bluuebunny/crossref_metadata_2025_split huggingface dataset:
P-Glycoprotein-Mediated Transport of Itraconazole across the Blood-Brain Barrier
Authors: Miyama Tetsuo , Takanaga Hitomi , Matsuo Hirotami , Yamano Katsuhiro , Yamamoto Koujirou , Iga Tatsuji , Naito Mikihiko , Tsuruo Takashi , Ishizuka Hitoshi , Kawahara Yukinori , Sawada Yasufumi 
Published: October 2018

ABSTRACTThe mechanism for the accumulation of itraconazole (ITZ) in its elimination from the brain was studied in rats and mice. The concentration of ITZ in liver tissue declined in parallel with the plasma ITZ concentration until 24 h after intravenous injection of the drug (half-life, 5 h); however, the ITZ in brain tissue rapidly disappeared (half-life, 0.4 h). The time profiles of the brain/plasma ITZ concentration ratio (Kpvalue) showed a marked overshooting, and theKpvalue increased with increasing dose; these phenomena were not observed in the liver tissue. This finding indicates the occurrence of a nonlinear efflux of ITZ from the brain to the blood. Moreover, based on a pharmacokinetic model which hypothesized processes for both nonlinear and linear effluxes of ITZ from the brain to the blood, we found that the efflux rate constant in the saturable process was approximately sevenfold larger than that in the nonsaturable process. TheKpvalue for the brain tissue was significantly increased in the presence of ketoconazole or verapamil. The brainKpvalue formdr1aknockout mice was also significantly increased compared with that of control mice. Moreover, the uptake of vincristine or vinblastine, both of which are substrates of the P glycoprotein (P-gp), into mouse brain capillary endothelial cells was also significantly increased by ITZ or verapamil. In conclusion, P-gp in the brain capillary endothelial cells participates in a process of active efflux of ITZ from the brain to the blood at the blood-brain barrier, and ITZ can be an inhibitor of various substrates of P-gp.
Anti-Hepatitis B Virus Activity and Metabolism of 2′,3′-Dideoxy-2′,3′-Didehydro-β-
l
(−)-5-Fluorocytidine
Authors: Zhu Yong-Lian , Dutschman Ginger E. , Liu Shwu-Huey , Bridges Edward G. , Cheng Yung-Chi 
Published: October 2018

ABSTRACT

2′,3′-Dideoxy-2′,3′-didehydro-β-
l
(−)-5-fluorocytidine [
l
(−)Fd4C] was found to be at least 10 times more potent than β-
l
-2′,3′-dideoxy-3′-thiacytidine [
l
(−)SddC; also called 3TC, or lamivudine]against hepatitis B virus (HBV) in culture. Its cytotoxicity against HepG2 growth in culture was also greater than that of
l
(−)SddC (3TC). There was no activity of this compound against mitochondrial DNA synthesis in cells at concentrations up to 10 μM. The dynamics of recovery of virus from the medium of cells pretreated with equal drug concentrations were slower with
l
(−)Fd4C than with
l
(−)SddC (3TC).
l
(−)Fd4C could be metabolized to mono-, di-, and triphosphate forms. The degree of
l
(−)Fd4C phosphorylation to the 5′-triphosphate metabolite was higher than the degree of
l
(−)SddC (3TC) phosphorylation when equal extracellular concentrations of the two drugs were used. The apparent

K
m

of
l
(−)Fd4C phosphorylated metabolites formed intracellularly was higher than that for
l
(−)SddC (3TC). This may be due in part to a difference in the behavior of
l
(−)Fd4C and
l
(−)SddC (3TC) towards cytosolic deoxycytidine kinase. Furthermore,
l
(−)Fd4C 5′-triphosphate was retained longer within cells than
l
(−)SddC (3TC) 5′-triphosphate.
l
(−)Fd4C 5′-triphosphate inhibited HBV DNA polymerase in competition with dCTP with a

K
i

of 0.069 ± 0.015 μM. Given the antiviral potency and unique pharmacodynamic properties of
l
(−)Fd4C, this compound should be considered for development as an expanded-spectrum anti-HBV drug.
Identification and Expression of Multidrug Transporters Responsible for Fluconazole Resistance in
Candida dubliniensis
Authors: Moran Gary P. , Sanglard Dominique , Donnelly Samantha M. , Shanley Diarmuid B. , Sullivan Derek J. , Coleman David C. 
Published: October 2018

ABSTRACT

Candida dubliniensis
is a recently described
Candida
species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [
3
H]fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-
N
-oxide and methotrexate. These findings suggested that
C. dubliniensis
may encode multidrug transporters similar to those encoded by the
C. albicans MDR1
,
CDR1
, and
CDR2
genes (
CaMDR1
,
CaCDR1
, and
CaCDR2
, respectively). A
C. dubliniensis
homolog of
CaMDR1
, termed
CdMDR1
, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding
CaMDR1
sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR,
C. dubliniensis
was also found to encode homologs of
CDR1
and
CDR2
, termed
CdCDR1
and
CdCDR2
, respectively. Expression of
CdMDR1
in a fluconazole-susceptible Δ
pdr5
null mutant of
Saccharomyces cerevisiae
conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [
3
H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of
C. dubliniensis
revealed that fluconazole resistance was associated with increased expression of
CdMDR1
mRNA. In contrast, most studies showed that overexpression of
CaCDR1
was associated with fluconazole resistance in
C. albicans
. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of
C. dubliniensis
generated in vitro, some of which also exhibited increased levels of
CdCDR1
mRNA and CdCdr1p protein. These results demonstrate that
C. dubliniensis
encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.
Effect of Rebamipide, a Novel Antiulcer Agent, on
Helicobacter pylori
Adhesion to Gastric Epithelial Cells
Authors: Hayashi Shunji , Sugiyama Toshiro , Amano Ken-Ichi , Isogai Hiroshi , Isogai Emiko , Aihara Miki , Kikuchi Mikio , Asaka Masahiro , Yokota Kenji , Oguma Keiji , Fujii Nobuhiro , Hirai Yoshikazu 
Published: October 2018

ABSTRACT

Helicobacter pylori
is a major etiological agent in gastroduodenal disorders. The adhesion of
H. pylori
to human gastric epithelial cells is the initial step of
H. pylori
infection. Inhibition of
H. pylori
adhesion is thus a therapeutic target in the prevention of
H. pylori
infection. Experiments were performed to evaluate the effect of rebamipide, a novel antiulcer agent, on
H. pylori
adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Ten
H. pylori
strains isolated from patients with chronic gastritis and gastric ulcer were used in the study. We evaluated the effect of rebamipide on
H. pylori
adhesion to MKN-28 and MKN-45 cells quantitatively using our previously established enzyme-linked immunosorbent assay. The adhesion of
H. pylori
to MKN-28 and MKN-45 cells was significantly inhibited by pretreatment of these cells with 100 μg of rebamipide per ml. However, the adhesion was not affected by the pretreatment of
H. pylori
with rebamipide. On the other hand, the viabilities of
H. pylori
, MKN-28 cells, and MKN-45 cells were not affected by rebamipide. Our studies suggest that rebamipide inhibits the adhesion of
H. pylori
to gastric epithelial cells.
Generation of BioluminescentStreptococcus mutansand Its Usage in Rapid Analysis of the Efficacy of Antimicrobial Compounds
Authors: Loimaranta Vuokko , Tenovuo Jorma , Koivisto Leeni , Karp Matti 
Published: October 2018

ABSTRACTThe oral bacteriumStreptococcus mutanswas transformed by electroporation with a shuttle vector (pCSS945) containing insect luciferase gene from a click beetle (Pyrophorus plagiophthalamus) resulting in a bioluminescent phenotype. ThisS. mutansstrain was used in experiments in which light emission was used as a rapid and, compared to conventional CFU counting, more convenient means of estimating the effects of various antimicrobial treatments. The basic parameters affecting in vivo light production by the strain were studied. It was found that pH 6.0 was optimal for incorporation of the substrated-luciferin for the luciferase reaction. The optimum concentration ofd-luciferin was approximately 150 μM at room temperature. Under optimum conditions the light emission in vivo increased rapidly to a constant level and thereafter had a decay of 0.6%/min when logarithmic-growth-phase cells were used. The light emission closely paralleled the numbers of CFU, giving a detectable signal from 30,000 cells and having a dynamic measurement range over 4 log CFU/relative light unit. The cells were treated with various antimicrobial agents, and the emitted bioluminescence was measured. With the bioluminescent measurements, the results were obtained within hours compared to the days required for agar plates, and also, the kinetics of the antibacterial actions could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents tested.
Characterization of the MexC-MexD-OprJ Multidrug Efflux System in Δ
mexA-mexB-oprM
Mutants of
Pseudomonas aeruginosa
Authors: Gotoh Naomasa , Tsujimoto Hideto , Tsuda Masataka , Okamoto Kiyomi , Nomura Atsuko , Wada Takaomi , Nakahashi Masaaki , Nishino Takeshi 
Published: October 2018

ABSTRACT

Expression of the multidrug efflux system MexC-MexD-OprJ in
nfxB
mutants of
Pseudomonas aeruginosa
contributes to resistance to fluoroquinolones and the “fourth-generation” cephems (cefpirome and cefozopran), but not to most β-lactams, including the ordinary cephems (ceftazidime and cefoperazone).
nfxB
mutants also express a second multidrug efflux system, MexA-MexB-OprM, due to incomplete transcriptional repression of this operon by the
mexR
gene product. To characterize the contribution of the MexC-MexD-OprJ system to drug resistance in
P. aeruginosa
, a site-specific deletion method was employed to remove the
mexA-mexB-oprM
region from the chromosome of wild-type and
nfxB
strains of
P. aeruginosa
. Characterization of mutants lacking the
mexA-mexB-oprM
region clearly indicated that the MexC-MexD-OprJ efflux system is involved in resistance to the ordinary cephems as well as fluoroquinolones and the fourth-generation cephems but not to carbenicillin and aztreonam. Rabbit polyclonal antisera and murine monoclonal antibody against the components of the MexA-MexB-OprM system were prepared and used to demonstrate the reduced production of this efflux system in the
nfxB
mutants. Consistent with this, transcription of the
mexA-mexB-oprM
operon decreased in an
nfxB
mutant. This reduction appears to explain the hypersusceptibility of the
nfxB
mutant to β-lactams, including ordinary cephems.
Site-Specific Mutations in the 23S rRNA Gene of
Helicobacter pylori
Confer Two Types of Resistance to Macrolide-Lincosamide-Streptogramin B Antibiotics
Authors: Wang Ge , Taylor Diane E. 
Published: October 2018

ABSTRACT

Clarithromycin resistance in
Helicobacter pylori
is mainly due to A-to-G mutations within the peptidyltransferase region of the 23S rRNA. In the present study, cross-resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (MLS phenotypes) has been investigated for several clinical isolates of
H. pylori
. Two major types of MLS resistance were identified and correlated with specific point mutations in the 23S rRNA gene. The A2142G mutation was linked with high-level cross-resistance to all MLS antibiotics (type I), and the A2143G mutation gave rise to an intermediate level of resistance to clarithromycin and clindamycin but no resistance to streptogramin B (type II). In addition, streptogramin A and streptogramin B were demonstrated to have a synergistic effect on both MLS-sensitive and MLS-resistant
H. pylori
strains. To further understand the mechanism of MLS resistance in
H. pylori
, we performed in vitro site-directed mutagenesis (substitution of G, C, or T for A at either position 2142 or 2143 of the 23S rRNA gene). The site-directed point mutations were introduced into a clarithromycin-susceptible strain,
H. pylori
UA802, by natural transformation followed by characterization of their effects on MLS resistance in an isogenic background. Strains with A-to-G and A-to-C mutations at the same position within the 23S rRNA gene had similar levels of clarithromycin resistance, and this level of resistance was higher than that for strains with the A-to-T mutation. Mutations at position 2142 conferred a higher level of clarithromycin resistance than mutations at position 2143. All mutations at position 2142 conferred cross-resistance to all MLS antibiotics, which corresponds to the type I MLS phenotype, whereas mutations at position 2143 were associated with a type II MLS phenotype with no resistance to streptogramin B. To explain that A-to-G transitions were predominantly observed in clarithromycin-resistant clinical isolates, we propose a possible mechanism by which A-to-G mutations are preferentially produced in
H. pylori
.
CTX-M-5, a Novel Cefotaxime-Hydrolyzing β-Lactamase from an Outbreak of
Salmonella typhimurium
in Latvia
Authors: Bradford Patricia A. , Yang Youjun , Sahm Daniel , Grope Ilze , Gardovska Dace , Storch Gregory 
Published: October 2018

ABSTRACT

At a children’s hospital in Riga, Latvia, isolates identified as
Salmonella typhimurium
were found to be resistant to expanded-spectrum cephalosporins. Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance. Isoelectric focusing revealed a common β-lactamase with a pI of 8.8. In addition, one of the strains produced a pI 7.6 β-lactamase. A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1. Transformants producing only the pI 8.8 β-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime. A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative
k
cat
value of 95 (benzylpenicillin equal to 100). The enzyme showed lower relative
k
cat
values for ceftazidime (3.3) and aztreonam (9.3). In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively. These results indicated the presence of an unusual extended-spectrum β-lactamase. The gene expressing the pI 8.8 β-lactamase was cloned. Nucleotide sequencing revealed a β-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from
S. typhimurium
, by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val. These results indicated the presence of a novel extended-spectrum β-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.
Sensitivity of
Leishmania viannia panamensis
to Pentavalent Antimony Is Correlated with the Formation of Cleavable DNA-Protein Complexes
Authors: Lucumi Armando , Robledo Sara , Gama Vivian , Saravia Nancy G. 
Published: October 2018

ABSTRACT

The emergence of
Leishmania
less sensitive to pentavalent antimonial agents (SbVs), the report of inhibition of purified topoisomerase I of
Leishmania donovani
by sodium stibogluconate (Pentostam), and the uncertain mechanism of action of antimonial drugs prompted an evaluation of SbVs in the stabilization of cleavable complexes in promastigotes of
Leishmania
(
Viannia
). The effect of camptothecin, an inhibitor of topoisomerase, and additive-free meglumine antimoniate (Glucantime) on the stabilization of cleavable DNA-protein complexes associated with the inhibition of topoisomerase was assessed in the human promonocytic cell line U-937, promastigotes of
L.
(
Viannia
)
panamensis
selected for SbV resistance in vitro, and the corresponding wild-type strain. The stabilization of cleavable complexes and the 50% effective dose (ED
50
) of SbVs for parasites isolated from patients with relapses were also evaluated. The median ED
50
for the wild-type strain was 16.7 μg of SbV/ml, while that of the line selected for resistance was 209.5 μg of SbV/ml. Treatment with both meglumine antimoniate and sodium stibogluconate (20 to 200 μg of SbV/ml) stabilized DNA-protein complexes in the wild-type strain but not the resistant line. The ED
50
s of the SbVs for
Leishmania
strains from patients with relapses was comparable to those for the line selected for in vitro resistance, and DNA-protein complexes were not stabilized by exposure to meglumine antimoniate. Cleavable complexes were observed in all
Leishmania
strains treated with camptothecin. Camptothecin stabilized cleavable complexes in U-937 cells; SbVs did not. The selective effect of the SbVs on the stabilization of DNA-protein complexes in
Leishmania
and the loss of this effect in naturally resistant or experimentally derived SbV-resistant
Leishmania
suggest that topoisomerase may be a target of antimonial drugs.
Disposition and Elimination of Meropenem in Cerebrospinal Fluid of Hydrocephalic Patients with External Ventriculostomy
Authors: Nau Roland , Lassek Christoph , Kinzig-Schippers Martina , Thiel Andreas , Prange Hilmar W. , Sörgel Fritz 
Published: October 2018

ABSTRACT

The broad antibacterial spectrum and the low incidence of seizures in meropenem-treated patients qualifies meropenem for therapy of bacterial meningitis. The present study evaluates concentrations in ventricular cerebrospinal fluid (CSF) in the absence of pronounced meningeal inflammation. Patients with occlusive hydrocephalus caused by cerebrovascular diseases, who had undergone external ventriculostomy (
n
= 10, age range 48 to 75 years), received 2 g of meropenem intravenously over 30 min. Serum and CSF were drawn repeatedly and analyzed by liquid chromatography-mass spectroscopy. Pharmacokinetics were determined by noncompartmental analysis. Maximum concentrations in serum were 84.7 ± 23.7 μg/ml. A CSF maximum (
C
maxCSF
) of 0.63 ± 0.50 μg/ml (mean ± standard deviation) was observed 4.1 ± 2.6 h after the end of the infusion. C
maxCSF
and the area under the curve for CSF (AUC
CSF
) depended on the AUC for serum (AUC
S
), the CSF-to-serum albumin ratio, and the CSF leukocyte count. Elimination from CSF was considerably slower than from serum (half-life at β phase [
t
1/2β
] of 7.36 ± 2.89 h in CSF versus
t
1/2β
of 1.69 ± 0.60 h in serum). The AUC
CSF
/AUC
S
ratio for meropenem, as a measure of overall CSF penetration, was 0.047 ± 0.022. The AUC
CSF
/AUC
S
ratio for meropenem was similar to that for other β-lactam antibiotics with a low binding to serum proteins. The concentration maxima of meropenem in ventricular CSF observed in this study are high enough to kill fully susceptible pathogens. They may not be sufficient to kill bacteria with a reduced sensitivity to carbapenems, although clinical success has been reported for patients with meningitis caused by penicillin-resistant pneumococci and
Pseudomonas aeruginosa
.
Molecular Characterization of OXA-20, a Novel Class D β-Lactamase, and Its Integron from
Pseudomonas aeruginosa
Authors: Naas Thierry , Sougakoff Wladimir , Casetta Anne , Nordmann Patrice 
Published: October 2018

ABSTRACT

The
Pseudomonas aeruginosa
Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum β-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188–2195, 1997). A second β-lactamase was cloned, and the recombinant
Escherichia coli
clone pPL10 expressed a pI 7.4 β-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D β-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar β-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of
oxa20
revealed the presence of the following integron features: (i) a second antibiotic resistance gene,
aacA4
; (ii) an
intI1
gene; and (iii) two 59-base elements, each associated with either
oxa20
or
aacA4
. This integron is peculiar because it lacks the 3′ conserved region, and therefore is not a
sul1
-associated integron like most of them, and because its 3′ end is located within
tnpR
, a gene involved in the transposition of Tn
5393
, a gram-negative transposon.
P. aeruginosa
Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.
Correlation between Quinolone Susceptibility Patterns and Sequences in the A and B Subunits of DNA Gyrase in Mycobacteria
Authors: Guillemin Isabelle , Jarlier Vincent , Cambau Emmanuelle 
Published: October 2018

ABSTRACT

The in vitro activities of seven quinolones and the sequences of the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase were determined for 14 mycobacterial species. On the basis of quinolone activity, quinolones were arranged from that with the greatest to that with the least activity as follows: sparfloxacin, levofloxacin, ciprofloxacin, ofloxacin, pefloxacin, flumequine, and nalidixic acid. Based on MICs, the species could be organized into three groups: resistant (
Mycobacterium avium
,
M. intracellulare
,
M. marinum
,
M. chelonae
,
M. abscessus
[ofloxacin MICs, ≥8 μg/ml]), moderately susceptible (
M. tuberculosis
,
M. bovis
BCG,
M. kansasii
,
M. leprae
,
M. fortuitum
third biovariant,
M. smegmatis
[ofloxacin MICs, 0.5 to 1 μg/ml]), and susceptible (
M. fortuitum
,
M. peregrinum
,
M. aurum
[ofloxacin MICs, ≤0.25 μg/ml]). Peptide sequences of the QRDR of GyrB were identical in all the species, including the amino acids at the three positions known to be involved in acquired resistance to quinolone, i.e., 426 (Asp), 447 (Arg), and 464 (Asn) (numbering system used for
Escherichia coli
). The last two residues could be involved in the overall low level of susceptibility of mycobacteria to quinolones since they differ from those found in the very susceptible
E. coli
(Lys-447 and Ser-464) but are identical to those found in the less susceptible
Staphylococcus aureus
and
Streptococcus pneumoniae
. Peptide sequences of the QRDR of GyrA were identical in all the species, except for the amino acid at position 83, which was an alanine in the two less susceptible groups and a serine in the most susceptible one, as in
E. coli
, suggesting that this amino acid is involved in the observed differences of quinolone susceptibility within the
Mycobacterium
genus.
Identification of Novel Mutation Patterns in the
parC
Gene of Ciprofloxacin-Resistant Isolates of
Neisseria gonorrhoeae
Authors: Trees David L. , Sandul Amy L. , Whittington William L. , Knapp Joan S. 
Published: October 2018

ABSTRACT

Of 65 ciprofloxacin-resistant, clinical isolates of
Neisseria gonorrhoeae
, 5 isolates exhibited ParC mutations previously undescribed in the gonococcus. For isolates containing two ParC mutations (the Ser-87→Ile and Glu-91→Gly mutations and the Gly-85→Cys and Arg116→Leu mutations) the MICs of ciprofloxacin (8.0 to 64.0 μg/ml) were higher than those for the isolate containing the single ParC mutation (Arg-116→Leu; MIC, 1.0 μg/ml).