bluuebunny/crossref_metadata_2025 huggingface dataset:
Full Three-Dimensional Photonic Bandgap Crystals at Near-Infrared Wavelengths
Authors: Noda Susumu , Tomoda Katsuhiro , Yamamoto Noritsugu , Chutinan Alongkarn
Published: July 2002

An artificial crystal structure has been fabricated exhibiting a full three-dimensional photonic bandgap effect at optical communication wavelengths. The photonic crystal was constructed by stacking 0.7-micrometer period semiconductor stripes with the accuracy of 30 nanometers by advanced wafer-fusion technique. A bandgap effect of more than 40 decibels (which corresponds to 99.99% reflection) was successfully achieved. The result encourages us to create an ultra-small optical integrated circuit including a three-dimensional photonic crystal waveguide with a sharp bend.
Twists in Catalysis: Alternating Conformations of
Escherichia coli
Thioredoxin Reductase
Authors: Lennon Brett W. , Williams Charles H. , Ludwig Martha L.
Published: July 2002

In thioredoxin reductase (TrxR) from
Escherichia coli
, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains. We describe the structure of the flavin-reducing conformation of
E. coli
TrxR at a resolution of 3.0 angstroms. The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin. The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide. Comparison of these structures demonstrates that switching between the two conformations involves a “ball-and-socket” motion in which the pyridine nucleotide–binding domain rotates by 67 degrees.
Learning-Induced LTP in Neocortex
Authors: Rioult-Pedotti Mengia-S. , Friedman Daniel , Donoghue John P.
Published: July 2002

The hypothesis that learning occurs through long-term potentiation (LTP)– and long-term depression (LTD)–like mechanisms is widely held but unproven. This hypothesis makes three assumptions: Synapses are modifiable, they modify with learning, and they strengthen through an LTP-like mechanism. We previously established the ability for synaptic modification and a synaptic strengthening with motor skill learning in horizontal connections of the rat motor cortex (MI). Here we investigated whether learning strengthened these connections through LTP. We demonstrated that synapses in the trained MI were near the ceiling of their modification range, compared with the untrained MI, but the range of synaptic modification was not affected by learning. In the trained MI, LTP was markedly reduced and LTD was enhanced. These results are consistent with the use of LTP to strengthen synapses during learning.
Impacts of Climatic Change and Fishing on Pacific Salmon Abundance Over the Past 300 Years
Authors: Finney Bruce P. , Gregory-Eaves Irene , Sweetman Jon , Douglas Marianne S. V. , Smol John P.
Published: July 2002

The effects of climate variability on Pacific salmon abundance are uncertain because historical records are short and are complicated by commercial harvesting and habitat alteration. We use lake sediment records of δ
15
N and biological indicators to reconstruct sockeye salmon abundance in the Bristol Bay and Kodiak Island regions of Alaska over the past 300 years. Marked shifts in populations occurred over decades during this period, and some pronounced changes appear to be related to climatic change. Variations in salmon returns due to climate or harvesting can have strong impacts on sockeye nursery lake productivity in systems where adult salmon carcasses are important nutrient sources.
Contributions of Land-Use History to Carbon Accumulation in U.S. Forests
Authors: Caspersen John P. , Pacala Stephen W. , Jenkins Jennifer C. , Hurtt George C. , Moorcroft Paul R. , Birdsey Richard A.
Published: July 2002

Carbon accumulation in forests has been attributed to historical changes in land use and the enhancement of tree growth by CO
2
fertilization, N deposition, and climate change. The relative contribution of land use and growth enhancement is estimated by using inventory data from five states spanning a latitudinal gradient in the eastern United States. Land use is the dominant factor governing the rate of carbon accumulation in these states, with growth enhancement contributing far less than previously reported. The estimated fraction of aboveground net ecosystem production due to growth enhancement is 2.0 ± 4.4%, with the remainder due to land use.
Coherence and Conservation
Authors: Earn David J. D. , Levin Simon A. , Rohani Pejman
Published: July 2002

A principal aim of current conservation policy is to reduce the impact of habitat fragmentation. Conservation corridors may achieve this goal by facilitating movement among isolated patches, but there is a risk that increased connectivity could synchronize local population fluctuations (causing coherent oscillations) and thereby increase the danger of global extinction. We identify general conditions under which populations can or cannot undergo coherent oscillations, and we relate these conditions to local and global extinction probabilities. We suggest a simple method to explore the potential success of conservation corridors and, more generally, any manipulations of dispersal patterns that aim to protect threatened species or control pests.
Range Shifts and Adaptive Responses to Quaternary Climate Change
Authors: Davis Margaret B. , Shaw Ruth G.
Published: July 2002

Tree taxa shifted latitude or elevation range in response to changes in Quaternary climate. Because many modern trees display adaptive differentiation in relation to latitude or elevation, it is likely that ancient trees were also so differentiated, with environmental sensitivities of populations throughout the range evolving in conjunction with migrations. Rapid climate changes challenge this process by imposing stronger selection and by distancing populations from environments to which they are adapted. The unprecedented rates of climate changes anticipated to occur in the future, coupled with land use changes that impede gene flow, can be expected to disrupt the interplay of adaptation and migration, likely affecting productivity and threatening the persistence of many species.
Arabidopsis
, the Rosetta Stone of Flowering Time?
Authors: Simpson Gordon G. , Dean Caroline
Published: July 2002

Multiple environmental and endogenous inputs regulate when plants flower. The molecular genetic dissection of flowering time control in
Arabidopsis
has identified an integrated network of pathways that quantitatively control the timing of this developmental switch. This framework provides the basis to understand the evolution of different reproductive strategies and how floral pathways interact through seasonal progression.
In Vivo Activity of Amphotericin B Lipid Complex in Immunocompromised Mice against Fluconazole-Resistant or Fluconazole-Susceptible
Candida tropicalis
Authors: Warn P. A. , Morrissey J. , Moore C. B. , Denning D. W.
Published: July 2002

We compared four doses of amphotericin B lipid complex (ABLC) with three doses of fluconazole in temporarily neutropenic mice in a murine model of disseminated candidiasis due to four different isolates of
Candida tropicalis
. The mice were infected with a 90% lethal dose of four strains of
C. tropicalis
for which the fluconazole MICs ranged from 1 to >125 mg/liter 3 days after receiving 200 mg of cyclophosphamide/kg of body weight. Treatment was started 18 h after infection and lasted for 7 days. ABLC (1, 2, 5, and 10 mg/kg) was administered once a day intravenously, fluconazole was administered by oral gavage once daily (25 and 50 mg/kg/day) or twice daily (125 mg/kg). MICs determined in five different ways with 24- and 48-h endpoints were also compared. The overall survival rates were controls, 14%; fluconazole, 64%; and ABLC, 82%. Treatment with ABLC at 2 to 10 mg/kg increased survival compared to controls (
P
= <0.0001) and was also superior to fluconazole at 25 and 50 mg/kg (
P
= 0.006). In the fluconazole-resistant
C. tropicalis
model (MIC, 128 μg/ml), ABLC at 2 to 10 mg/kg was superior to fluconazole at 250 mg/kg and ABLC at 10 mg/kg was superior to all fluconazole doses (
P
= <0.05). Fluconazole at 250 mg/kg daily was superior to both 25 and 50 mg/kg at reducing mortality with most isolates. ABLC was superior to fluconazole (
P
= <0.01), and fluconazole at 250 mg/kg was superior to fluconazole at both 25 and 50 mg/kg (
P
= 0.02) in all models at reducing
C. tropicalis
counts in the kidneys. Neither drug consistently sterilized the brain or kidneys. A 48-h endpoint reading with the NCCLS susceptibility testing microtiter variation overestimates resistance to fluconazole. ABLC is an effective treatment for fluconazole-resistant
C. tropicalis
at all doses tested.
Antipneumococcal Activities of Gemifloxacin Compared to Those of Nine Other Agents
Authors: Davies Todd A. , Kelly Linda M. , Pankuch Glenn A. , Credito Kim L. , Jacobs Michael R. , Appelbaum Peter C.
Published: July 2002

The activities of gemifloxacin compared to those of nine other agents was tested against a range of penicillin-susceptible and -resistant pneumococci by agar dilution, microdilution, time-kill, and post-antibiotic effect (PAE) methods. Against 64 penicillin-susceptible, 68 penicillin-intermediate, and 75 penicillin-resistant pneumococci (all quinolone susceptible), agar dilution MIC
50
s (MICs at which 50% of isolates are inhibited)/MIC
90
s (in micrograms per milliliter) were as follows: gemifloxacin, 0.03/0.06; ciprofloxacin, 1.0/4.0; levofloxacin, 1.0/2.0; sparfloxacin, 0.5/1.0; grepafloxacin, 0.125/0.5; trovafloxacin, 0.125/0.25; amoxicillin, 0.016/0.06 (penicillin-susceptible isolates), 0.125/1.0 (penicillin-intermediate isolates), and 2.0/4.0 (penicillin-resistant isolates); cefuroxime, 0.03/0.25 (penicillin-susceptible isolates), 0.5/2.0 (penicillin-intermediate isolates), and 8.0/16.0 (penicillin-resistant isolates); azithromycin, 0.125/0.5 (penicillin-susceptible isolates), 0.125/>128.0 (penicillin-intermediate isolates), and 4.0/>128.0 (penicillin-resistant isolates); and clarithromycin, 0.03/0.06 (penicillin-susceptible isolates), 0.03/32.0 (penicillin-intermediate isolates), and 2.0/>128.0 (penicillin-resistant isolates). Against 28 strains with ciprofloxacin MICs of ≥8 μg/ml, gemifloxacin had the lowest MICs (0.03 to 1.0 μg/ml; MIC
90
, 0.5 μg/ml), compared with MICs ranging between 0.25 and >32.0 μg/ml (MIC
90
s of 4.0 to >32.0 μg/ml) for other quinolones. Resistance in these 28 strains was associated with mutations in
parC
,
gyrA
,
parE
, and/or
gyrB
or efflux, with some strains having multiple resistance mechanisms. For 12 penicillin-susceptible and -resistant pneumococcal strains (2 quinolone resistant), time-kill results showed that levofloxacin at the MIC, gemifloxacin and sparfloxacin at two times the MIC, and ciprofloxacin, grepafloxacin, and trovafloxacin at four times the MIC were bactericidal for all strains after 24 h. Gemifloxacin was uniformly bactericidal after 24 h at ≤0.5 μg/ml. Various degrees of 90 and 99% killing by all quinolones were detected after 3 h. Gemifloxacin and trovafloxacin were both bactericidal at two times the MIC for the two quinolone-resistant pneumococci. Amoxicillin at two times the MIC and cefuroxime at four times the MIC were uniformly bactericidal after 24 h, with some degree of killing at earlier time points. Macrolides gave slower killing against the seven susceptible strains tested, with 99.9% killing of all strains at two to four times the MIC after 24 h. PAEs for five quinolone-susceptible strains were similar (0.3 to 3.0 h) for all quinolones, and significant quinolone PAEs were found for the quinolone-resistant strain.
Characterization of TEM-56, a Novel β-Lactamase Produced by a
Klebsiella pneumoniae
Clinical Isolate
Authors: Neuwirth Catherine , Labia Roger , Siebor Eliane , Pechinot Andre , Madec Stephanie , Chaibi El Bachir , Kazmierczak Antoine
Published: July 2002

TEM-56 produced by a
Klebsiella pneumoniae
clinical isolate is a novel β-lactamase of isoelectric point 6.4 that confers a moderate resistance level to expanded-spectrum cephalosporins. The amino acid sequence deduced from the corresponding
bla
gene showed two amino acid replacements with respect to the TEM-2 sequence: Glu-104 to Lys and His-153 to Arg. This enzyme showed catalytic properties close to those of TEM-18. Thus, TEM-56 appears as a new TEM mutant, an intermediary between TEM-18 and the extended-spectrum β-lactamase TEM-21.
Mechanism of Uptake of a Cationic Water-Soluble Pyridinium Zinc Phthalocyanine across the Outer Membrane of
Escherichia coli
Authors: Minnock Andrew , Vernon David I. , Schofield Jack , Griffiths John , Parish J. Howard , Brown Stanley B.
Published: July 2002

Previous studies have shown that a cationic water-soluble pyridinium zinc phthalocyanine (PPC) is a powerful photosensitizer that is able to inactivate
Escherichia coli
. In the current work incubation of
E. coli
cells with PPC in the dark caused alterations in the outer membrane permeability barrier of the cells, rendering the bacteria much more sensitive to hydrophobic compounds, with little effect seen with hydrophilic compounds. Addition of Mg
2+
to the medium prior to incubation of the cells with PPC prevented these alterations in the outer membrane permeability barrier. The presence of Mg
2+
in the medium also prevented the photoinactivation of
E. coli
cells with PPC. These results are consistent with the hypothesis that PPC gains access across the outer membrane of
E. coli
cells via the self-promoted uptake pathway, a mechanism of uptake postulated for the uptake of other cationic compounds across the outer membranes of gram-negative bacteria.
Role of Antibiotic Penetration Limitation in
Klebsiella pneumoniae
Biofilm Resistance to Ampicillin and Ciprofloxacin
Authors: Anderl Jeff N. , Franklin Michael J. , Stewart Philip S.
Published: July 2002

The penetration of two antibiotics, ampicillin and ciprofloxacin, through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria to killing by the antibiotics were also measured. Biofilms of
Klebsiella pneumoniae
were developed on microporous membranes resting on agar nutrient medium. The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined. Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant
K. pneumoniae
strain in which β-lactamase activity was eliminated. For wild-type
K. pneumoniae
grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 μg/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 ± 0.33 and 4.14 ± 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of −0.06 ± 0.06 and 1.02 ± 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type
K. pneumoniae
biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a β-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 ± 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 ± 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.
Itraconazole Oral Solution for Primary Prophylaxis of Fungal Infections in Patients with Hematological Malignancy and Profound Neutropenia: a Randomized, Double-Blind, Double-Placebo, Multicenter Trial Comparing Itraconazole and Amphotericin B
Authors: Harousseau J. L. , Dekker A. W. , Stamatoullas-Bastard A. , Fassas A. , Linkesch W. , Gouveia J. , De Bock R. , Rovira M. , Seifert W. F. , Joosen H. , Peeters M. , De Beule K.
Published: July 2002

Systemic and superficial fungal infections are a major problem among immunocompromised patients with hematological malignancy. A double-blind, double-placebo, randomized, multicenter trial was performed to compare the efficacy and safety of itraconazole oral solution (2.5 mg/kg of body weight twice a day) with amphotericin B capsules (500 mg orally four times a day) for prophylaxis of systemic and superficial fungal infection. Prophylactic treatment was initiated on the first day of chemotherapy and was continued until the end of the neutropenic period (>0.5 × 109neutrophils/liter) or up to a maximum of 3 days following the end of neutropenia, unless a systemic fungal infection was documented or suspected. The maximum treatment duration was 56 days. In the intent-to-treat population, invasive aspergillosis was noted in 5 (1.8%) of the 281 patients assigned to itraconazole oral solution and in 9 (3.3%) of the 276 patients assigned to oral amphotericin B; of these, 1 and 4 patients died, respectively. Proven systemic fungal infection (including invasive aspergillosis) occurred in 8 patients (2.8%) who received itraconazole, compared with 13 (4.7%) who received oral amphotericin B. Itraconazole significantly reduced the incidence of superficial fungal infections as compared to oral amphotericin B (2 [1%] versus 13 [5%];P= 0.004). Although the incidences of suspected fungal infection (including fever of unknown origin) were not different between the groups, fewer patients were administered intravenous systemic antifungals (mainly intravenous amphotericin B) in the group receiving itraconazole than in the group receiving oral amphotericin B (114 [41%] versus 132 [48%];P= 0.066). Adequate plasma itraconazole levels were achieved in about 80% of the patients from 1 week after the start of treatment. In both groups, the trial medication was safe and well tolerated. Prophylactic administration of itraconazole oral solution significantly reduces superficial fungal infection in patients with hematological malignancies and neutropenia. The incidence of proven systemic fungal infections, the number of deaths due to deep fungal infections, and the use of systemic antifungals tended to be lower in the itraconazole-treated group than in the amphotericin B-treated group, without statistical significance. Itraconazole oral solution is a broad-spectrum systemic antifungal agent with prophylactic activity in neutropenic patients, especially for those at high risk of prolonged neutropenia.
Anti-
Toxoplasma
Activities of Antiretroviral Drugs and Interactions with Pyrimethamine and Sulfadiazine In Vitro
Authors: Derouin Francis , Santillana-Hayat Maud
Published: July 2002

The anti-
Toxoplasma
activities of nine antiretroviral drugs were examined in vitro. Nucleoside analogs had no effect on parasite growth, whereas ritonavir and nelfinavir were inhibitory for
Toxoplasma
, with 50% inhibitory concentrations of 5.4 and 4.0 μg/ml, respectively. None of the antiviral drugs affected the anti-
Toxoplasma
activity of pyrimethamine or sulfadiazine.
Effectiveness of Once-Weekly Rifapentine and Moxifloxacin Regimens against
Mycobacterium tuberculosis
in Mice
Authors: Lounis Nacer , Bentoucha Abdelhalim , Truffot-Pernot Chantal , Ji Baohong , O'Brien Richard J. , Vernon Andrew , Roscigno Giorgio , Grosset Jacques
Published: July 2002

Mice infected with 1.6 × 10
7
CFU of
Mycobacterium tuberculosis
were treated 14 days later for 6 months with a regimen of once-weekly 10 mg of rifapentine and 75 mg of isoniazid per kg of body weight supplemented with either 150 mg of streptomycin per kg or 100 mg of moxifloxacin per kg during either both the 2-week daily initial and once-weekly continuation phases or only in the daily 2-week initial phase. On completion of treatment, all lung cultures were negative, except for three mice, each with a single colony: two whose rifapentine-isoniazid regimen was supplemented with streptomycin during the whole course of therapy and one whose rifapentine-isoniazid regimen had no initial daily phase, but was supplemented with streptomycin and moxifloxacin during the whole course of therapy. After 3 months of follow-up, positive lung cultures were obtained from 61 and 56% of mice supplemented with streptomycin during either the full course of therapy or only the daily 2-week initial phase, respectively, and 15 and 50% of mice supplemented with moxifloxacin during either the full course of therapy or only the daily 2-week initial phase, respectively. These results suggest that moxifloxacin has sterilizing activity against
M. tuberculosis
.
Safety, Tolerance, and Pharmacokinetics of High-Dose Liposomal Amphotericin B (AmBisome) in Patients Infected withAspergillusSpecies and Other Filamentous Fungi: Maximum Tolerated Dose Study
Authors: Walsh Thomas J. , Goodman Jesse L. , Pappas Peter , Bekersky Ihor , Buell Donald N. , Roden Maureen , Barrett John , Anaissie Elias J.
Published: July 2002

We conducted a phase I-II study of the safety, tolerance, and plasma pharmacokinetics of liposomal amphotericin B (L-AMB; AmBisome) in order to determine its maximally tolerated dosage (MTD) in patients with infections due toAspergillusspp. and other filamentous fungi. Dosage cohorts consisted of 7.5, 10.0, 12.5, and 15.0 mg/kg of body weight/day; a total of 44 patients were enrolled, of which 21 had a proven or probable infection (13 aspergillosis, 5 zygomycosis, 3 fusariosis). The MTD of L-AMB was at least 15 mg/kg/day. Infusion-related reactions of fever occurred in 8 (19%) and chills and/or rigors occurred in 5 (12%) of 43 patients. Three patients developed a syndrome of substernal chest tightness, dyspnea, and flank pain, which was relieved by diphenhydramine. Serum creatinine increased two times above baseline in 32% of the patients, but this was not dose related. Hepatotoxicity developed in one patient. Steady-state plasma pharmacokinetics were achieved by day 7. The maximum concentration of drug in plasma (Cmax) of L-AMB in the dosage cohorts of 7.5, 10.0, 12.5, and 15.0 mg/kg/day changed to 76, 120, 116, and 105 μg/ml, respectively, and the mean area under the concentration-time curve at 24 h (AUC24) changed to 692, 1,062, 860, and 554 μg · h/ml, respectively, while mean CL changed to 23, 18, 16, and 25 ml/h/kg, respectively. These data indicate that L-AMB follows dose-related changes in disposition processing (e.g., clearance) at dosages of ≥7.5 mg/kg/day. Because several extremely ill patients had early death, success was determined for both the modified intent-to-treat and evaluable (7 days of therapy) populations. Response rates (defined as complete response and partial response) were similar for proven and probable infections. Response and stabilization, respectively, were achieved in 36 and 16% of the patients in the modified intent-to-treat population (n= 43) and in 52 and 13% of the patients in the 7-day evaluable population (n= 31). These findings indicate that L-AMB at dosages as high as 15 mg/kg/day follows nonlinear saturation-like kinetics, is well tolerated, and can provide effective therapy for aspergillosis and other filamentous fungal infections.
Plasmid Location and Molecular Heterogeneity of the L1 and L2 β-Lactamase Genes of
Stenotrophomonas maltophilia
Authors: Avison Matthew B. , Higgins Catherine S. , von Heldreich Charlotte J. , Bennett Peter M. , Walsh Timothy R.
Published: July 2002

An approximately 200-kb plasmid has been purified from clinical isolates of
Stenotrophomonas maltophilia
. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 β-lactamase genes. The location of
L1
and
L2
on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes,
L1c, L1d
, and
L1e
, with 8, 12, and 20% divergence from the published strain IID 1275
L1
(
L1a
), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower
k
cat
/

K
m

ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (
L2b, L2c
, and
L2d
) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275
L2
(
L2a
) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.
Drug Targeting
Mycobacterium tuberculosis
Cell Wall Synthesis: Genetics of dTDP-Rhamnose Synthetic Enzymes and Development of a Microtiter Plate-Based Screen for Inhibitors of Conversion of dTDP-Glucose to dTDP-Rhamnose
Authors: Ma Yufang , Stern Richard J. , Scherman Michael S. , Vissa Varalakshmi D. , Yan Wenxin , Jones Victoria Cox , Zhang Fangqiu , Franzblau Scott G. , Lewis Walter H. , McNeil Michael R.
Published: July 2002

An
l
-rhamnosyl residue plays an essential structural role in the cell wall of
Mycobacterium tuberculosis
. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics.
M. tuberculosis
genes encoding RmlA, RmlC, and RmlD have been identified and expressed in
Escherichia coli
. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the
M. tuberculosis
genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP
+
with a concomitant decrease in optical density at 340 nm (OD
340
). Inhibitor candidates were monitored for their ability to lower the rate of OD
340
change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 μM were identified; four of these showed activities against whole
M. tuberculosis
cells, with MICs from 128 to 16 μg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole
M. tuberculosis
cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of
M. tuberculosis
in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 μg/ml.
Effect of Drug Concentration on Emergence of Macrolide Resistance in
Mycobacterium avium
Authors: Nash Kevin A.
Published: July 2002

The emergence of antibiotic resistance in mycobacteria involves the selection of mutant variants within a susceptible bacterial population. However, it is unclear whether antimycobacterial drugs act just as selective agents or can influence the rate of appearance of resistant mutants. The present study was initiated to address this issue by monitoring the effects of antimicrobial agents on the appearance and growth of clarithromycin (CLR)-resistant (CLR
r
) bacilli in broth cultures of
Mycobacterium avium
. Preexposure of
M. avium
to CLR had a significant dose effect on the emergence of resistance, with concentrations of 4 to 8 μg/ml resulting in a maximal (∼10
4
-fold) increase in the number of CLR
r
bacilli after a 4-day incubation. In addition, a dose effect was found with azithromycin. The use of combinations of CLR with either ethambutol (EMB) or rifabutin (RFB) resulted in fewer resistant bacilli compared to the use of CLR alone. The lowest active concentration of EMB (4 μg/ml) was equivalent to the EMB MIC (4 to 8 μg/ml) for the parental CLR
s
strain and the emergent CLR
r
variants, and thus, the antiresistance effect was probably the result of the bacteriostatic effect of EMB on CLR
r
bacilli. However, RFB was an order of magnitude more active (0.05 μg/ml) at reducing resistance than suggested by the MIC of this agent (0.5 to 1 μg/ml). These results indicate that the emergence of resistance was not simply the selection of a preexisting subpopulation of resistant bacilli. Further analysis suggested that early events in the emergence of resistance involved organisms (progenitors) that acquired a resistance phenotype. In addition, the progenitors appeared to be in a transient state, able to develop into a stable resistant lineage in the presence of CLR, or able to revert to the wild type in nonselective conditions.
Expression of Multidrug Efflux Pump SmeDEF by Clinical Isolates of
Stenotrophomonas maltophilia
Authors: Alonso Ana , Martinez Jose L.
Published: July 2002

The presence of the multidrug efflux pump SmeDEF was assessed in a collection of clinical isolates of
Stenotrophomonas maltophilia
. All isolates encoded this pump, as demonstrated by PCR. Forty-seven percent of the strains overproduced a protein of the same size that was immunoreactive against an anti-SmeF antibody, and 33% overexpressed the gene
semD
when they were tested by reverse transcription-PCR. A correlation between
smeDEF
overexpression and antibiotic resistance was observed.
Mutational Replacement of Leu-293 in the Class C
Enterobacter cloacae
P99 β-Lactamase Confers Increased MIC of Cefepime
Authors: Vakulenko Sergei B. , Golemi Dasantila , Geryk Bruce , Suvorov Maxim , Knox James R. , Mobashery Shahriar , Lerner Stephen A.
Published: July 2002

The class C β-lactamase from
Enterobacter cloacae
P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the
E. cloacae
P99
ampC
gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified
E. cloacae
P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (
k
cat
/

K
m

) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.
Biodegradation ofcis-1,4-Polyisoprene Rubbers by Distinct Actinomycetes: Microbial Strategies and Detailed Surface Analysis
Authors: Linos Alexandros , Berekaa Mahmoud M. , Reichelt Rudolf , Keller Ulrike , Schmitt Jürgen , Flemming Hans-Curt , Kroppenstedt Reiner M. , Steinbüchel Alexander
Published: July 2002

Several actinomycetes isolated from nature were able to use both natural rubber (NR) and syntheticcis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed twoGordoniasp. strains, VH2 and Kb2, and oneMycobacterium fortuitumstrain, NF4, belonging to the first group as well as oneMicromonospora aurantiacastrain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of theGordoniastrains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number ofcis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.
Bile Salt Hydrolase of
Bifidobacterium longum
—Biochemical and Genetic Characterization
Authors: Tanaka Hiroshi , Hashiba Honoo , Kok Jan , Mierau Igor
Published: July 2002

A bile salt hydrolase (BSH) was isolated from
Bifidobacterium longum
SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (
bsh
) in a member of the genus
Bifidobacterium
. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of
B. longum
BSH is between 5 and 7, and the temperature optimum is 40°C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH of
B. longum
can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the

K
m

values. The nucleotide sequence of
bsh
was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of
Bacillus sphaericus
were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that
bsh
is part of an operon containing at least two genes,
bsh
and
glnE
(GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from
B. longum
SBT2928 cultures and characterized. These mutants had point mutations that inactivated
bsh
by premature termination, frameshift, or amino acid exchange.
Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic
Escherichia coli
O157:H7 Strains in Environmental Samples
Authors: Kimura Richard , Mandrell Robert E. , Galland John C. , Hyatt Doreene , Riley Lee W.
Published: July 2002

Enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting
E. coli
O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of
E. coli
isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be
E. coli
O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish
E. coli
O157:H7 from a large number of
E. coli
isolates from environmental samples.
The Common Nodulation Genes of
Astragalus sinicus
Rhizobia Are Conserved despite Chromosomal Diversity
Authors: Zhang Xue-Xian , Turner Sarah L. , Guo Xian-Wu , Yang Hui-Jun , Debellé Frédéric , Yang Guo-Ping , Dénarié Jean , Young J. Peter W. , Li Fu-Di
Published: July 2002

The nodulation genes of
Mesorhizobium
sp. (
Astragalus sinicus
) strain 7653R were cloned by functional complementation of
Sinorhizobium meliloti nod
mutants. The common
nod
genes,
nodD
,
nodA
, and
nodBC
, were identified by heterologous hybridization and sequence analysis. The
nodA
gene was found to be separated from
nodBC
by approximately 22 kb and was divergently transcribed. The 2.0-kb
nodDBC
region was amplified by PCR from 24 rhizobial strains nodulating
A. sinicus
, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of
nodA
from
nodBC
is a common feature of
A. sinicus
rhizobia. Sequence analysis of the PCR-amplified
nodA
gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical
nodA
sequences. These data indicate that, whereas microsymbionts of
A. sinicus
exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of
nod
genes among diverse recipient bacteria.
Fungal Colonization and Biodeterioration of Plasticized Polyvinyl Chloride
Authors: Webb Jeremy S. , Nixon Marianne , Eastwood Ian M. , Greenhalgh Malcolm , Robson Geoffrey D. , Handley Pauline S.
Published: July 2002

Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics.Aureobasidium pullulanswas the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, includingRhodotorula aurantiacaandKluyveromycesspp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominatedA. pullulansafter 95 weeks, with a mean viable count ± standard error of 1,000 ± 200 yeast CFU cm−2, compared to 390 ± 50A. pullulansCFU cm−2. No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains ofA. pullulanstested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and thatA. pullulansis critical to the establishment of a microbial community on pPVC.
Analysis of
gyrB
and
toxR
Gene Sequences of
Vibrio hollisae
and Development of
gyrB
- and
toxR
-Targeted PCR Methods for Isolation of
V. hollisae
from the Environment and Its Identification
Authors: Vuddhakul Varaporn , Nakai Toshihiro , Matsumoto Chiho , Oh Takanori , Nishino Takeshi , Chen Chien-Hsien , Nishibuchi Mitsuaki , Okuda Jun
Published: July 2002

Isolation of
Vibrio hollisae
strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of
V. hollisae
. The two genes are presumed to be conserved among the bacterial species (
gyrB
) or among the species of the genus
Vibrio
(
toxR
). A portion of the
gyrB
sequence of
V. hollisae
was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding
Vibrio parahaemolyticus gyrB
sequence. The
toxR
gene of
V. hollisae
was cloned utilizing a
htpG
gene probe derived from the
V. parahaemolyticus htpG
gene, which is known to be linked to the
toxR
gene in
V. hollisae
. The coding sequence of the cloned
V. hollisae toxR
gene had 59% identity with the
V. parahaemolyticus toxR
coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of
V. hollisae
indicated that these gene sequences could be utilized for differentiation of
V. hollisae
from other
Vibrio
species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of
V. hollisae
but not other organisms. A strain of
V. hollisae
added at a concentration of 1 to 10
2
CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37°C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the
V. hollisae toxR
gene. We conclude that screening of seafood samples by this 35-cycle,
V. hollisae toxR
-specific PCR, followed by isolation on a differential medium and identification by the above
htpG
- and
toxR
-targeted PCR methods, can be useful for isolation from the environment and identification of
V. hollisae
.
Purification and Characterization of Novel Antifungal Compounds from the Sourdough
Lactobacillus plantarum
Strain 21B
Authors: Lavermicocca Paola , Valerio Francesca , Evidente Antonio , Lazzaroni Silvia , Corsetti Aldo , Gobbetti Marco
Published: July 2002

Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of
Lactobacillus plantarum
21B grown in wheat flour hydrolysate almost completely inhibited
Eurotium repens
IBT18000,
Eurotium rubrum
FTDC3228,
Penicillium corylophilum
IBT6978,
Penicillium roqueforti
IBT18687,
Penicillium expansum
IDM/FS2,
Endomyces fibuliger
IBT605 and IDM3812,
Aspergillus niger
FTDC3227 and IDM1,
Aspergillus flavus
FTDC3226,
Monilia sitophila
IDM/FS5, and
Fusarium graminearum
IDM623. The nonconcentrated culture filtrate of
L. plantarum
21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml
−1
was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to
L. plantarum
21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of
L. plantarum
21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml
−1
except for
P. roqueforti
IBT18687 and
P. corylophilum
IBT6978 (inhibitory concentration, 166 mg ml
−1
).
L. plantarum
20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of
A. niger
FTDC3227 occurred after 2 days in breads started with
Saccharomyces cerevisiae
141 alone or with
S. cerevisiae
and
Lactobacillus brevis
1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with
S. cerevisiae
and selected
L. plantarum
21B.
The Sigma Factor AlgU (AlgT) Controls Exopolysaccharide Production and Tolerance towards Desiccation and Osmotic Stress in the Biocontrol Agent
Pseudomonas fluorescens
CHA0
Authors: Schnider-Keel Ursula , Lejbølle Kirsten Bang , Baehler Eric , Haas Dieter , Keel Christoph
Published: July 2002

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and ς
22
) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent
Pseudomonas fluorescens
CHA0. The
algU
-
mucA
-
mucB
gene cluster of strain CHA0 was similar to that of the pathogens
Pseudomonas aeruginosa
and
Pseudomonas syringae
. Strain CHA0 is naturally nonmucoid, whereas a
mucA
deletion mutant or
algU
-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An
algU
deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the
mucA
mutant were. Expression of an
algU
′
-
′
lacZ
reporter fusion was induced severalfold in the wild type and in the
mucA
mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the
algU
mutant. Overexpression of
algU
did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of
P. fluorescens
towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the
algU
mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of
P. fluorescens
towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of
P. fluorescens
to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.