bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Repair of Mutations in Plant Cell-Free Extracts Directed by Specific Chimeric Oligonucleotides
Authors: Rice Michael C. , May Gregory D. , Kipp Peter B. , Parekh Hetal , Kmiec Eric B. 
Published: July 2002

Abstract
Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.
Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry
Authors: Wang Lintao , Lane Leslie C. , Smith David L. 
Published: July 2002

AbstractAmide hydrogen exchange and mass spectrometry have been used to study the pH‐induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D2O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi‐threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo‐electron microscopy and image reconstruction. Structural changes in the extended N‐ and C‐terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids.
Backbone dynamics of the regulatory domain of calcium vector protein, studied by 15N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker
Authors: Théret Isabelle , Cox Jos A. , Mispelter Joel , Craescu Constantin T. 
Published: July 2002

AbstractCaVP is a calcium‐binding protein from amphioxus. It has a modular composition with two domains, but only the two EF‐hand motifs localized in the C‐terminal domain are functional. We recently determined the solution structure of this regulatory half (C‐CaVP) in the Ca2+‐saturated form and characterized the stepwise ion binding. This paper reports the 15N nuclear relaxation rates of the Ca2+‐saturated C‐CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide HN‐N vectors. Fitting the spectral density values at eight frequencies by a model‐free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF‐hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca2+ binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C‐CaVP characterized here is discussed in relation with other well‐characterized Ca2+‐binding proteins.
Pressure Conversion Constants for Magnetron Ionization Gauges
Authors: Pelz David , Newton George 
Published: July 2002

The gas calibrations of the Explorer 32 satellite atmospheric density experiment provided comparisons of the operation of the following five kinds of ionization gauges: (1) flight type, glass enveloped, cold cathode, magnetron gauge (model NRC-528), (2) flight type, metal enveloped, cold cathode, magnetron gauge (model GCA-R5), (3) commercially available, glass enveloped, “Redhead gauge” (model NRC-552), (4) flight type, metal enveloped, warm cathode, magnetron gauge (WCM), and (5) commercially available, metal enveloped, Penning-type gauge (model GE-22GT210).
The nitrogen pressure responses of these gauges are compared in terms of the conversion constant (C). C is the ratio of the cathode current to the pressure and is related to the number of ions produced per neutral particle in the gauge. The 552 and R5 gauges exhibit values of C which axe maximum between 10−10 and 10−9 Torr and decrease at higher pressures. In contrast to this behavior the 528 and 22GT210 gauges exhibit primarily increasing C values with pressure, and the C values of the warm cathode magnetron are constant at low pressures. Experimental results which can be interpreted in terms of the behavior of the negative space charge in the gauges are briefly discussed. A high sensitivity of the R5 gauge response characteristics to anode voltage is demonstrated in terms of C values, oscillation regions, and discontinuities.
Fluoroquinolone Transport by Human Monocytes: Characterization and Comparison to Other Cells of Myeloid Lineage
Authors: Bounds Steven J. , Nakkula Robin , Walters John D. 
Published: July 2002

ABSTRACT

Human monocytes transport and accumulate ciprofloxacin and other fluoroquinolones. Although little is known about the mechanisms of transport, we expected monocytes to be similar to other cells of myeloid lineage. In the present study, monocyte fluoroquinolone transport was characterized and compared to the corresponding transport pathways of human polymorphonuclear leukocytes (PMNs) and HL-60 cells. Ciprofloxacin transport by monocytes was saturable, temperature dependent, sodium independent, and relatively insensitive to pH. Quiescent monocytes transported ciprofloxacin with a

K
m

of 171 μg/ml and a
V
max
of 32.7 ng/min/10
6
cells. Adenine competitively inhibited ciprofloxacin transport by quiescent monocytes (

K
i

= 3.8 mM), but nucleosides had no significant inhibitory effect. In all of these respects, transport by monocytes was similar to that observed for quiescent PMNs and immature HL-60 cells. Unlike PMNs, however, monocytes and immature HL-60 cells did not exhibit dramatically enhanced ciprofloxacin transport when activated by phorbol myristate acetate (PMA). Consistent with this finding, HL-60 cells committed to granulocytic differentiation exhibited a significant component of PMA-inducible ciprofloxacin transport activity, while HL-60 cells committed to monocytic differentiation did not. In PMNs, the PMA-inducible component of transport appeared to be mobilized from a granule compartment, since its activity could be modulated by agents that enhance or inhibit stimulated degranulation. Thus, quiescent monocytes, PMNs, and HL-60 cells take up ciprofloxacin via similar energy-dependent transport mechanisms. Unlike granulocytes, monocytes do not express a second, higher-affinity pathway for ciprofloxacin accumulation when they are activated by PMA.
Toward Antiviral Strategies That Resist Viral Escape
Authors: Endy Drew , Yin John 
Published: July 2002

ABSTRACT
We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance.
Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin in
Staphylococcus aureus
Authors: Ince Dilek , Hooper David C. 
Published: July 2002

ABSTRACT

Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two- to fourfold more active against wild-type
Staphylococcus aureus
ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single
grlBA
mutations caused two- to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in
gyrA
and
grlA
or
grlB
caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100- to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.
Differential Antifungal Activity of Isomeric Forms of Nystatin
Authors: Ostrosky-Zeichner Luis , Bazemore Scott , Paetznick Victor L. , Rodriguez Jose R. , Chen Enuo , Wallace Tom , Cossum Paul , Rex John H. 
Published: July 2002

ABSTRACT

When nystatin is placed in RPMI and other biological fluids, there is loss of pure nystatin, with the development of two distinguishable chromatographic peaks, 1 and 2. Peak 1 appears identical to commercially prepared nystatin. By nuclear magnetic resonance (NMR) and mass spectral analysis, peak 2 appears to be an isomer of peak 1. The isomers are quantitatively and fully interconvertible. Formation of peak 2 is accelerated at a pH of >7.0 and ultimately reaches a near 55:45 (peak 1/peak 2 ratio) mixture. We sought to determine the relative activities of peaks 1 and 2 against
Candida
spp. Peak 2 consistently showed higher MICs when it was the predominant form during the experiment. Time-kill analyses showed that peak 2 required ≥8× the concentration of peak 1 to produce a modest and delayed killing effect, which was never of the same magnitude as that produced by peak 1. In both types of assays, the activity of peak 2 corresponded with intra-assay formation of peak 1. Both MIC measurements and time-kill analysis suggest that peak 2 has considerably less activity, if any at all, against
Candida
spp. Peak 2 may serve as a reservoir for peak 1.
Multiple-Dose Pharmacokinetics and Tolerability of Gemifloxacin Administered Orally to Healthy Volunteers
Authors: Allen Ann , Bygate Elizabeth , Vousden Marika , Oliver Stuart , Johnson Martin , Ward Christopher , Cheon Ae-Jin , Choo Youn Sung , Kim In-Chull 
Published: July 2002

ABSTRACT

Gemifloxacin mesylate (SB-265805-S, LB-20304a) is a potent, novel fluoroquinolone agent with a broad spectrum of antibacterial activity. The pharmacokinetics and tolerability of oral gemifloxacin were characterized in two parallel group studies in healthy male volunteers after doses of 160, 320, 480, and 640 mg once daily for 7 days. Multiple serum or plasma and urine samples were collected on days 1 and 7 and were analyzed for gemifloxacin by high-performance liquid chromatography (HPLC)–fluorescence (study 1) or HPLC-mass spectrometry (study 2). Safety assessments included vital signs, 12-lead electrocardiogram (ECG) readings, hematology, clinical chemistry, urinalysis, and adverse experience monitoring. Gemifloxacin was rapidly absorbed, with a time to maximum concentration of approximately 1 h after dosing followed by a biexponential decline in concentration. Generally, maximum concentration and area under the concentration-time curve (AUC) increased linearly with dose after either single or repeat doses. Mean ± standard deviation values of AUC
0–τ
on day 7 were 4.92 ± 1.08, 9.06 ± 2.20, 12.2 ± 3.69, and 20.1 ± 3.67 μg·h/ml following 160-, 320-, 480-, and 640-mg doses, respectively. The terminal-phase half-life was approximately 7 to 8 h, independent of dose, and was similar following single and repeated administrations. There was minimal accumulation of gemifloxacin after multiple dosing. Approximately 20 to 30% of the administered dose was excreted unchanged in the urine. The renal clearance was 160 ml/min on average after single and multiple doses, which was slightly greater than the accepted glomerular filtration rate (approximately 120 ml/min). These data show that the pharmacokinetics of gemifloxacin are linear and independent of dose. Gemifloxacin was generally well tolerated, although one subject was withdrawn from the study after 6 days at 640 mg for mild, transient elevations of alanine aminotransferase and aspartate aminotransferase not associated with any clinical signs or symptoms. There were no other significant changes in clinical chemistry, hematology or urinalysis parameters, vital signs, or ECG readings. In conclusion, the results of these studies, combined with the antibacterial spectrum and potency, support the further investigation of once-daily administration of gemifloxacin for indications such as respiratory tract and urinary tract infections.
Structural Comparison of Three Types of Staphylococcal Cassette Chromosome
mec
Integrated in the Chromosome in Methicillin-Resistant
Staphylococcus aureus
Authors: Ito Teruyo , Katayama Yuki , Asada Kazumi , Mori Namiko , Tsutsumimoto Kanae , Tiensasitorn Chuntima , Hiramatsu Keiichi 
Published: July 2002

ABSTRACT

The β-lactam resistance gene
mecA
of
Staphylococcus aureus
is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome
mec
(SCC
mec
), identified in the chromosome of a Japanese methicillin-resistant
S. aureus
(MRSA) strain. We now report identification of two additional types of
mecA
-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCC
mec
. However, new elements shared the chromosomal integration site with the SCC
mec
. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCC
mec
: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the
mecA
gene, and the presence of cassette chromosome recombinase (
ccr
) genes responsible for the movements of SCC
mec
. The elements, therefore, were considered to comprise the SCC
mec
family of staphylococcal mobile genetic elements together with the previously identified SCC
mec
. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCC
mec
elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCC
mec
in their chromosome.
Phenotypic Susceptibilities to Tenofovir in a Large Panel of Clinically Derived Human Immunodeficiency Virus Type 1 Isolates
Authors: Harrigan P. R. , Miller M. D. , McKenna P. , Brumme Z. L. , Larder B. A. 
Published: July 2002

ABSTRACTTenofovir is a nucleotide analogue human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor, and its oral prodrug, tenofovir disoproxil fumarate, has recently been approved for the treatment of HIV-1 infection in the United States. The objective of this study was to characterize the in vitro susceptibility profiles of a large panel of clinically derived HIV-1 isolates for tenofovir. The distribution of tenofovir susceptibilities in over 1,000 antiretroviral-naive, HIV-1-infected individuals worldwide was determined using the Virco Antivirogram assay. In addition, phenotypic susceptibilities to tenofovir and other RT inhibitors were determined in a panel of nearly 5,000 recombinant HIV-1 clinical isolates from predominantly treatment-experienced patients analyzed as a part of routine drug resistance testing. Greater than 97.5% of isolates from treatment-naive patients had tenofovir susceptibilities <3-fold above those of the wild-type controls by the Antivirogram. The clinically derived panel of 5,000 samples exhibited a broad range of antiretroviral drug susceptibilities, including 69, 43, and 16% having >10-fold-decreased susceptibilities to at least one, two, and three antiretroviral drug classes, respectively. Greater than 88% of these 5,000 clinical isolates were within the threefold susceptibility range for tenofovir, and >99% exhibited <10-fold-reduced susceptibilities to tenofovir. Decreased susceptibility to tenofovir was not directly associated with resistance to other RT inhibitors;r2values of log-log linear regression plots of susceptibility to tenofovir versus susceptibility to other RT inhibitors were <0.4. The results suggest that the majority of treatment-naive and treatment-experienced individuals harbor HIV that remains within the normal range of tenofovir susceptibilities and may be susceptible to tenofovir disoproxil fumarate therapy.
Method for Measuring Postantifungal Effect inAspergillusSpecies
Authors: Vitale Roxana G. , Mouton Johan W. , Afeltra Javier , Meis Jacques F. G. M. , Verweij Paul E. 
Published: July 2002

ABSTRACTAn in vitro method for determination of postantifungal effect (PAFE) in molds was developed by using three isolates each ofAspergillus fumigatus,A. flavus,A. terreus,A. nidulans, andA. ustus. MICs of amphotericin B and itraconazole were determined by using National Committee for Clinical Laboratory Standards guidelines (M38-P). The inoculum was prepared in RPMI 1640 broth buffered with MOPS (morpholinepropanesulfonic acid) at pH 7.0, and conidia were exposed to amphotericin B and itraconazole at concentrations of 4, 1, and 0.25 times the MIC, each for 4, 2, and 1 h at 37°C. The same procedure was followed for controls with drug-free medium. Following exposure, the conidia were washed three times in saline and the numbers of CFU per milliliter were determined. Exposed and control conidia were then inoculated into microtitration plates and incubated at 37°C for 48 h in a spectrophotometer reader. The optical density (OD) was measured automatically at 10-min intervals, resulting in growth curves. PAFE was quantified by comparing three arbitrary points in the control growth curve, the first increase of OD and the points when 20 and 50% of the maximal growth were reached, with the growth curve of drug-exposed conidia. Amphotericin B induced PAFE inA. fumigatusat four times the MIC after 2 and 4 h of exposure ranging from 1.83 to 6.00 h and 9.33 to 10.80 h, respectively. Significantly shorter PAFEs or lack of PAFE was observed forA. terreus,A. ustus, andA. nidulans. Itraconazole did not induce measurable PAFE in theAspergillusisolates at any concentration or exposure time tested. Further studies are warranted to investigate the implications of PAFE in relation to clinical efficacy and dosing frequency.
Effects of Synthetic Siderophores on Proliferation ofPlasmodium falciparumin Infected Human Erythrocytes
Authors: Rotheneder Andrea , Fritsche Gernot , Heinisch Lothar , Möllmann Ute , Heggemann Susanne , Larcher Clara , Weiss Günter 
Published: July 2002

ABSTRACTBecause iron is essential forPlasmodium falciparum, we investigated the in vitro potential of various synthetic siderophores to killP. falciparumin infected human erythrocytes. The substances with the most promising profile, i.e., low 50% lethal doses for plasmodia and minimum toxicity towards mammalian cells, were siderophores with an acylated monocatecholate or a triscatecholate as substituent.
Development of Real-Time PCR Assays for Rapid Detection of
Pfiesteria piscicida
and Related Dinoflagellates
Authors: Bowers Holly A. , Tengs Torstein , Glasgow Howard B. , Burkholder JoAnn M. , Rublee Parke A. , Oldach David W. 
Published: July 2002

ABSTRACT

Pfiesteria
complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far,
Pfiesteria piscicida
and
P. shumwayae
(sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized
Pfiesteria
species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.