bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition
Authors: Ray Kallol , Hines Christina S. , Rodgers David W. 
Published: October 2002

AbstractThe highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate‐binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.
Characterization of Herpes Simplex Virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity
Authors: Kokoris Mark S. , Black Margaret E. 
Published: October 2002

AbstractHerpes Simplex Virus type 1 (HSV‐1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV‐1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild‐type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14‐fold decrease in Km compared to the wild‐type enzyme. The most dramatic change is displayed by mutant SR26, with a 124‐fold decrease in Km with acyclovir as the substrate. Such new “prodrug kinases” could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.
Plant annexins form calcium‐independent oligomers in solution
Authors: Hofmann Andreas , Ruvinov Sergei , Hess Sonja , Schantz Rodolphe , Delmer Deborah P. , Wlodawer Alexander 
Published: October 2002

AbstractThe oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino‐terminal Hisn fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer–trimer equilibria for each protein with association constants in the range of 0.9 × 1010−1.7 × 1011 M−2. All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer–dimer equilibrium in solution.
Assessment of Mechanical Properties of Adherent Living Cells by Bead
Micromanipulation: Comparison of Magnetic Twisting Cytometry vs Optical Tweezers
Authors: Laurent Valérie M. , Hénon Sylvie , Planus Emmanuelle , Fodil Redouane , Balland Martial , Isabey Daniel , Gallet Franc¸ois 
Published: October 2002

Abstract
We compare the measurements of viscoelastic properties of adherent alveolar epithelial cells by two micromanipulation techniques: (i) magnetic twisting cytometry and (ii) optical tweezers, using microbeads of same size and similarly attached to F-actin. The values of equivalent Young modulus E, derived from linear viscoelasticity theory, become consistent when the degree of bead immersion in the cell is taken into account. E-values are smaller in (i) than in (ii): ∼34–58 Pa vs ∼29–258 Pa, probably because higher stress in (i) reinforces nonlinearity and cellular plasticity. Otherwise, similar relaxation time constants, around 2 s, suggest similar dissipative mechanisms.
Predicting the Failure Response of Cement-Bone Constructs Using a
Non-Linear Fracture Mechanics Approach
Authors: Mann Kenneth A. , Damron Leatha A. 
Published: October 2002

Abstract
A non-linear fracture mechanics approach was used to predict the failure response of complex cement-bone constructs. A series of eight mechanical tests with a combination of tensile and shear loading along the cement-bone interface was performed. Each experiment was modeled using the finite element method with non-linear constitutive models at the cement-bone interface. Interface constitutive parameters were assigned based on the quantity of bone interdigitated with the cement. There was a strong correlation r2=0.80 between experimentally measured and finite element predicted ultimate loads. The average error in predicted ultimate load was 23.9 percent. In comparison to the ultimate load predictions, correlations and errors for total energy to failure (r2=0.24, avg. error=38.2 percent) and displacement at 50 percent of the ultimate load (r2=0.27, avg. error=52.2 percent) were poor. The results indicate that the non-linear constitutive laws could be useful in predicting the initiation and progression of interface failure of cemented bone-implant systems. However, improvements in the estimation of post-yield interface properties from the quantity of bone interdigitated with cement are needed to enhance predictions of the overall failure response.
Heterologous Expression of Epothilone Biosynthetic Genes in
Myxococcus xanthus
Authors: Julien Bryan , Shah Sanjay 
Published: October 2002

ABSTRACT

Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol). Epothilones are produced from the myxobacterium
Sorangium cellulosum
, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B. Furthermore, genetic manipulation of
S. cellulosum
is difficult. To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria
Myxococcus xanthus
. We inserted 65.4 kb of
S. cellulosum
DNA that encompassed the entire epothilone gene cluster into the chromosome of
M. xanthus
by a series of homologous recombination events. The resulting strain produced epothilones A and B. Construction of a strain that contained a mutation in
epoK
, the P450 epoxidase, resulted in production of epothilones C and D.
TheCandida dubliniensis CdCDR1Gene Is Not Essential for Fluconazole Resistance
Authors: Moran Gary , Sullivan Derek , Morschhäuser Joachim , Coleman David 
Published: October 2002

ABSTRACTThe present study investigated the role of theCandida dubliniensis CdCDR1andCdCDR2genes in the development of fluconazole resistance. TheC. dubliniensis CdCDR1gene was 92% identical at the nucleotide sequence level to the correspondingC. albicansgene. However, 58% (14 of 24) ofC. dubliniensisgenotype 1 isolates tested harbored a nonsense mutation in theCdCDR1open reading frame that converted codon 756 (TAT) to a TAG translational stop codon. Analysis of five of theseC. dubliniensisisolates by Western immunoblotting showed that they expressed a truncated 85-kDa CdCdr1p compared to the full-length 170-kDa CdCdr1p. Expression ofCdCDR1alleles from sixC. dubliniensisisolates in apdr5 Saccharomyces cerevisiaestrain revealed thatCdCDR1alleles from three isolates that encoded truncated proteins were unable to confer resistance to drugs and antifungals. However, reassignment of the TAG sequence at codon 756 to TAT (encoding tyrosine) in an allele from strain CD36 conferred the ability to mediate resistance to multiple drugs. Fluconazole-resistant isolates ofC. dubliniensisharboring functional alleles ofCdCDR1were found to exhibit two- to ninefold-higher levels ofCdCDR1mRNA than did matched fluconazole-susceptible isolates. By comparison, levels ofCdMDR1expression ranged from approximately 50- to 100-fold greater in resistant isolates. Fluconazole resistance was also identified in isolates harboring nonfunctionalCdCDR1alleles, but resistance in these isolates was only associated with increasedCdMDR1expression. Targeted disruption of two functional alleles ofCdCDR1in a fluconazole-resistant derivative ofC. dubliniensisthat overexpressed bothCdCDR1andCdMDR1revealed that althoughCdCDR1was important for mediating reduced susceptibility to itraconazole and ketoconazole, there was no affect on fluconazole susceptibility in the double mutant. Evidence presented in this study reveals thatCdCDR1is not essential for the development of fluconazole resistance inC. dubliniensis.
Effect of 5-Iodo-2′-Deoxyuridine on Vaccinia Virus (Orthopoxvirus) Infections in Mice
Authors: Neyts Johan , Verbeken Erik , De Clercq Erik 
Published: October 2002

ABSTRACT

There is a concern that there may be unregistered stocks of smallpox that can be used for bioterrorism or biological warfare. According to the WHO Advisory Committee on Variola Research, there is a need to develop strategies to treat smallpox infections should they reappear. It would also be important to have an effective drug at hand for the treatment of monkeypox disease in humans. We show here that 5-iodo-2′-deoxyuridine (IDU) is a potent inhibitor of vaccinia virus (VV) replication and that IDU inhibits VV DNA synthesis in a dose-dependent way. The in vivo protective effect of IDU was assessed in the VV tail lesion model in immunocompetent mice and in a lethal model for VV infection in SCID (severe combined immune deficiency) mice that had been infected either intranasally, intraperitoneally, or intravenously. Subcutaneous treatment with IDU at 150 and 100 mg/kg of body weight markedly reduced the number of tail lesions in immunocompetent NMRI mice. Untreated intranasally VV-infected SCID mice died at 20.8 ± 3.1 days after infection (mean ± standard deviation). Treatment with IDU (subcutaneously, 150 mg/kg/day [from day 0 to 4] and 75 mg/kg/day [from day 6 to 11]) delayed-virus induced mortality by 15 days (mean day of death ± standard deviation, 35.8 ± 6.7;
P
< 0.0001). This protective effect was associated with (i) an improvement of lung histology and (ii) a marked reduction in lung viral titers. IDU also delayed VV-induced mortality when mice had either been infected intraperitoneally or intravenously. Even when the start of treatment with IDU (in intraperitoneally VV-infected mice) was postponed until 2 or 4 days after infection, an important delay in virus-induced mortality was noted.
Ertapenem versus Ceftriaxone Followed by Appropriate Oral Therapy for Treatment of Complicated Urinary Tract Infections in Adults: Results of a Prospective, Randomized, Double-Blind Multicenter Study
Authors: Tomera Kevin M. , Burdmann Emmanuel A. , Reyna Oscar G. Pamo , Jiang Qi , Wimmer Wendy M. , Woods Gail L. , Gesser Richard M. 
Published: October 2002

ABSTRACT

The efficacy and safety of intravenous (i.v.) ertapenem (1 g once a day) with the option to switch to an oral agent for treatment of adults with complicated urinary tract infections (UTIs) were compared with that of i.v. ceftriaxone (1 g daily) with the same oral switch option in a multicenter, double-blind, prospective, randomized study. At entry, 592 patients were assigned to one of two strata: acute pyelonephritis or other complicated UTI without acute pyelonephritis. After a minimum of 3 days, patients could be switched to an oral antimicrobial agent. A total of 159 patients in the ertapenem group and 171 patients in the ceftriaxone group were microbiologically evaluable. Approximately 95% of patients in each treatment group were switched to oral therapy. The most common pathogens were
Escherichia coli
and
Klebsiella pneumoniae
. At the primary efficacy endpoint 5 to 9 days after treatment, 91.8% of patients who received ertapenem and 93.0% of those who received ceftriaxone had a favorable microbiological response (95% confidence interval for the difference, adjusting for strata, −7.6 to 5.1%), indicating that outcomes in the two treatment groups were equivalent. Microbiological success rates for the two treatment groups were similar when compared by stratum and also by severity of infection. The frequency and severity of drug-related adverse events were generally similar in both treatment groups. In this study, ertapenem was as effective as ceftriaxone for the initial treatment of complicated UTIs in adults, was generally well tolerated, and had a similar overall safety profile.
Characterization of Sparsomycin Resistance in
Streptomyces sparsogenes
Authors: Lázaro E. , Sanz E. , Remacha M. , Ballesta J. P. G. 
Published: October 2002

ABSTRACT

The antitumor antibiotic sparsomycin, produced by
Streptomyces sparsogenes,
is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive
Streptomyces lividans
with an
S. sparsogenes
library. Resistance was linked to the presence of a plasmid containing an
S. sparsogenes
5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to
srd
, an open reading frame of 1,800 nucleotides. Ribosomes from
S. sparsogenes
and the
S. lividans
-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.
Consumption of Imipenem Correlates with β-Lactam Resistance in
Pseudomonas aeruginosa
Authors: Lepper Philipp M. , Grusa Eberhard , Reichl Helga , Högel Josef , Trautmann Matthias 
Published: October 2002

ABSTRACT

It is generally assumed that the antibiotic prescription policy of a hospital has a significant impact on bacterial resistance rates; however, few studies are available to support this concept with valid statistical data. During a 3-year period from 1997 to 2000, we monitored the consumption of β-lactam and other antibiotics with known activity against
Pseudomonas aeruginosa
in a 600-bed community hospital. Monthly isolations of
P. aeruginosa
were assessed, and resistance rates were recorded. Partial correlation coefficients between consumption and resistance rates were determined, taking into account possible associations with other variables such as seasonal effects and transfers from other hospitals. A total of 30 ± 7 novel
P. aeruginosa
strains per month were isolated without epidemic clustering. Prescriptions of imipenem varied significantly during the study period, while prescriptions of other antipseudomonal agents were stable, with the exception of an increase in piperacillin-tazobactam prescriptions. Rates of resistance of
P. aeruginosa
to the antimicrobial agents used showed a time course similar to figures for imipenem consumption. Monthly rates of resistance to imipenem (partial correlation coefficient [cc], 0.63), piperacillin-tazobactam (cc, 0.57), and ceftazidime (cc, 0.56) were significantly associated with imipenem prescription rates in the same or the preceding month, while consumption of ceftazidime or piperacillin-tazobactam had no apparent association with resistance. Among the variables investigated, imipenem consumption was identified as the major factor associated with both carbapenem and β-lactam resistance in endemic
P. aeruginosa
. Periods of extensive imipenem use were associated with significant increases in resistance. Our data support the concept that a written antibiotic policy which balances the use of various antibiotic classes may help to avoid disturbances of a hospital's microbial sensitivity patterns.
Human Immunodeficiency Virus Type 1 Genotypic and Pharmacokinetic Determinants of the Virological Response to Lopinavir-Ritonavir-Containing Therapy in Protease Inhibitor-Experienced Patients
Authors: Masquelier Bernard , Breilh Dominique , Neau Didier , Lawson-Ayayi Sylvie , Lavignolle Valérie , Ragnaud Jean-Marie , Dupon Michel , Morlat Philippe , Dabis F. , Fleury H. 
Published: October 2002

ABSTRACT

The response to regimens including lopinavir-ritonavir (LPV/r) in patients who have received multiple protease (PR) inhibitors (PI) can be analyzed in terms of human immunodeficiency virus type 1 (HIV-1) genotypic and pharmacokinetic (pK) determinants. We studied these factors and the evolution of HIV-1 resistance in response to LPV/r in a prospective study of patients receiving LPV/r under a temporary authorization in Bordeaux, France. HIV-1 PR and reverse transcriptase sequences were determined at baseline LPV/r for all the patients and at month 3 (M3) and M6 in the absence of response to treatment. pK measurements were determined at M1 and M3. Virological failure (VF) was defined as a plasma viral load ≥400 copies/ml at M3. A multivariate analysis of the predictors of VF, including clinical and biological characteristics and the treatment history of the patients, was performed. The PR gene sequence at M0, including individual mutations or a previously defined LPV mutation score (D. J. Kempf, J. D. Isaacson, M. S. King, S. C. Brun, Y. Xu, K. Real, B. M. Bernstein, A. J. Japour, E. Sun, and R. A. Rode, J. Virol.
75:
7262-7269, 2001), and the individual exposure to LPV were also included covariates. Sixty-eight patients were enrolled. Thirty-four percent had a virological response at M3. An LPV mutation score of >5 mutations, the presence of the PR I54V mutation at baseline, a high number of previous PIs, prior therapy with ritonavir or indinavir, absence of coprescription of efavirenz, and a lower exposure to LPV or lower LPV trough concentrations were independently associated with VF on LPV/r. Additional PI resistance mutations, including primary mutation I50V, could be selected in patients failing on LPV/r. Genotypic and pK parameters should be used to optimize the virological response to LPV/r in PI-experienced patients and to avoid further viral evolution.
Application of Real-Time PCR for Determination of Antiviral Drug Susceptibility of Herpes Simplex Virus
Authors: Stránská Růžena , van Loon Anton M. , Polman Merjo , Schuurman Rob 
Published: October 2002

ABSTRACT

A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 well-characterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (
r
= 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory.
Occurrence of Antibiotic Resistance Gene Cassettes
aac
(
6
′)
-Ib
,
dfrA5
,
dfrA12
, and
ereA2
in Class I Integrons in Non-O1, Non-O139
Vibrio cholerae
Strains in India
Authors: Thungapathra M. , Amita  , Sinha Kislay K. , Chaudhuri Saumya Ray , Garg Pallavi , Ramamurthy Thandavarayan , Nair Gopinath Balakrish , Ghosh Amit 
Published: October 2002

ABSTRACT

Molecular mechanisms of multidrug resistance in
Vibrio cholerae
belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were
dfrA1
,
dfrA15
,
dfrA5
, and
dfrA12
for trimethoprim;
aac
(
6
′)
-Ib
for amikacin and tobramycin;
aadA1
and
aadA2
for streptomycin and spectinomycin; and
ereA2
for erythromycin resistance. To our knowledge, this is the first report of the presence of
dfrA5
,
dfrA12
,
aac
(
6
′)
-Ib,
and
ereA2
cassettes in class I integrons of
V. cholerae
. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.