduced amino acid sequences of CMY-8, MOX-1, CMY-1, and the AmpC β-lactamase of
Aeromonas sobria
, respectively.
Fluorescence Assay for Studying the Ability of Macrolides To Induce Production of Ribosomal Methylase
Authors: Clarebout Gervais , Leclercq Roland 
Published: July 2002

ABSTRACT

A screening assay to test the inducing capacity of macrolides by fusing the attenuator of the inducible
erm
(B) gene from
Streptococcus pneumoniae
HM28 with the
gfpmut1
gene has been designed. Fluorescence was detected under UV light around disks impregnated with inducer macrolides (erythromycin or azithromycin) but not with noninducer ketolides. Induction could be quantified by fluorometry.
Structure-Function Relationship Studies on the Frog Skin Antimicrobial Peptide Tigerinin 1: Design of Analogs with Improved Activity and Their Action on Clinical Bacterial Isolates
Authors: Sitaram Narasimhaiah , Sai Korrapati Purna , Singh Shashi , Sankaran Krishnan , Nagaraj Ramakrishnan 
Published: July 2002

ABSTRACT
Structure-function relationships in antimicrobial peptides have been extensively investigated in order to obtain improved analogs. Most of these studies have targeted either α-helical peptides or β-sheet peptides with multiple disulfide bridges. Tigerinins are short, nonhelical antimicrobial peptides with a single disulfide bridge. In this study, we have synthesized several analogs of tigerinin 1 with an aim to understand the structural basis of activity as well as improve its activity. The studies demonstrate that the loop structure of tigerinin 1 is essential for its optimal activity. However, linearization with increased cationic charges can compensate for loss of loop structure to some extent. Morphology of the cells after treatment with the active analogs shows extensive leakage of cytoplasmic contents. Tigerinin 1 and two of its analogs exhibit impressive activity against a variety of clinical bacterial isolates.
GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats
Authors: Edwards Jeffrey E. , Brouwer Kenneth R. , McNamara Patrick J. 
Published: July 2002

ABSTRACT

The goal of this study was to determine the distribution of unbound amprenavir in the central nervous system (CNS) of rats. The concentration of unbound amprenavir in the extracellular fluid of the brain and the blood was examined in the presence and absence of the MDR modulator GF120918 by microdialysis. The brain-to-blood ratio of amprenavir in the absence and presence of GF120918 was found to be significantly different (
P
< 0.003; 0.076 and 0.617, respectively). The use of the MDR modulator GF120918 could potentially increase the penetration of human immunodeficiency virus protease inhibitors into the CNS.
Inhibition of Cyclin-Dependent Kinase 1 by Purines and Pyrrolo[2,3-d]Pyrimidines Does Not Correlate with Antiviral Activity
Authors: Evers David L. , Breitenbach Julie M. , Borysko Katherine Z. , Townsend Leroy B. , Drach John C. 
Published: July 2002

ABSTRACTWe have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC50; 14 μM) similar to that of roscovitine; it was competitive with respect to ATP (Ki= 14 μM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC50s, 32 to 100 μM) but not its antiviral efficacy (IC50s, 0.02 to 0.3 μM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC50, 38 μM) and HCMV (IC50, 40 μM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.
Comparison of Visual and Spectrophotometric Methods of Broth Microdilution MIC End Point Determination and Evaluation of a Sterol Quantitation Method for In Vitro Susceptibility Testing of Fluconazole and Itraconazole against Trailing and Nontrailing
Candida
Isolates
Authors: Arthington-Skaggs Beth A. , Lee-Yang Wendy , Ciblak Meral A. , Frade Joao P. , Brandt Mary E. , Hajjeh Rana A. , Harrison Lee H. , Sofair Andre N. , Warnock and David W. 
Published: July 2002

ABSTRACT

Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven
Candida
spp., obtained through active population-based surveillance between 1998 and 2000. Of 429
C. albicans
isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118
C. tropicalis
isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five
Candida
spp. tested (
C. dubliniensis
,
C. glabrata
,
C. krusei
,
C. lusitaniae
, and
C. parapsilosis
). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.