bluuebunny/crossref_metadata_2025 huggingface dataset:
Carrier-Mediated Delivery of 9-(2-Phosphonylmethoxyethyl)Adenine to Parenchymal Liver Cells: a Novel Therapeutic Approach for Hepatitis B
Authors: de Vrueh Remco L. A. , Rump Erik T. , van de Bilt Erika , van Veghel Richard , Balzarini Jan , Biessen Erik A. L. , van Berkel Theo J. C. , Bijsterbosch Martin K.
Published: July 2002

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3α-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [
3
H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% ± 7.7% of the dose (free [
3
H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [
3
H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% ± 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.
In Vivo Pharmacodynamic Activities of Two Glycylcyclines (GAR-936 and WAY 152,288) against Various Gram-Positive and Gram-Negative Bacteria
Authors: van Ogtrop M. L. , Andes D. , Stamstad T. J. , Conklin B. , Weiss W. J. , Craig W. A. , Vesga O.
Published: July 2002

The in vivo pharmacodynamic activities of two glycylcyclines (GAR-936 and WAY 152,288) were assessed in an experimental murine thigh infection model in neutropenic mice. Mice were infected with one of several strains of
Streptococcus pneumoniae
,
Staphylococcus aureus
,
Escherichia coli
, or
Klebsiella pneumoniae
. Most infections were treated with a twice-daily dosing schedule, with administration of 0.75 to 192 mg of GAR-936 or WAY 152,288 per kg of body weight. A maximum-effect dose-response model was used to calculate the dose that produced a net bacteriostatic effect over 24 h of therapy. This dose was called the bacteriostatic dose. More extensive dosing studies were performed with
S. pneumoniae
1199,
E. coli
ATCC 25922, and
K. pneumoniae
ATCC 43816, with doses being given as one, two, four, or eight equal doses over a period of 24 h. The dosing schedules were designed in order to minimize the interrelationship between the various pharmacokinetic and pharmacodynamic parameters studied. These parameters were time above 0.03 to 32 times the MIC, area under the concentration-time curve (AUC), and maximum concentration of drug in serum (
C
max
). The bacteriostatic dose remained essentially the same, irrespective of the dosing frequency, for
S. pneumoniae
1199 (0.3 to 0.9 mg/kg/day). For
E. coli
ATCC 25922 and
K. pneumoniae
ATCC 43816, however, more frequent dosing led to lower bacteriostatic doses. Pharmacokinetic studies demonstrated dose-dependent elimination half-lives of 1.05 to 2.34 and 1.65 to 3.36 h and serum protein bindings of 59 and 71% for GAR-936 and WAY 152,288, respectively. GAR-936 and WAY 152,288 were similarly effective against the microorganisms studied, with small differences in maximum effect and 50% effective dose. The glycylcyclines were also similarly effective against tetracycline-sensitive and tetracycline-resistant bacteria. Time above a certain factor (range, 0.5 to 4 times) of the MIC was a better predictor of in vivo efficacy than
C
max
or AUC for most organism-drug combinations. The results demonstrate that in order to achieve 80% maximum efficacy, the concentration of unbound drug in serum should be maintained above the MIC for at least 50% of the time for GAR-936 and for at least 75% of the time for WAY 152,288. The results of these experiments will aid in the rational design of dose-finding studies for these glycylcyclines in humans.
Effect of Azithromycin plus Rifampin versus Amoxicillin Alone on Eradication and Inflammation in the Chronic Course of
Chlamydia pneumoniae
Pneumonitis in Mice
Authors: Bin Xie Xiao , Wolf Katerina , Schaffner Thomas , Malinverni Raffaele
Published: July 2002

The effects of treatment with azithromycin plus rifampin (A+R), amoxicillin (A), or placebo (P) on the chronic course of experimental
Chlamydia pneumoniae
pneumonitis in mice were assessed by culture, PCR, and immunocytochemistry as well as by degree of inflammation in lung tissue. Eradication of the pathogen was significantly more frequent and inflammation in tissue was significantly reduced after treatment with A+R compared to after treatment with A or P. Combination therapy with azithromycin plus rifampin showed favorable effects in the chronic course of
C. pneumoniae
pneumonitis.
BMS-284756 in Experimental Cephalosporin-Resistant Pneumococcal Meningitis
Authors: Rodriguez-Cerrato Violeta , Ghaffar Faryal , Saavedra Jesus , Michelow Ian C. , Hardy Robert D. , Iglehart Janie , Olsen Kurt , McCracken George H.
Published: July 2002

BMS-284756 is a novel des-fluoro(6) quinolone with a broad antimicrobial activity, including
Streptococcus pneumoniae
. The purpose of this study was to evaluate the pharmacodynamic profile and effectiveness of BMS-284756 for therapy of experimental meningitis caused by penicillin- and cephalosporin-resistant
S. pneumoniae
(CRSP). Meningitis was induced in rabbits by intracisternal inoculation of CRSP. BMS-284756 was given intravenously 16 h after intracisternal inoculation in single doses of 2.5 (
n
= 5 animals), 5 (
n
= 6), 10 (
n
= 6), 20 (
n
= 8), and 30 mg/kg (
n
= 6), in two doses of 10 mg/kg each separated by 5 h (
n
= 4), and as a 20-mg/kg dose followed 5 h later by 10 mg/kg (
n
= 5). The MICs and MBCs of BMS-284756, ceftriaxone, and vancomycin were 0.06 and 0.06, 4 and 4, and 0.25 and 0.25 μg/ml, respectively. After single doses of 10, 20, and 30 mg/kg, the maximum concentrations in cerebrospinal fluid (CSF) (mean ± standard deviation) were 0.32 ± 0.12, 0.81 ± 0.38, and 1.08 ± 0.43 μg/ml, respectively; the elimination half-life in CSF was 4.5 to 6.3 h. The CSF bacterial killing rates (BKR) at 5 h of the single-dose regimens of 10, 20 and 30 mg/kg were −0.84 ± 0.48, −1.09 ± 0.32, and −1.35 ± 0.05 Δlog
10
CFU/ml/h. The BKR
0–5
of the divided regimens (10 mg/kg twice and 20 mg/kg followed by 10 mg/kg) was −0.82 ± 0.52 and −1.24 ± 0.34 Δlog
10
CFU/ml/h, respectively. The BKR
0–5
of the combined therapy with vancomycin and ceftriaxone was −1.09 ± 0.39 Δlog
10
CFU/ml/h. The penetration of BMS-284756 into purulent CSF relative to plasma was 14 to 25%. The bactericidal effect of BMS-284756 in CSF was concentration dependent. BMS-284756 at 30 mg/kg as a single or divided dose was as effective as standard therapy with vancomycin and ceftriaxone.
Analysis of Antimalarial Synergy between Bestatin and Endoprotease Inhibitors Using Statistical Response-Surface Modelling
Authors: Gavigan Clare S. , Machado Stella G. , Dalton John P. , Bell Angus
Published: July 2002

The pathway of hemoglobin degradation by erythrocytic stages of the human malarial parasite
Plasmodium falciparum
involves initial cleavages of globin chains, catalyzed by several endoproteases, followed by liberation of amino acids from the resulting peptides, probably by aminopeptidases. This pathway is considered a promising chemotherapeutic target, especially in view of the antimalarial synergy observed between inhibitors of aspartyl and cysteine endoproteases. We have applied response-surface modelling to assess antimalarial interactions between endoprotease and aminopeptidase inhibitors using cultured
P. falciparum
parasites. The synergies observed were consistent with a combined role of endoproteases and aminopeptidases in hemoglobin catabolism in this organism. As synergies between antimicrobial agents are often inferred without proper statistical analysis, the model used may be widely applied in studies of antimicrobial drug interactions.
Synergistic Activities of Gatifloxacin in Combination with Other Antimicrobial Agents against
Pseudomonas aeruginosa
and Related Species
Authors: Gradelski Elizabeth , Valera Lourdes , Bonner Daniel , Fung-Tomc Joan
Published: July 2002

Drug combinations have been used to treat serious infections caused by
Pseudomonas, Burkholderia, Stenotrophomonas
, and
Acinetobacter
. In this study, the combined drug effects of gatifloxacin (GAT) and nonquinolones were determined by time-kill analysis at clinically achievable drug concentrations. Synergy (≥2 log
10
-enhanced killing at 24 h) was observed with GAT plus amikacin or a β-lactam against 50 to 75% of strains, including strains nonsusceptible to one or both drugs.
Functional Expression of
Candida albicans
Drug Efflux Pump Cdr1p in a
Saccharomyces cerevisiae
Strain Deficient in Membrane Transporters
Authors: Nakamura Kenjirou , Niimi Masakazu , Niimi Kyoko , Holmes Ann R. , Yates Jenine E. , Decottignies Anabelle , Monk Brian C. , Goffeau Andre , Cannon Richard D.
Published: July 2002

Analysis of the transport functions of individual
Candida albicans
plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed
C. albicans
Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic
PDR5
locus in a
Saccharomyces cerevisiae
mutant (AD1-8u
−
) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the
S. cerevisiae PDR5
promoter and driven by
S. cerevisiae
Pdr1p transcriptional regulator mutation
pdr1-3
, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions.
S. cerevisiae
AD1-8u
−
was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC
80
s] were 0.625 μg/ml for fluconazole, <0.016 μg/ml for ketoconazole, and <0.016 μg/ml for itraconazole), whereas the strain (AD1002) that overexpressed
C. albicans
Cdr1p was resistant to azoles (MIC
80
s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 μg/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of
C. albicans
Cdr1p in an
S. cerevisiae
background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.
Characterization of
emeA
, a
norA
Homolog and Multidrug Resistance Efflux Pump, in
Enterococcus faecalis
Authors: Jonas Brandie M. , Murray Barbara E. , Weinstock George M.
Published: July 2002

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the
Enterococcus faecalis
V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene
emeA
(enterococcal multidrug resistance efflux).
Impact of the Order of Initiation of Fluconazole and Amphotericin B in Sequential or Combination Therapy on Killing of
Candida albicans
In Vitro and in a Rabbit Model of Endocarditis and Pyelonephritis
Authors: Louie Arnold , Kaw Pamela , Banerjee Partha , Liu Weiguo , Chen George , Miller Michael H.
Published: July 2002

In vitro time-kill studies and a rabbit model of endocarditis and pyelonephritis were used to define the impact that the order of exposure of
Candida albicans
to fluconazole (FLC) and amphotericin B (AMB), as sequential and combination therapies, had on the susceptibility of
C. albicans
to AMB and on the outcome. The contribution of FLC-induced resistance to AMB for
C. albicans
also was assessed. In vitro, AMB monotherapy rapidly killed each of four
C. albicans
strains; FLC alone was fungistatic. Preincubation of these fungi with FLC for 18 h prior to exposure to AMB decreased their susceptibilities to AMB for 8 to >40 h. Induced resistance to AMB was transient, but the duration of resistance increased with the length of FLC preincubation. Yeast sequentially incubated with FLC followed by AMB plus FLC (FLC→AMB+FLC) showed fungistatic growth kinetics similar to that of fungi that were exposed to FLC alone. This antagonistic effect persisted for at least 24 h. Simultaneous exposure of
C. albicans
to AMB and FLC [AMB+FLC(simult)] demonstrated activity similar to that with AMB alone for AMB concentrations of ≥1 μg/ml; antagonism was seen using an AMB concentration of 0.5 μg/ml. The in vitro findings accurately predicted outcomes in our rabbit infection model. In vivo, AMB monotherapy and treatment with AMB for 24 h followed by AMB plus FLC (AMB→AMB+FLC) rapidly sterilized kidneys and cardiac vegetations. AMB+FLC(simult) and FLC→AMB treatments were slower in clearing fungi from infected tissues. FLC monotherapy and FLC→AMB+FLC were both fungistatic and were the least active regimens. No adverse interaction was observed between AMB and FLC for the AMB→FLC regimen. However, FLC→AMB treatment was slower than AMB alone in clearing fungi from tissues. Thus, our in vitro and in vivo studies both demonstrate that preexposure of
C. albicans
to FLC reduces fungal susceptibility to AMB. The length of FLC preexposure and whether AMB is subsequently used alone or in combination with FLC determine the duration of induced resistance to AMB.
Antiviral Activity of β-
l
-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus
Authors: Le Guerhier F. , Pichoud C. , Jamard C. , Guerret S. , Chevallier M. , Peyrol S. , Hantz O. , King I. , Trépo C. , Cheng Y.-C. , Zoulim F.
Published: July 2002

The
l
-nucleoside analog β-
l
-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-
l
-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-
l
-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-
l
-Fd4C-treated groups. Moreover, β-
l
-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-
l
-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-
l
-Fd4C treatment. There was no evidence that lamivudine or β-
l
-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-
l
-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
Enhanced Expression of the Multidrug Efflux Pumps AcrAB and AcrEF Associated with Insertion Element Transposition in
Escherichia coli
Mutants Selected with a Fluoroquinolone
Authors: Jellen-Ritter A. S. , Kern W. V.
Published: July 2002

The development of fluoroquinolone resistance in
Escherichia coli
may be associated with mutations in regulatory gene loci such as
marRAB
that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from
marRAB
- or
acrAB
-inactivated
E. coli
K-12 strains having a single mutation in the quinolone-resistance-determining region of
gyrA
. Mutant 3-AG100MKX, obtained from a
mar
knockout strain after two selection steps, showed enhanced expression of
acrB
in a reverse transcriptase PCR associated with insertion of IS
186
into the AcrAB repressor gene
acrR
. In vitro selection experiments with
acrAB
knockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of
acrEF
and associated with insertion of IS2 into the upstream region of
acrEF
, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in
E. coli
.
Selection and Characterization of Varicella-Zoster Virus Variants Resistant to (
R
)-9-[4-Hydroxy-2-(Hydroxymethy)Butyl]Guanine
Authors: Ng Teresa I. , Shi Yan , Huffaker H. Janette , Kati Warren , Liu Yaya , Chen Chih-Ming , Lin Zhen , Maring Clarence , Kohlbrenner William E. , Molla Akhteruzzaman
Published: July 2002

(
R
)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G) is a potent and selective inhibitor of herpesvirus replication. It is a nucleoside analog, and its triphosphate derivative (H2G-TP) is a competitive inhibitor of herpesvirus DNA polymerases. In this study, the antiviral activities of H2G and acyclovir (ACV) and the development of viral resistance to these agents were compared in varicella-zoster virus (VZV)-infected cells. In plaque reduction assays, the 50% effective concentration of H2G for VZV was 60- to 400-fold lower than that of ACV, depending on the virus strain and the cell line tested. The enhanced efficacy of H2G against VZV can be accounted for in part by the fact that the intaracellular H2G-TP level (>170 pmol/10
6
cells) is higher than the intracellular ACV-TP level (<1 pmol/10
6
cells). In addition, H2G-TP has extended half-lives of 3.9 and 8.6 h in VZV-infected MRC-5 and MeWo cells, respectively. To assess the emergence of H2G-resistant VZV in vitro, VZV was passaged in the presence of increasing concentrations of H2G. Earlier in the passage, when the concentration of H2G was relatively low, the predominant variant had the (A)76 deletion in the viral thymidine kinase (TK) gene. This mutant was identical to an ACV-resistant mutant generated in parallel experiments. However, higher concentrations of H2G appeared to favor a novel mutant, which had deletions of two consecutive nucleotides at positions 805 and 806 of the TK gene. All of these changes introduced frameshift mutations in the TK gene resulting in the expression of truncated polypeptides. H2G-resistant viruses were cross-resistant to ACV, and vice versa.
Uptake, Transport, and Delivery of Antimicrobial Agents by Human Polymorphonuclear Neutrophils
Authors: Mandell Gerald L. , Coleman Elizabeth
Published: July 2002

Polymorphonuclear neutrophils (PMN) are attracted to sites of infection. They have the potential to deliver antimicrobial agents to these sites if the agents enter the cells and do not alter migration. Penicillin G did not enter cells and was not transported by PMN. We found that azithromycin, ciprofloxacin, levofloxacin, moxifloxacin, and telithromycin were concentrated in PMN and transported toward a chemoattractant. These antimicrobial agents were released from the PMN and inhibited the growth of bacteria on test plates.
In Vitro Activities of Moxifloxacin and Other Fluoroquinolones against
Mycoplasma pneumoniae
Authors: Hamamoto Kumiko , Shimizu Takashi , Fujimoto Naoyuki , Zhang Ye , Arai Sumio
Published: July 2002

A total of 105 isolates of
Mycoplasma pneumoniae
were evaluated for susceptibility to moxifloxacin, sparfloxacin, levofloxacin, and ciprofloxacin. Moxifloxacin, a newly synthesized compound, showed the greatest activity. The MICs and MBCs at which 50 and 90% of isolates were affected were 0.15 (MIC
50
and MBC
50
) and 0.3 μg/ml (MIC
90
and MBC
90
) respectively. The results indicate that moxifloxacin might be promising an antimycoplasmal agent.
Biochemical Mechanism of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistance to Stavudine
Authors: Lennerstrand Johan , Stammers David K. , Larder Brendan A.
Published: July 2002

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.
Telithromycin Is Active against
Mycobacterium avium
in Mice despite Lacking Significant Activity in Standard In Vitro and Macrophage Assays and Is Associated with Low Frequency of Resistance during Treatment
Authors: Bermudez Luiz E. , Inderlied Clark B. , Kolonoski Peter , Wu Martin , Aralar Priscilla , Young Lowell S.
Published: July 2002

The activity of telithromycin, a new ketolide, was evaluated in vitro and in vivo against
Mycobacterium avium
complex (MAC) strains. The MIC of telithromycin for several
M. avium
isolates obtained from the blood of AIDS patients ranged from 16 to >128 μg/ml (MIC at which 90% of isolates are inhibited, >128 μg/ml), and the compound did show activity in the macrophage system at concentrations greater than 8 or 16 μg/ml, but this was dependent on the MAC strain used. Telithromycin was then administered to mice infected with MAC strain 101 for 4 weeks at doses of 100, 200, or 400 mg/kg of body weight/day. Treatment with 100 and 200 mg/kg/day was bacteriostatic, but at 400 mg/kg/day telithromycin was bactericidal for MAC strains. The frequency of the emergence of resistance to telithromycin was low despite prolonged usage (12 weeks). This study demonstrates that telithromycin is active in vivo against MAC and warrants further evaluation.
Kappacin, a Novel Antibacterial Peptide from Bovine Milk
Authors: Malkoski Marina , Dashper Stuart G. , O'Brien-Simpson Neil M. , Talbo Gert H. , Macris Mary , Cross Keith J. , Reynolds Eric C.
Published: July 2002

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens
Streptococcus mutans
and
Porphyromonas gingivalis
and against
Escherichia coli
. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against
S. mutans
in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated κ-casein (residues 106 to 169) [κ-casein(106–169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against
S. mutans
. The peptide Ser(
P
)
149
κ-casein-A(138–158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(
P
)
149
κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(
P
)
149
κ-casein-A(138–158) displayed growth-inhibitory activity against
S. mutans
(MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.
Antigen Clearance during Treatment of Disseminated Histoplasmosis with Itraconazole versus Fluconazole in Patients with AIDS
Authors: Wheat L. Joseph , Connolly Patricia , Haddad Nicholas , Le Monte Ann , Brizendine Edward , Hafner Richard
Published: July 2002

Antifungal treatment reduces the concentration of
Histoplasma
antigen in blood and urine, supporting a hypothesis that antigen clearance could be used to compare the activity of new agents for treating histoplasmosis. In separate trials in patients with AIDS, clinical response was similar with itraconazole (85%) and fluconazole (74%). Fungal blood cultures at week 4, however, were negative in a significantly higher proportion of patients treated with itraconazole (92.3%) than in those treated with fluconazole (61.9%) (
P
= 0.017). Baseline antigen concentrations were similar in the two groups: serum,
P
= 0.7235; and urine,
P
= 0.1360. After 4 weeks of treatment, the decline in antigen from baseline in serum was similar in the two treatment groups (
P
= 0.5237), as it was in urine (
P
= 0.4679). At week 12, the decline in antigen from baseline in serum also was similar in the two groups (
P
= 0.4911) and in urine (
P
= 0.2786). More rapid clearance of fungemia suggests that itraconazole is more effective than fluconazole in treating histoplasmosis. This study demonstrates that clearance of fungemia is a better measure of antifungal effect than clearance of antigen.
Discrepancy between Uptake and Intracellular Activity of Moxifloxacin in a
Staphylococcus aureus
-Human THP-1 Monocytic Cell Model
Authors: Paillard Delphine , Grellet Jean , Dubois Véronique , Saux Marie-Claude , Quentin Claudine
Published: July 2002

The correlation between uptake of moxifloxacin by THP-1, a continuous line of monocytic cells devoid of intrinsic bactericidal properties, and its activity against
Staphylococcus aureus
ATCC 25923, a susceptible reference strain (MIC and minimal bactericidal concentration of moxifloxacin, 0.1 mg/liter), was studied in a 5-h assay. The uptake of the drug, added to the culture medium at 0.2 to 32 mg/liter, was evaluated by high-performance liquid chromatography. The ratio of the cellular to extracellular concentration of moxifloxacin reached, at equilibrium, 4.36 ± 0.39 in uninfected cells and 6.25 ± 0.41 in infected cells. The intracellular activity of moxifloxacin, introduced into the extracellular fluid at 0.06 to 8 mg/liter, was determined by the enumeration of viable bacteria. At concentrations ≤0.2 mg/liter, the drug inhibited only the intracellular bacterial growth, while at concentrations ≥0.5 mg/liter, it decreased the bacterial inoculum by less than 1 log
10
unit, with a maximum effect at 3 to 4 h, followed by regrowth of surviving bacteria to 80 to 120% of the original level at 5 h. In contrast, when killing curves were determined by using Mueller-Hinton broth with a similar inoculum (10
7
CFU/ml), moxifloxacin at concentrations ≥0.2 mg/liter reduced the inoculum by at least 3 log
10
units at 3 to 4 h, leaving ≤0.1% survival at 24 h. Persisters exhibited a fluoroquinolone susceptibility identical to that of
S. aureus
ATCC 25923. Our data indicate that moxifloxacin at therapeutic extracellular concentrations accumulates approximately sixfold in infected THP-1 cells and remains active intracellularly, but significantly less active than under in vitro conditions.
Pharmacodynamics of Abacavir in an In Vitro Hollow-Fiber Model System
Authors: Drusano G. L. , Bilello P. A. , Symonds W. T. , Stein D. S. , McDowell J. , Bye A. , Bilello J. A.
Published: July 2002

Abacavir is a potent new carbocyclic nucleoside analogue. We employed our hollow-fiber pharmacodynamic modeling system to examine the antiretroviral effects of different abacavir exposures, as well as the impact of the schedule of drug administration on efficacy. Dose ranging of abacavir revealed that a concentration of four times the 50% effective concentration (EC
50
) (approximately the EC
95
) was required to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) (strain MN) either in a continuous-infusion hollow-fiber experiment or in a classical tissue culture flask experiment. In contrast to earlier work with another drug class (HIV-1 protease inhibitors), addition of physiological amounts of the human drug binding proteins albumin and α
1
acid glycoprotein revealed that there was little impact on the antiviral effect of the drug. Comparison of equivalent exposures (an area under the concentration-time curve [AUC] developed by approximately 500 mg per day of orally administered abacavir), either in a continuous-infusion mode or as a single oral dose of abacavir, demonstrated no difference in the ability to suppress either strain III
B
or strain MN. Comparison of administration of 250 mg every 12 h (q12h) versus once-daily administration of 500 mg for strain MN again showed no significant difference in suppressive effect. These experiments were carried out over 8 to 15 days. Because of these promising initial results, we extended the experiment to 30 days and examined three different schedules of administration that generated the same AUC at 24 h (AUC
24
): 300 mg q12h, 600 mg q24h, and 1,200 mg q48h. The aim of the last of these regimens was to definitively demonstrate schedule failure. There was little difference between the 1,200-mg q48h treatment group and the untreated control at 30 days. Likewise, there was little difference between the 600-mg q24h and 300-mg q12h treatment groups. However, at circa day 18 of the experiment, there was a small increase in viral output of p24 in the once-daily dosing unit. Examination of virus from all groups demonstrated no phenotypic or genotypic differences. The small difference in hollow-fiber unit p24 in the once-daily dosing group was not due to emergence of resistance over the 30-day single-drug exposure. We conclude that the dose of abacavir currently being studied in clinical trials (300 mg orally q12h) will be efficacious for the majority of sensitive clinical isolates of HIV-1. These in vitro data also suggest that this drug may be able to be administered to patients on a once-daily basis at a dose of 600 mg.
Azole Cross-Resistance in
Aspergillus fumigatus
Authors: Mosquera J. , Denning D. W.
Published: July 2002

We susceptibility tested 17 clinical isolates of
Aspergillus fumigatus
, for most of which MICs of itraconazole were elevated (MIC at which 50% of the isolates tested are inhibited, 16 μg/ml), against itraconazole, posaconazole, ravuconazole, and voriconazole. Posaconazole was the most active against itraconazole-susceptible isolates. A complex pattern of cross-resistance and hypersusceptibility was seen with voriconazole and ravuconazole, suggesting marked differences in activity and mechanisms of resistance.
Synergistic Activities of Macrolide Antibiotics against
Pseudomonas aeruginosa
,
Burkholderia cepacia
,
Stenotrophomonas maltophilia
, and
Alcaligenes xylosoxidans
Isolated from Patients with Cystic Fibrosis
Authors: Saiman Lisa , Chen Yunhua , Gabriel Pablo San , Knirsch Charles
Published: July 2002

Azithromycin and clarithromycin were paired with other antibiotics to test synergistic activity against 300 multidrug-resistant pathogens isolated from cystic fibrosis (CF) patients. Clarithromycin-tobramycin was most active against
Pseudomonas aeruginosa
and inhibited 58% of strains. Azithromycin-trimethoprim-sulfamethoxazole, azithromycin-ceftazidime, and azithromycin-doxycycline or azithromycin-trimethoprim-sulfamethoxazole inhibited 40, 20, and 22% of
Stenotrophomonas maltophilia
,
Burkholderia cepacia
complex, and
Achromobacter
(
Alcaligenes
)
xylosoxidans
strains, respectively.
30S Ribosomal Subunit Assembly Is a Target for Inhibition by Aminoglycosides in
Escherichia coli
Authors: Mehta Roopal , Champney W. Scott
Published: July 2002

The aminoglycosides paromomycin and neomycin were examined in
Escherichia coli
cells for an inhibitory effect on 30S ribosomal subunit assembly. Both compounds inhibited the growth rate, viable cell number, and protein synthesis rate with similar 50% inhibitory concentrations. Each drug also showed a concentration-dependent inhibition of 30S subunit formation. The inhibitory effect on 30S particle formation was approximately equivalent to the inhibitory effect on translation for these antibiotics.
rpoB
Mutation Conferring Rifampin Resistance in
Streptococcus pyogenes
Authors: Aubry-Damon Hélène , Galimand Marc , Gerbaud Guy , Courvalin Patrice
Published: July 2002

Streptococcus pyogenes
BM4478 and
Staphylococcus aureus
BM4479 were isolated from a patient undergoing rifampin therapy. High-level resistance to rifampin was due to the following mutations in the
rpoB
gene: Ser
522
Leu in strain BM4478 and His
526
Asn and Ser
574
Leu in strain BM4479.
In Vitro Activities of Ravuconazole and Voriconazole Compared with Those of Four Approved Systemic Antifungal Agents against 6,970 Clinical Isolates of
Candida
spp
Authors: Pfaller M. A. , Messer S. A. , Hollis R. J. , Jones R. N. , Diekema D. J.
Published: July 2002

The in vitro activities of ravuconazole and voriconazole were compared with those of amphotericin B, flucytosine (5FC), itraconazole, and fluconazole against 6,970 isolates of
Candida
spp. obtained from over 200 medical centers worldwide. Both ravuconazole and voriconazole were very active against all
Candida
spp. (MIC at which 90% of the isolates tested are inhibited [MIC
90
], 0.25 μg/ml; 98% of MICs were ≤1 μg/ml); however, a decrease in the activities of both of these agents was noted among isolates that were susceptible-dose dependent (fluconazole MIC, 16 to 32 μg/ml) and resistant (MIC, ≥ 64 μg/ml) to fluconazole.
Candida albicans
was the most susceptible species (MIC
90
of both ravuconazole and voriconazole, 0.03 μg/ml), and
C. glabrata
was the least susceptible species (MIC
90
, 1 to 2 μg/ml). Ravuconazole and voriconazole were each more active in vitro than amphotericin B, 5FC, itraconazole, and fluconazole against all
Candida
spp. and were the only agents with good in vitro activity against
C. krusei
. These results provide further evidence for the spectrum and potency of ravuconazole and voriconazole against a large and geographically diverse collection of
Candida
spp.
Cold Shock and Its Effect on Ribosomes and Thermal Tolerance in
Listeria monocytogenes
Authors: Bayles Darrell O. , Tunick Michael H. , Foglia Thomas A. , Miller Arthur J.
Published: July 2002

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of
Listeria monocytogenes
. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0°C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 ± 0.1°C (mean ± standard deviation) to 72.1 ± 0.5°C (
P
≤ 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 ± 0.4°C to 66.8 ± 0.5°C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in
D
value observed when
L. monocytogenes
is cold shocked.
Internal Trehalose Protects Endocytosis from Inhibition by Ethanol inSaccharomyces cerevisiae
Authors: Lucero P. , Peñalver E. , Moreno E. , Lagunas R.
Published: July 2002

Endocytosis inSaccharomyces cerevisiaeis inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.
Advances in Development of a Genetic System for
Thermoanaerobacterium
spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome
Authors: Mai Volker , Wiegel Juergen
Published: July 2002

Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system,
Thermoanaerobacterium saccharolyticum
JW/SL-YS485. Transformed
Thermoanaerobacterium
expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for
T. saccharolyticum
JW/SL-YS485, two improved
Escherichia coli-Thermoanaerobacterium
shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (
cbhA
) from
Clostridium thermocellum
JW20 were integrated into the xylanase gene (
xynA
) region of the
Thermoanaerobacterium
chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.
Isolation, Purification, and Characterization of a Killer Protein fromSchwanniomyces occidentalis
Authors: Chen Wen-Bao , Han Yuh-Fehng , Jong Shung-Chang , Chang Shenq-Chyi
Published: July 2002

The yeastSchwanniomyces occidentalisproduces a killer toxin lethal to sensitive strains ofSaccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 fromS. cerevisiaeat positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40°C. The killer protein was chromosomally encoded. Mannan, but not β-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain ofS. occidentalismay help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.
Construction and Characterization of an
Escherichia coli
Strain Genetically Engineered for Ni(II) Bioaccumulation
Authors: Krishnaswamy Rahul , Wilson David B.
Published: July 2002

An
Escherichia coli
strain that accumulated Ni(II) was constructed by introducing the
nixA
gene (coding for a nickel transport system) from
Helicobacter pylori
into JM109 cells that expressed a glutathione
S
-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.
Chemoraces and Habitat Specialization of
Claviceps purpurea
Populations
Authors: Pažoutová Sylvie , Olšovská Jana , Linka Marek , Kolı́nská Renata , Flieger Miroslav
Published: July 2002

We studied genetic variability of 100 isolates of
Claviceps purpurea
by using randomly amplified polymorphic DNA (RAPD), an
Eco
RI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from
Spartina anglica
from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an
Eco
RI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of
C. purpurea
are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.
Dissimilatory Metal Reduction by the Facultative Anaerobe
Pantoea agglomerans
SP1
Authors: Francis Chris A. , Obraztsova Anna Y. , Tebo Bradley M.
Published: July 2002

Anaerobic enrichments with acetate as the electron donor and Fe(III) as the terminal electron acceptor were obtained from sediments of Salt Pond, a coastal marine basin near Woods Hole, Mass. A pure culture of a facultatively anaerobic Fe(III) reducer was isolated, and 16S rRNA analysis demonstrated that this organism was most closely related to
Pantoea
(formerly
Enterobacter
)
agglomerans
, a member of the family
Enterobacteriaceae
within the gamma subdivision of the
Proteobacteria
. This organism, designated strain SP1, can grow by coupling the oxidation of acetate or H
2
to the reduction of a variety of electron acceptors, including Fe(III), Mn(IV), Cr(VI), and the humic substance analog 2,6-anthraquinone disulfonate, but not sulfate. To our knowledge, this is the first mesophilic facultative anaerobe reported to couple acetate oxidation to dissimilatory metal reduction.
Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway of
Aspergillus niger
Authors: Ngiam Celina , Jeenes David J. , Punt Peter J. , Van Den Hondel Cees A. M. J. J. , Archer David B.
Published: July 2002

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene,
pdiA
, encoding PDIA was previously isolated from
Aspergillus niger
, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A.
pdiA
also complemented PDI function in a
Saccharomyces cerevisiae Δpdi1
mutant in a yeast-based killer toxin assay. Levels of
pdiA
mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of
pdiA
mRNA levels was slower and lower in magnitude than that of
A. niger bipA
, suggesting that the induction of
pdiA
is not part of the primary stress response. An increased level of
pdiA
transcripts was also observed in two
A. niger
strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in
S. cerevisiae
, overexpression of PDIA did not increase secreted yields of HEWL in
A. niger
, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of
pdiA
by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.
Phosphate Inhibits Acetotrophic Methanogenesis on Rice Roots
Authors: Conrad Ralf , Klose Melanie , Claus Peter
Published: July 2002

The contribution of acetate- and H
2
/CO
2
-dependent methanogenesis to total CH
4
production was determined in excised washed rice roots by radiolabeling, methyl fluoride inhibition, and stable carbon isotope fractionation. Addition of ≥20 mM phosphate inhibited methanogenesis, which then was exclusively from H
2
/CO
2
. Otherwise, acetate contributed about 50 to 60% of the total methanogenesis, demonstrating that phosphate specifically inhibited acetotrophic methanogens on rice roots.