bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ethanol Vapor Is an Efficient Inducer of the alc Gene Expression System in Model and Crop Plant Species
Authors: Sweetman Justin P. , Chu Chengcai , Qu Nan , Greenland Andrew J. , Sonnewald Uwe , Jepson Ian 
Published: July 2002

Abstract
We have demonstrated that low concentrations of ethanol vapor efficiently induce the alc gene expression system in tobacco (Nicotiana tabacum cv Samsun NN), potato (Solanum tuberosum cv Solara), and oilseed rape (Brassica napus cv Westar). For many situations, this may be the preferred method of induction because it avoids direct application of comparatively high concentrations of an ethanol solution. Although induction was seen with less than 0.4 μm ethanol vapor, maximal induction of the chloramphenicol acetyl transferase gene was achieved after 48 h in leaves of tobacco plants enclosed with 4.5 μm ethanol vapor. In the absence of ethanol, there is no detectable gene expression. Treatment of potato tubers with ethanol vapor results in uniform β-glucoronidase (GUS) expression. Vapor treatment of a single oilseed rape leaf resulted in induction of GUS in the treated leaf only and 14C-ethanol labeling in tobacco confirmed that the inducer was not translocated. In contrast, enclosure of the roots, aerial parts, or whole plant with ethanol vapor resulted in induction of GUS activity in leaves and roots. The data reported here broaden the utility of the alc system for research and crop biotechnology.
Effect of Yeast CTA1 Gene Expression on Response of Tobacco Plants to Tobacco Mosaic Virus Infection
Authors: Talarczyk Andrzej , Krzymowska Magdalena , Borucki Wojciech , Hennig Jacek 
Published: July 2002

Abstract
The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase geneCTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3′-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H2O2) in the TMV infected tissue. Presence of H2O2 could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H2O2 and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.
Plant Nuclei Can Contain Extensive Grooves and Invaginations [W]
Authors: Collings David A. , Carter Crystal N. , Rink Jochen C. , Scott Amie C. , Wyatt Sarah E. , Allen Nina Strömgren 
Published: July 2002

AbstractPlant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.
Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase
Authors: Schneider Benoit , Norda Ameli , Karlsson Anna , Veron Michel , Deville‐Bonne Dominique 
Published: July 2002

AbstractNucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000‐fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.
Insights into the alkaline transformation of ferricytochrome c from 1H NMR studies in 30% acetonitrile–water
Authors: Sivakolundu Sivashankar G. , Mabrouk Patricia Ann 
Published: July 2002

AbstractRecently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous–nonaqueous media, resulting in the formation of a mixture of alkaline‐like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One‐dimensional (1D) and two‐dimensional (2D) 1H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline‐like forms in 30% acetonitrile–water solution. A native‐like (M80‐ligated) III* form, two lysine‐ligated forms (IVa* and IVb*), and a hydroxide‐ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D 1H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20°C and 35°C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8‐ and 3‐methyl heme protons were made for the hydroxide‐ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.
Peptide‐plane flipping in proteins
Authors: Hayward Steven 
Published: July 2002

AbstractA peptide‐plane flip is a large‐scale rotation of the peptide plane that takes the ϕ,ψ angles at residues i and i + 1 to different structural regions in the Ramachandran plot with a comparatively small effect on the relative orientation of their side chains. This phenomenon, which is expected to play an important role during the early stages of protein folding, has been investigated using 76 proteins for which two high‐resolution X‐ray conformations are available. Peptide‐plane flips are identified by looking for those cases where changes in |ψi| + |ϕi + 1| are large (>200°), but changes in |ψi + ϕi + 1| are comparatively small (<50°). Of a total of 23 cases, the most common peptide‐plane flip was identified to be the type I to type II β‐turn interconversion. Although individually rarer, there are many other types of flips that are collectively more common. Given the four main accessible regions αR, αL, β and ε, identified from the ϕ,ψ distribution corresponding to non‐hydrogen‐bonded peptide planes, 32 main types of peptide‐plane flip are identified. Only 8 of these are “passive,” in that they require only relatively minor adjustments in the orientation of adjacent peptide planes. Of these, only the type I to type II β‐turn interconversion, denoted, β(i) + αL(i + 1) ― αR(i) + αR(i + 1), and the rarer αR(i) + αL(i + 1) ― β(i) + αR(i + 1), do not involve the ε region. “Active” peptide‐plane flips affect the orientation of adjacent peptide planes. The flip, αL(i) + αL(i + 1) ― β(i) + β(i + 1), of which one example was found, shows how concerted peptide‐plane flips can convert the αL structure to the β structure without affecting the relative orientations of the side chains.
Chaperone‐like activity of peptidyl‐prolyl cis‐trans isomerase during creatine kinase refolding
Authors: Ou Wen‐Bin , Luo Wei , Park Yong‐Doo , Zhou Hai‐Meng 
Published: July 2002

AbstractPorcine kidney 18 kD peptidyl‐prolyl cis‐trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 μM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration‐dependent manner. PPIase exhibited chaperone‐like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.
Modeling of denatured state for calculation of the electrostatic contribution to protein stability
Authors: Kundrotas Petras J. , Karshikoff Andrey 
Published: July 2002

AbstractExisting models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this article, a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi‐random distribution restricted by the protein sequence. The model was applied for the calculations of electrostatic interactions in two proteins, barnase and N‐terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, ΔG(pH), reproduces the experimental data better than the commonly used null approximation (NA). It was shown that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pairwise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.
Inhibition of Nanobacteria by Antimicrobial Drugs as Measured by a Modified Microdilution Method
Authors: Çíftçíoglu N. , Miller-Hjelle M. A. , Hjelle J. T. , Kajander E. O. 
Published: July 2002

ABSTRACT
Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.
Molecular Characterization of Recombinant
Pneumocystis carinii
Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine
Authors: Van Dross Rukiyah T. , Sanders Marilyn M. 
Published: July 2002

ABSTRACT

The
Pneumocystis carinii
topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The
P. carinii
topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg
2+
requirement. Cleavage assays reveal that both the human and
P. carinii
enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4′,6′-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that
P. carinii
topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme,
P. carinii
topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of
P. carinii
and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with
P. carinii
topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.
Experimental Acute Otitis Media Due to NontypeableHaemophilus influenzae: Comparison of High and Low Azithromycin Doses with Placebo
Authors: Babl Franz E. , Pelton Stephen I. , Li Zhong 
Published: July 2002

ABSTRACTTreatment of acute otitis media (AOM) with azithromycin results in apparent clinical success, but tympanocentesis performed 4 to 6 days after initiation of therapy in children with nontypeableHaemophilus influenzae(NTHI) recovered from initial middle ear cultures demonstrates persistence of infection in more than 50% of episodes. We sought to determine the effect of azithromycin at different doses on the density of middle ear infection due to NTHI to provide additional understanding of this dichotomy between clinical and microbiologic outcome measures in AOM. In a chinchilla model of experimental otitis media (EOM), animals treated with placebo were compared to animals receiving a single daily dose 30 or 120 mg of azithromycin per kg of body weight per day for 5 days. Microbiologic outcome was assessed by obtaining quantitative cultures from the middle ear during a 5-day course and for 1 week following therapy. Azithromycin concentrations were measured to ascertain whether a concentration-dependent effect was present. Azithromycin at 30 and 120 mg/kg/day demonstrated a dose-dependent effect on the quantitative assessment of middle ear infection due to NTHI. A 30-mg/kg dose of azithromycin daily resulted in levels in serum and areas under the serum concentration-time curve at 24 h comparable to published data obtained with children given azithromycin at 5 to 10 mg/kg in multiday regimens. Increased doses of azithromycin (120 mg/kg) achieved 2.5- to 4-fold-higher levels in serum and 3- to 6-fold-higher total levels and levels in extracellular middle ear fluid as well as more rapid reduction in bacterial density and a greater proportion of middle ears with complete sterilization than either placebo or the 30-mg/kg/day regimen.
Comparative Stability Studies of Antipseudomonal β-Lactams for Potential Administration through Portable Elastomeric Pumps (Home Therapy for Cystic Fibrosis Patients) and Motor-Operated Syringes (Intensive Care Units)
Authors: Viaene Eric , Chanteux Hugues , Servais Hélène , Mingeot-Leclercq Marie-Paule , Tulkens Paul M. 
Published: July 2002

ABSTRACTThe stability of antipseudomonal β-lactams in concentrated solutions was examined in view of their potential administration by continuous infusion with external pumps (for intensive care patients) or with portable pumps carried under clothing (for cystic fibrosis patients). Aztreonam (100 g/liter), piperacillin (128 g/liter, with tazobactam), and azlocillin (128 g/liter) remained 90% stable for up to more than 24 h at 37°C (mezlocillin [128 g/liter] was stable at 25°C but not at 37°C). Ceftazidime (120 g/liter), cefpirome (32 g/liter), and cefepime (50 g/liter) remained 90% stable for up to 24, 23.7, and 20.5 h at 25°C but only for 8, 7.25, and 13 h at 37°C, respectively. The control of temperature therefore appears to be critical for all three cephalosporins that cannot be recommended for use in portable pumps carried under clothes for prolonged periods for reasons of stability. Cefpirome and cefepime solutions developed an important color change (from light yellow to dark red) upon exposure when stored at 30°C or higher. Degradation of ceftazidime was accompanied by the liberation of pyridine which, at 37°C, was in excess of what is allowed by the U.S. Pharmacopeia, i.e., 1.1 mg/liter, after 8 and 12 h for drug concentrations of 12 and 8.3%, respectively. Imipenem and meropenem are too unstable (10% degradation at 25°C after 3.5 and 5.15 h, respectively) to be recommended for use by continuous infusion. Faropenem, examined in comparison with imipenem and meropenem, proved as stable as aztreonam or piperacillin.
Stress-Based Identification and Classification of Antibacterial Agents: Second-Generation
Escherichia coli
Reporter Strains and Optimization of Detection
Authors: Shapiro Elyse , Baneyx François 
Published: July 2002

ABSTRACT

Escherichia coli
strains bearing single-copy fusions between the
lacZ
reporter gene and the
cspA
,
ibp
, or P3
rpoH
stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A. A. Bianchi and F. Baneyx, Appl. Environ. Microbiol.
65:
5023-5027, 1999). Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible
sulA
promoter and
lacZ
is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the
tolC
gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.
PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for
Streptococcus pneumoniae
Authors: Belanger Aimee E. , Lai Angel , Brackman Marcia A. , LeBlanc Donald J. 
Published: July 2002

ABSTRACT

Described here are the development and validation of a novel approach to identify genes encoding drug targets in
Streptococcus pneumoniae
. The method relies on the use of an ordered genomic library composed of PCR amplicons that were generated under error-prone conditions so as to introduce random mutations into the DNA. Since some of the mutations occur in drug target-encoding genes and subsequently affect the binding of the drug to its respective cellular target, amplicons containing drug targets can be identified as those producing drug-resistant colonies when transformed into
S. pneumoniae
. Examination of the genetic content of the amplicon giving resistance coupled with bioinformatics and additional genetic approaches could be used to rapidly identify candidate drug target genes. The utility of this approach was verified by using a number of known antibiotics. For drugs with single protein targets, amplicons were identified that rendered
S. pneumoniae
drug resistant. Assessment of amplicon composition revealed that each of the relevant amplicons contained the gene encoding the known target for the particular drug tested. Fusidic acid-resistant mutants that resulted from the transformation of
S. pneumoniae
with amplicons containing
fusA
were further characterized by sequence analysis. A single mutation was found to occur in a region of the
S. pneumoniae
elongation factor G protein that is analogous to that already implicated in other bacteria as being associated with fusidic acid resistance. Thus, in addition to facilitating the identification of genes encoding drug targets, this method could provide strains that aid future mechanistic studies.