bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity
Authors: Gaillard Richard K. , Barnard Jennifer , Lopez Vincent , Hodges Paula , Bourne Eric , Johnson Lance , Allen Marchelle I. , Condreay Patrick , Miller Wayne H. , Condreay Lynn D. 
Published: July 2002

ABSTRACT

Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent

K
m

values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased

K
i

values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent

K
m

values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 μM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased

K
m

values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.
Nonparametric Population Pharmacokinetic Analysis of Amikacin in Neonates, Infants, and Children
Authors: Tréluyer J. M. , Merlé Y. , Tonnelier S. , Rey E. , Pons G. 
Published: July 2002

ABSTRACTThe therapeutic and toxic effects of amikacin are known to depend on its concentration in plasma, but the pharmacokinetics of this drug in neonates, infants, and children and the influences of clinical and biological variables have been only partially assessed. Therapeutic drug monitoring data collected from 155 patients (49 neonates, 77 infants, and 29 children) receiving amikacin were analyzed by a nonparametric population-based approach, the nonparametric maximum-likelihood method. We assessed the effects of gestational and postnatal age, weight, Apgar score, and plasma creatinine and urea concentrations on pharmacokinetic parameters. There is no specific formulation of amikacin for neonates and infants. We therefore used an error model to account for errors due to dilution during preparation of the infusion. The covariates that reduced the variance of clearance from plasma and the volume of distribution by more than 10% were postnatal age (43 and 28%, respectively) and body weight (30.4 and 17.4%, respectively). The expected reduction of clearance was about 10% for the plasma creatinine concentration. The other covariates studied (Apgar scores, plasma urea concentration, gestational age, sex) were found to have little effect. Simulations showed that a smaller percentage of patients had a maximum concentration in plasma/MIC ratio greater than 8 with a regimen of 7.5 mg/kg of body weight twice daily than with a regimen of 15 mg/kg once a day for MICs of 1 to 8 mg/liter.
High Prevalence of KatG Ser315Thr Substitution among Isoniazid-Resistant
Mycobacterium tuberculosis
Clinical Isolates from Northwestern Russia, 1996 to 2001
Authors: Mokrousov Igor , Narvskaya Olga , Otten Tatiana , Limeschenko Elena , Steklova Lidia , Vyshnevskiy Boris 
Published: July 2002

ABSTRACT

A total of 204 isoniazid (INH)-resistant strains of
Mycobacterium tuberculosis
isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses
Hap
II cleavage of an amplified fragment of the
katG
gene to detect the transversion 315AGC→ACC (Ser→Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the
katG
S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS
6110
-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The
katG
S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the
katG
315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.
Bactericidal Activity of Increasing Daily and Weekly Doses of Moxifloxacin in Murine Tuberculosis
Authors: Yoshimatsu Tetsuyuki , Nuermberger Eric , Tyagi Sandeep , Chaisson Richard , Bishai William , Grosset Jacques 
Published: July 2002

ABSTRACT

Moxifloxacin (MXF) is a new 8-methoxyquinolone with potent activity against
Mycobacterium tuberculosis
and a half-life of 9 to 12 h in humans. Previous in vivo studies using daily doses of 100 mg/kg of body weight have demonstrated bactericidal activity comparable to that of isoniazid (INH) in a murine model of tuberculosis (TB). Recent pharmacokinetic data suggest that MXF may have been underadministered in these studies and that a 400-mg/kg dose in mice better approximates the area under the concentration-time curve obtained in humans after a 400-mg oral dose. Therefore, the bactericidal activity of MXF in doses up to 400 mg/kg given daily or weekly for 28 days was assessed in mice infected intravenously with 5 × 10
6
CFU of
M. tuberculosis
. INH was used as a positive control. After 3 days of daily therapy, the CFU counts from splenic homogenates for mice treated with MXF in doses of 100 to 400 mg/kg/day were lower than those from pretreatment controls. No significant differences in CFU counts were seen when mice receiving INH or MXF at 50 mg/kg/day were compared to pretreatment controls. After 28 days of therapy, dose-dependent reductions in CFU counts in splenic homogenates were seen for daily MXF therapy. The maximum bactericidal effect was seen with daily doses of 400 mg/kg, which resulted in a reduction in CFU counts of 1 log
10
greater than that with INH treatment, although the difference was not statistically significant. CFU counts from lung homogenates after 28 days of therapy were significantly lower in all treatment groups than in untreated controls. The weekly administration of MXF in doses ranging from 50 to 400 mg/kg resulted in no significant bactericidal activity. Mice receiving daily MXF doses of 200 and 400 mg/kg/day failed to gain weight and appeared ill after 28 days of therapy, findings suggestive of drug toxicity. In conclusion, MXF has dose-dependent bactericidal activity against
M. tuberculosis
in the mouse when given in doses up to 400 mg/kg, where its pharmacokinetic profile better approximates that of standard human dosages. Combination regimens which take advantage of the enhanced pharmacodynamic profile of MXF at these doses have the potential to shorten the course of antituberculous therapy or allow more intermittent (i.e., once-weekly) therapy and should be evaluated in the mouse model of TB.
Chromosome-Encoded Class D β-Lactamase OXA-23 in
Proteus mirabilis
Authors: Bonnet R. , Marchandin H. , Chanal C. , Sirot D. , Labia R. , De Champs C. , Jumas-Bilak E. , Sirot J. 
Published: July 2002

ABSTRACT

Ten nonrepetitive
Proteus mirabilis
isolates, which were collected over 4 years (1996 to 1999) at the teaching hospital of Clermont-Ferrand, France, produced class D carbapenemase OXA-23. MICs of imipenem were 0.25 to 0.5 μg/ml for these clinical isolates. Molecular typing revealed that the 10
P. mirabilis
isolates originated from the same clonal strain. Hybridization of I-
Ceu
I-generated chromosome fragments with a
bla
OXA-23
probe showed that the gene was chromosome encoded in the
P. mirabilis
strain.
Inactivation of
Escherichia coli
and
Listeria innocua
in Milk by Combined Treatment with High Hydrostatic Pressure and the Lactoperoxidase System
Authors: García-Graells Cristina , Valckx Caroline , Michiels Chris W. 
Published: July 2002

ABSTRACT

We have studied inactivation of four strains each of
Escherichia coli
and
Listeria innocua
in milk by the combined use of high hydrostatic pressure and the lactoperoxidase-thiocyanate-hydrogen peroxide system as a potential mild food preservation method. The lactoperoxidase system alone exerted a bacteriostatic effect on both species for at least 24 h at room temperature, but none of the strains was inactivated. Upon high-pressure treatment in the presence of the lactoperoxidase system, different results were obtained for
E. coli
and
L. innocua
. For none of the
E. coli
strains did the lactoperoxidase system increase the inactivation compared to a treatment with high pressure alone. However, a strong synergistic interaction of both treatments was observed for
L. innocua
. Inactivation exceeding 7 decades was achieved for all strains with a mild treatment (400 MPa, 15 min, 20°C), which in the absence of the lactoperoxidase system caused only 2 to 5 decades of inactivation depending on the strain. Milk as a substrate was found to have a considerable effect protecting
E. coli
and
L. innocua
against pressure inactivation and reducing the effectiveness of the lactoperoxidase system under pressure on
L. innocua
. Time course experiments showed that
L. innocua
counts continued to decrease in the first hours after pressure treatment in the presence of the lactoperoxidase system.
E. coli
counts remained constant for at least 24 h, except after treatment at the highest pressure level (600 MPa, 15 min, 20°C), in which case, in the presence of the lactoperoxidase system, a transient decrease was observed, indicating sublethal injury rather than true inactivation.
Zoospore Homing and Infection Events: Effects of the Biocontrol Bacterium
Burkholderia cepacia
AMMDR1 on Two Oomycete Pathogens of Pea (
Pisum sativum
L.)
Authors: Heungens K. , Parke J. L. 
Published: July 2002

ABSTRACT

Burkholderia cepacia
AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of
B. cepacia
AMMDR1 on
Pythium aphanidermatum
and
Aphanomyces euteiches
zoospore homing events and on infection of pea seeds or roots. In vitro,
B. cepacia
AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes.
B. cepacia
AMMDR1 also reduced the attractiveness of seed exudates to
Pythium
zoospores to nondetectable levels. However, when present at high levels on seeds,
B. cepacia
AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied
B. cepacia
AMMDR1 dramatically reduced the incidence of infection by
Pythium
zoospores in situ compared with an antibiosis-deficient Tn
5
mutant strain. This mutant strain also decreased
Pythium
infection incidence to some extent, but only when the pathogen inoculum potential was low.
B. cepacia
AMMDR1 did not affect attraction of
Aphanomyces
zoospores or Aphanomyces root rot incidence. These results suggest that
B. cepacia
AMMDR1 controls
P. aphanidermatum
largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of
Aphanomyces
and
Pythium
are discussed in relation to differences in the ecology of the two pathogens.
Degradation ofn-Hexadecane and Its Metabolites byPseudomonas aeruginosaunder Microaerobic and Anaerobic Denitrifying Conditions
Authors: Chayabutra Chawala , Ju Lu-Kwang 
Published: July 2002

ABSTRACTA strategy for sequential hydrocarbon bioremediation is proposed. The initial O2-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examiningPseudomonas aeruginosafermentation. The effects of dissolved oxygen (DO) concentration onn-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O2-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO2−-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO2−-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO3−-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation byP. aeruginosawas then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N2). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.
Survival of
Bifidobacterium longum
Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution
Authors: Lee Ki-Yong , Heo Tae-Ryeon 
Published: July 2002

ABSTRACT

Bifidobacterium longum
KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.
Selective Accumulation May Account for Shellfish-Associated Viral Illness
Authors: Burkhardt William , Calci Kevin R. 
Published: July 2002

ABSTRACT

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (
Crassostrea virginica
) to accumulate indicator microorganisms (i.e., fecal coliforms,
Escherichia coli
,
Clostridium perfringens
, and F
+
coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F
+
coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.
Characterization of a Novel β-Galactosidase from
Bifidobacterium adolescentis
DSM 20083 Active towards Transgalactooligosaccharides
Authors: Van Laere Katrien M. J. , Abee Tjakko , Schols Henk A. , Beldman Gerrit , Voragen Alphons G. J. 
Published: July 2002

ABSTRACT

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of
Bifidobacterium adolescentis
DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown
B. adolescentis
by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highest
V
max
values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by
B. adolescentis
.
Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by
Azotobacter vinelandii
Authors: Cornish Anthony S. , Page William J. 
Published: July 2002

ABSTRACT

Both molybdate and iron are metals that are required by the obligately aerobic organism
Azotobacter vinelandii
to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of
A. vinelandii
siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe
3+
. Thus, competition between Fe
3+
and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn
2+
, and Mn
2+
. We found that all five of these metal ions partially inhibited uptake of
55
Fe-protochelin and
55
Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.
Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from
Morganella morganii
Authors: Mihara Yasuhiro , Utagawa Takashi , Yamada Hideaki , Asano Yasuhisa 
Published: July 2002

ABSTRACT

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The
Morganella morganii
gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the
M. morganii
PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into
Escherichia coli
, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With
E. coli
overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased

K
m

value for inosine was responsible for the increased productivity.
Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture
Authors: Sutherland Tara D. , Horne Irene , Lacey Michael J. , Harcourt Rebecca L. , Russell Robyn J. , Oakeshott John G. 
Published: July 2002

ABSTRACTAn endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored thealphaisomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.