bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Daily Clouds on Titan
Authors: Griffith Caitlin A. , Hall Joseph L. , Geballe Thomas R.
Published: July 2002

We have discovered frequent variations in the near-infrared spectrum of Titan, Saturn's largest moon, which are indicative of the daily presence of sparse clouds covering less than 1% of the area of the satellite. The thermodynamics of Titan's atmosphere and the clouds' altitudes suggest that convection governs their evolutions. Their short lives point to the presence of rain. We propose that Titan's atmosphere resembles Earth's, with clouds, rain, and an active weather cycle, driven by latent heat release from the primary condensible species.
A Low Temperature Transfer of ALH84001 from Mars to Earth
Authors: Weiss Benjamin P. , Kirschvink Joseph L. , Baudenbacher Franz J. , Vali Hojatollah , Peters Nick T. , Macdonald Francis A. , Wikswo John P.
Published: July 2002

The ejection of material from Mars is thought to be caused by large impacts that would heat much of the ejecta to high temperatures. Images of the magnetic field of martian meteorite ALH84001 reveal a spatially heterogeneous pattern of magnetization associated with fractures and rock fragments. Heating the meteorite to 40°C reduces the intensity of some magnetic features, indicating that the interior of the rock has not been above this temperature since before its ejection from the surface of Mars. Because this temperature cannot sterilize most bacteria or eukarya, these data support the hypothesis that meteorites could transfer life between planets in the solar system.
β-Arrestin 2: A Receptor-Regulated MAPK Scaffold for the Activation of JNK3
Authors: McDonald Patricia H. , Chow Chi-Wing , Miller William E. , Laporte Stéphane A. , Field Michael E. , Lin Fang-Tsyr , Davis Roger J. , Lefkowitz Robert J.
Published: July 2002

β-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein–coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of β-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal–regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with β-arrestin 2. Cellular transfection of β-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of β-arrestin 2 and active JNK3 to intracellular vesicles. Thus, β-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.
Accumulation of Dietary Cholesterol in Sitosterolemia Caused by Mutations in Adjacent ABC Transporters
Authors: Berge Knut E. , Tian Hui , Graf Gregory A. , Yu Liqing , Grishin Nick V. , Schultz Joshua , Kwiterovich Peter , Shan Bei , Barnes Robert , Hobbs Helen H.
Published: July 2002

In healthy individuals, acute changes in cholesterol intake produce modest changes in plasma cholesterol levels. A striking exception occurs in sitosterolemia, an autosomal recessive disorder characterized by increased intestinal absorption and decreased biliary excretion of dietary sterols, hypercholesterolemia, and premature coronary atherosclerosis. We identified seven different mutations in two adjacent, oppositely oriented genes that encode new members of the adenosine triphosphate (ATP)–binding cassette (ABC) transporter family (six mutations in
ABCG8
and one in
ABCG5
) in nine patients with sitosterolemia. The two genes are expressed at highest levels in liver and intestine and, in mice, cholesterol feeding up-regulates expressions of both genes. These data suggest that ABCG5 and ABCG8 normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and that mutated forms of these transporters predispose to sterol accumulation and atherosclerosis.
Orchestrated Transcription of Key Pathways in
Arabidopsis
by the Circadian Clock
Authors: Harmer Stacey L. , Hogenesch John B. , Straume Marty , Chang Hur-Song , Han Bin , Zhu Tong , Wang Xun , Kreps Joel A. , Kay Steve A.
Published: July 2002

Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in
Arabidopsis
and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.
Guidance of Cell Migration by EGF Receptor Signaling During
Drosophila
Oogenesis
Authors: Duchek Peter , Rørth Pernille
Published: July 2002

Directed cell migration is important for many aspects of normal animal development, but little is known about how cell migrations are guided or the mechanisms by which guidance cues are translated into directed cell movement. Here we present evidence that signaling mediated by the epidermal growth factor receptor (EGFR) guides dorsal migration of border cells during
Drosophila
oogenesis. The transforming growth factor–α (TGF-α)–like ligand Gurken appears to serve as the guidance cue. To mediate this guidance function, EGFR signals via a pathway that is independent of Raf-MAP kinase and receptor-specific.
Migration of
Plasmodium
Sporozoites Through Cells Before Infection
Authors: Mota Maria M. , Pradel Gabriele , Vanderberg Jerome P. , Hafalla Julius C. R. , Frevert Ute , Nussenzweig Ruth S. , Nussenzweig Victor , Rodrı́guez Ana
Published: July 2002

Intracellular bacteria and parasites typically invade host cells through the formation of an internalization vacuole around the invading pathogen.
Plasmodium
sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, have an alternative mechanism to enter cells. We observed breaching of the plasma membrane of the host cell followed by rapid repair. This mode of entry did not result in the formation of a vacuole around the sporozoite, and was followed by exit of the parasite from the host cell. Sporozoites traversed the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, in which they developed into the next infective stage. Sporozoite migration through several cells in the mammalian host appears to be essential for the completion of the life cycle.
A Role for Flavin Monooxygenase-Like Enzymes in Auxin Biosynthesis
Authors: Zhao Yunde , Christensen Sioux K. , Fankhauser Christian , Cashman John R. , Cohen Jerry D. , Weigel Detlef , Chory Joanne
Published: July 2002

Although auxin is known to regulate many processes in plant development and has been studied for over a century, the mechanisms whereby plants produce it have remained elusive. Here we report the characterization of a dominant
Arabidopsis
mutant,
yucca
, which contains elevated levels of free auxin.
YUCCA
encodes a flavin monooxygenase–like enzyme and belongs to a family that includes at least nine other homologous
Arabidopsis
genes, a subset of which appears to have redundant functions. Results from tryptophan analog feeding experiments and biochemical assays indicate that YUCCA catalyzes hydroxylation of the amino group of tryptamine, a rate-limiting step in tryptophan-dependent auxin biosynthesis.
Microstructured Magnetic Materials for RF Flux Guides in Magnetic Resonance Imaging
Authors: Wiltshire M. C. K. , Pendry J. B. , Young I. R. , Larkman D. J. , Gilderdale D. J. , Hajnal J. V.
Published: July 2002

Magnetic resonance imaging and spectroscopy systems use coils, either singly or as arrays, to intercept radio-frequency (RF) magnetic flux from regions of interest, often deep within the body. Here, we show that a new magnetic material offers novel possibilities for guiding RF flux to the receiver coil, permitting a clear image to be obtained where none might otherwise be detectable. The new material contains microstructure designed according to concepts taken from the field of photonic band gap materials. In the RF range, it has a magnetic permeability that can be produced to specification while exhibiting negligible direct-current magnetism. The latter property is vital to avoid perturbing the static and audio-frequency magnetic fields needed to obtain image and spectral data. The concept offers a new paradigm for the manipulation of RF flux in all nuclear magnetic resonance systems.
DNA Methylation and Epigenetic Inheritance in Plants and Filamentous Fungi
Authors: Martienssen Robert A. , Colot Vincent
Published: July 2002

Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.
Self-Organized Discotic Liquid Crystals for High-Efficiency Organic Photovoltaics
Authors: Schmidt-Mende L. , Fechtenkötter A. , Müllen K. , Moons E. , Friend R. H. , MacKenzie J. D.
Published: July 2002

Self-organization of liquid crystalline and crystalline-conjugated materials has been used to create, directly from solution, thin films with structures optimized for use in photodiodes. The discotic liquid crystal hexa-peri-hexabenzocoronene was used in combination with a perylene dye to produce thin films with vertically segregated perylene and hexabenzocoronene, with large interfacial surface area. When incorporated into diode structures, these films show photovoltaic response with external quantum efficiencies of more than 34 percent near 490 nanometers. These efficiencies result from efficient photoinduced charge transfer between the hexabenzocoronene and perylene, as well as from effective transport of charges through vertically segregated perylene and hexabenzocoronene π systems. This development demonstrates that complex structures can be engineered from novel materials by means of simple solution-processing steps and may enable inexpensive, high-performance, thin-film photovoltaic technology.
Calcium Signaling by HBx Protein in Hepatitis B Virus DNA Replication
Authors: Bouchard Michael J. , Wang Li-Hua , Schneider Robert J.
Published: July 2002

Hepatitis B virus (HBV) infects more than 300 million people and is a leading cause of liver cancer and disease. The HBV HBx protein is essential for infection; HBx activation of Src is important for HBV DNA replication. In our study, HBx activated cytosolic calcium-dependent proline-rich tyrosine kinase–2 (Pyk2), a Src kinase activator. HBx activation of HBV DNA replication was blocked by inhibiting Pyk2 or calcium signaling mediated by mitochondrial calcium channels, which suggests that HBx targets mitochondrial calcium regulation. Reagents that increased cytosolic calcium substituted for HBx protein in HBV DNA replication. Thus, alteration of cytosolic calcium was a fundamental requirement for HBV replication and was mediated by HBx protein.
Resistance to Multiple Fluoroquinolones in a Clinical Isolate of
Streptococcus pyogenes
: Identification of
gyrA
and
parC
and Specification of Point Mutations Associated with Resistance
Authors: Yan S. Steve , Fox Melissa L. , Holland Steven M. , Stock Frida , Gill Vee J. , Fedorko Daniel P.
Published: July 2002

A strain of
Streptococcus pyogenes
resistant to multiple fluoroquinolones was isolated from the blood of an immunocompromised patient. Resistance to fluoroquinolones in
S. pyogenes
has not been previously studied. Compared to 10 sensitive strains of
S. pyogenes
, the fluoroquinolone-resistant clinical isolate of
S. pyogenes
presented point mutations in
gyrA
, predicting that serine-81 was changed to phenylalanine and that methionine-99 was changed to leucine, and in
parC
, predicting that serine-79 was changed to tyrosine. The mechanism of fluoroquinolone resistance in this isolate of
S. pyogenes
appears to be analogous to previously reported mechanisms for
Streptococcus pneumoniae
.
Immunization with the
Candida albicans
Membrane Fraction and in Combination with Fluconazole Protects against Systemic Fungal Infections
Authors: Mizutani Shigetoshi , Endo Masahiro , Ino-ue Toshiaki , Kurasawa Masahiro , Uno Yoko , Saito Hideharu , Kato Ikunoshin , Takesako Kazutoh
Published: July 2002

We studied the immunogenicity of a membrane fraction prepared from
Candida albicans
cells called
C. albicans
membrane antigen (CMA). The present study revealed that CMA immunization has antifungal activity in mouse models of systemic fungal infection. Immunization of mice by subcutaneous injections of CMA with incomplete Freund adjuvant induced resistance to infections caused not only by
C. albicans
but also by
Aspergillus fumigatus
. The level of resistance to candidiasis was as high as that induced by whole-cell immunization. The acquired resistance to candidiasis in the mice immunized with CMA was not diminished by immunosuppressive treatment with cyclophosphamide. The level of resistance to fungal infections was superior to that given by fluconazole (FLC) treatment alone and highly enhanced by the combination with FLC. When CD4
+
cells in CMA-immunized mice were depleted by a monoclonal antibody, the antifungal activity induced by the combination of CMA and FLC was significantly reduced. These results indicate that immunization with CMA is useful for preventing systemic fungal infections and in combination with FLC for increasing resistance after infection.
Multiple Antibiotic Resistance in
Stenotrophomonas maltophilia
: Involvement of a Multidrug Efflux System
Authors: Zhang Li , Li Xian-Zhi , Poole Keith
Published: July 2002

Clinical strains of
Stenotrophomonas maltophilia
are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of
Pseudomonas aeruginosa
hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these
S. maltophilia
MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of
P. aeruginosa
. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the
P. aeruginosa
pump. The outer and cytoplasmic membranes of several clinical
S. maltophilia
strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (
Stenotrophomonas
multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of
P. aeruginosa
and
Pseudomonas putida
. Deletion of the L1 and L2 β-lactamase genes confirmed that these enzymes were responsible for the bulk of the β-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few β-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of
S. maltophilia
.
Inhibition of Inositol Phosphorylceramide Synthase by Aureobasidin A in
Candida
and
Aspergillus
Species
Authors: Zhong Wenyan , Jeffries Matthew W. , Georgopapadakou Nafsika H.
Published: July 2002

Inositol phosphorylceramide (IPC) synthase is an enzyme common to fungi and plants that catalyzes the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC. The reaction is a key step in fungal sphingolipid biosynthesis and the target of the antibiotics galbonolide A, aureobasidin A, and khafrefungin. As a first step toward understanding the antifungal spectrum of IPC synthase inhibitors, we examined the sensitivity of IPC synthase to aureobasidin A in membrane preparations of
Candida
species (
Candida albicans
,
C. glabrata
,
C. tropicalis
,
C. parapsilosis
, and
C. krusei
) and
Aspergillus
species (
Aspergillus fumigatus
,
A. flavus
,
A. niger
, and
A. terreus
). As expected, preparations from the five
Candida
species, all exquisitely susceptible to aureobasidin A (MICs, <2 μg/ml), had IPC synthase activity (specific activity, 50 to 400 pmol/min/mg of protein) sensitive to aureobasidin A (50% inhibitory concentrations [IC
50
s], 2 to 4 ng/ml). Surprisingly, preparations from the four
Aspergillus
species, including
A. fumigatus
and
A. flavus
, which are intrinsically resistant to aureobasidin A (MICs, >50 μg/ml), had IPC synthase activity (specific activity, 1 to 3 pmol/min/mg of protein) also sensitive to aureobasidin A (IC
50
s, 3 to 5 ng/ml). The mammalian multidrug resistance modulators verapamil, chlorpromazine, and trifluoperazine lowered the MIC of aureobasidin A for
A. fumigatus
from >50 μg/ml to 2 to 3 μg/ml, suggesting that the resistance of this major fungal pathogen is the result of increased efflux.
Sodium Dodecyl Sulfate and C31G as Microbicidal Alternatives to Nonoxynol 9: Comparative Sensitivity of Primary Human Vaginal Keratinocytes
Authors: Krebs Fred C. , Miller Shendra R. , Catalone Bradley J. , Welsh Patricia A. , Malamud Daniel , Howett Mary K. , Wigdahl Brian
Published: July 2002

A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides.
In Vitro and In Vivo Activities of SCH 56592 (Posaconazole), a New Triazole Antifungal Agent, against
Aspergillus
and
Candida
Authors: Cacciapuoti Anthony , Loebenberg David , Corcoran Erik , Menzel Fred , Moss Eugene L. , Norris Christine , Michalski Monika , Raynor Kimberly , Halpern Judith , Mendrick Cara , Arnold Brian , Antonacci Barry , Parmegiani Raulo , Yarosh-Tomaine Taisa , Miller George H. , Hare Roberta S.
Published: July 2002

SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39
Aspergillus
strains and to that of fluconazole against 275
Candida
and 9
Cryptococcus
strains. The SCH 56592 MICs for
Aspergillus
ranged from ≤0.002 to 0.5 μg/ml, and those of itraconazole ranged from ≤0.008 to 1 μg/ml. The SCH 56592 MICs for
Candida
and
Cryptococcus
strains ranged from ≤0.004 to 16 μg/ml, and those of fluconazole ranged from ≤0.062 to >64 μg/ml. SCH 56592 showed excellent activity against
Aspergillus fumigatus
and
Aspergillus flavus
in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD
50
s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD
50
s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD
50
s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistant
Candida albicans
strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD
50
s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD
50
s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against serious
Aspergillus
or
Candida
infections.
Comparative Activities of Ciprofloxacin, Clinafloxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Moxifloxacin, and Trovafloxacin against Epidemiologically Defined
Acinetobacter baumannii
Strains
Authors: Heinemann Barbara , Wisplinghoff Hilmar , Edmond Michael , Seifert Harald
Published: July 2002

In vitro activities of seven fluoroquinolones against 140 clinical
Acinetobacter baumannii
isolates representing 138 different strain types were determined. The rank order of activity was clinafloxacin > gatifloxacin > levofloxacin > trovafloxacin > gemifloxacin = moxifloxacin > ciprofloxacin. The 31 outbreak-related
A. baumannii
strains were significantly more resistant than were 109 sporadic strains.
Sodium Stibogluconate (Pentostam) Potentiates Oxidant Production in Murine Visceral Leishmaniasis and in Human Blood
Authors: Rais Samira , Perianin Axel , Lenoir Monique , Sadak Abderrahim , Rivollet Daniele , Paul Muriel , Deniau Michele
Published: July 2002

Sodium stibogluconate (Sbb), a leishmanicidal drug, was studied for its in vivo effect on the formation of reactive oxygen species (ROS), assessed by chemiluminescence (CL) in the whole blood of mice infected withLeishmania infantum. Stimulation of ROS formation induced ex vivo by zymosan particles or the protein kinase C activator phorbol myristate acetate (PMA) was reduced by approximately 25% (P< 0.05) after infection of mice. Treatment of infected mice with Sbb (50 to 400 mg/kg of body weight) enhanced the blood CL induced by zymosan and PMA (47 to 96%,P< 0.01). The drug potentiation effect also occurred in uninfected mice. In vitro treatment of normal human blood with Sbb (1, 10, or 100 μg/ml) for 1 h primed the CL response to PMA (29 to 54%). The priming effect of Sbb was also observed on the production of superoxide by isolated polymorphonuclear leukocytes stimulated either by PMA and zymosan or by the chemoattractantsN-formyl-Met-Leu-Phe and platelet-activating factor. These data provide the first evidence of priming of the phagocyte respiratory burst by Sbb. This novel property of Sbb may contribute to the drug's leishmanicidal effect.
Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-Resistant
Streptococcus pneumoniae
Authors: Maskell Jeffrey P. , Sefton Armine M. , Hall Lucinda M. C.
Published: July 2002

Trimethoprim resistance in
Streptococcus pneumoniae
can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC
50
], 4.2 μM) than was the DHFR from strain CP1015 (IC
50
, 0.09 μM). However,
K

m

values indicated a lower affinity for the enzyme's natural substrates (
K

m

for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme,
K

m

values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC
50
of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the
Escherichia coli
DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates.
In Vitro Growth of
Leishmania amazonensis
Promastigotes Resistant to Pentamidine Is Dependent on Interactions among Strains
Authors: Agnew P. , Holzmuller P. , Michalakis Y. , Sereno D. , Lemesre J. L. , Renaud F.
Published: July 2002

The in vitro growth of promastigote cells of
Leishmania amazonensis
was found to strongly depend on interactions among strains that differed in their pentamidine resistance. In particular, the growth of resistant strains was reduced when they shared the same environment with a less-resistant strain.
Identification of
Proteus mirabilis
Mutants with Increased Sensitivity to Antimicrobial Peptides
Authors: McCoy Andrea J. , Liu Hongjian , Falla Timothy J. , Gunn John S.
Published: July 2002

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example,
Proteus mirabilis
is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate
P. mirabilis
mutants sensitive to APs. Four unique PM-sensitive mutants of
P. mirabilis
were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an
O
-acetyltransferase (PM and protegrin sensitive) and ATP synthase and
sap
loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in
P. mirabilis
swarming motility.
Mutation in the DNA Gyrase A Gene of
Escherichia coli
That Expands the Quinolone Resistance-Determining Region
Authors: Friedman S. Marvin , Lu Tao , Drlica Karl
Published: July 2002

In three
Escherichia coli
mutants, a change (Ala-51 to Val) in the gyrase A protein outside the standard quinolone resistance-determining region (QRDR) lowered the level of quinolone susceptibility more than changes at amino acids 67, 82, 84, and 106 did. Revision of the QRDR to include amino acid 51 is indicated.
Mutant TEM β-Lactamase Producing Resistance to Ceftazidime, Ampicillins, and β-Lactamase Inhibitors
Authors: Vakulenko Sergei , Golemi Dasantila
Published: July 2002

A derivative of the TEM-1 β-lactamase producing clinically significant levels of resistance to ceftazidime and β-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection. This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to β-lactamase inhibitors. Although the Glu104Lys substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime. Kinetic studies of the purified β-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (
k
cat
/

K
m

) of ceftazidime. The increases in the

K
i

values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibility of β-lactam resistance with these inhibitors.
Pharmacokinetic Modeling and Simulations of Interaction of Amprenavir and Ritonavir
Authors: Sale Mark , Sadler Brian M. , Stein Daniel S.
Published: July 2002

Data from three pharmacokinetic drug interaction studies of amprenavir and ritonavir were used to develop a pharmacokinetic interaction model using NONMEM (nonlinear mixed-effect model). A two-compartment linear model with first-order absorption best fit the amprenavir data, while a one-compartment model was used to describe the ritonavir data. The inhibition of elimination of amprenavir by ritonavir was modeled with a maximum effect (
E
max
) inhibition model and the observed ritonavir concentration. Monte Carlo simulation was then used to predict amprenavir concentrations for various combinations of amprenavir and ritonavir in twice-daily and once-daily dosing regimens. Simulated minimum amprenavir concentrations in plasma (
C
min
) in twice-daily and once-daily dosing regimens were compared with protein binding-adjusted 50% inhibitory concentrations (IC
50
s) for clinical human immunodeficiency virus isolates with different susceptibilities to protease inhibitors (central tendency ratios). The model based on the first two studies predicted the results of the third study. Data from all three studies were then combined to refine the final model. The observed and simulated noncompartmental pharmacokinetic parameters agreed well. From this model, several candidate drug regimens were simulated. These simulations suggest that, in patients who have clinically failed a traditional amprenavir regimen, a regimen of 600 mg of amprenavir with 100 mg of ritonavir twice daily would result in
C
min
-to-IC
50
ratios similar to that of 1,200 mg of amprenavir twice daily alone for wild-type viruses. In addition, once-daily regimens that result in
C
min
s above the protein binding-corrected IC
50
s for wild-type virus are clearly feasible.
Neurotoxic Mode of Action of Artemisinin
Authors: Schmuck Gabriele , Roehrdanz Elke , Haynes Richard K. , Kahl Regine
Published: July 2002

We recently described a screening system designed to detect neurotoxicity of artemisinin derivatives based on primary neuronal brain stem cell cultures (G. Schmuck and R. K. Haynes, Neurotoxicity Res. 2:37-49, 2000). Here, we probe possible mechanisms of this brain stem-specific neurodegeneration, in which artemisinin-sensitive neuronal brain stem cell cultures are compared with nonsensitive cultures (cortical neurons, astrocytes). Effects on the cytoskeleton of brain stem cell cultures, but not that of cortical cell cultures, were visible after 7 days. However, after a recovery period of 7 days, this effect also became visible in cortical cells and more severe in brain stem cell cultures. Neurodegeneration appears to be induced by effects on intracellular targets such as the cytoskeleton, modulation of the energy status by mitochondrial or metabolic defects, oxidative stress or excitotoxic events. Artemisinin reduces intracellular ATP levels and the potential of the inner mitochondrial membrane below the cytotoxic concentration range in all three cell cultures, with these effects being most dominant in the brain stem cultures. Surprisingly, there were substantial effects on cortical neurons after 7 days and on astrocytes after 1 day. Artemisinin additionally induces oxidative stress, as observed as an increase of reactive oxygen species and of lipid peroxidation in both neuronal cell types. Interestingly, an induction of expression of AOE was only seen in astrocytes. Here, manganese superoxide dismutase (MnSOD) expression was increased more than 3-fold and catalase expression was increased more than 1.5-fold. In brain stem neurons, MnSOD expression was dose dependently decreased. Copper-zinc superoxide dismutase and glutathione peroxidase, two other antioxidant enzymes that were investigated, did not show any changes in their mRNA expression in all three cell types after exposure to artemisinin.
In Vitro Low-Level Resistance to Azoles in
Candida albicans
Is Associated with Changes in Membrane Lipid Fluidity and Asymmetry
Authors: Kohli Avmeet , Smriti NFN , Mukhopadhyay Kasturi , Rattan Ashok , Prasad Rajendra
Published: July 2002

The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of
Candida albicans
, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 μg ml
−1
) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 μg ml
−1
) and YO1-64 (MIC, 64 μg ml
−1
). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g.,
CDR1
and
CDR2
, and the azole target enzyme,
ERG11
. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values,
P
value) in the adapted strain (
P
value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in
C. albicans
.
In Vitro Antiplasmodium Effects of Dermaseptin S4 Derivatives
Authors: Dagan Arie , Efron Leah , Gaidukov Leonid , Mor Amram , Ginsburg Hagai
Published: July 2002

The 13-residue dermaseptin S4 derivative K
4
S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of
Plasmodium falciparum
. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 μM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.
Rapid Mineralization of Benzo[a]pyrene by a Microbial Consortium Growing on Diesel Fuel
Authors: Kanaly Robert A. , Bartha Richard , Watanabe Kazuya , Harayama Shigeaki
Published: July 2002

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-14C]benzo[a]pyrene to14CO2when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter−1and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter−1. A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the classProteobacteriabut that the consortium also included members which were related to the generaMycobacteriumandSphingobacterium.
Metabolic Engineering of an Aerobic Sulfate Reduction Pathway and Its Application to Precipitation of Cadmium on the Cell Surface
Authors: Wang Clifford L. , Maratukulam Priya D. , Lum Amy M. , Clark Douglas S. , Keasling J. D.
Published: July 2002

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.
Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems
Authors: Bond Philip L. , Druschel Greg K. , Banfield Jillian F.
Published: July 2002

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ∼0.5 to ∼1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “
Ferroplasma
” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength.
Leptospirillum
spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured
Leptospirillum
sp. dominated.
Sulfobacillus
spp. were detected as a prominent inhabitant in warmer (∼43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.
Influence of Infected Cell Growth State on Bacteriophage Reactivation Levels
Authors: Kadavy Dana R. , Shaffer Julie J. , Lott Susan E. , Wolf Thomas A. , Bolton Cathy E. , Gallimore William H. , Martin Eugene L. , Nickerson Kenneth W. , Kokjohn Tyler A.
Published: July 2002

Reactivation of UV-C-inactivated
Pseudomonas aeruginosa
bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of
P. aeruginosa
host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of
P. aeruginosa
cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1.5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.
Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants
Authors: Kim Yong-Sung , Jung Heung-Chae , Pan Jae-Gu
Published: July 2002

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.