bluuebunny/crossref_metadata_2025 huggingface dataset:
Paleolithic Population Growth Pulses Evidenced by Small Animal Exploitation
Authors: Stiner Mary C. , Munro Natalie D. , Surovell Todd A. , Tchernov Eitan , Bar-Yosef Ofer
Published: July 2002

Variations in small game hunting along the northern and eastern rims of the Mediterranean Sea and results from predator-prey simulation modeling indicate that human population densities increased abruptly during the late Middle Paleolithic and again during the Upper and Epi-Paleolithic periods. The demographic pulses are evidenced by increasing reliance on agile, fast-reproducing partridges, hares, and rabbits at the expense of slow-reproducing but easily caught tortoises and marine shellfish and, concurrently, climate-independent size diminution in tortoises and shellfish. The results indicate that human populations of the early Middle Paleolithic were exceptionally small and highly dispersed.
Pulmonary Function and Metabolic Physiology of Theropod Dinosaurs
Authors: Ruben John A. , Dal Sasso Cristiano , Geist Nicholas R. , Hillenius Willem J. , Jones Terry D. , Signore Marco
Published: July 2002

Ultraviolet light analysis of a fossil of the theropod dinosaur
Scipionyx samniticus
revealed that the liver subdivided the visceral cavity into distinct anterior pleuropericardial and posterior abdominal regions. In addition,
Scipionyx
apparently had diaphragmatic musculature and a dorsally attached posterior colon. These features provide evidence that diaphragm-assisted lung ventilation was present in theropods and that these dinosaurs may have used a pattern of exercise physiology unlike that in any group of living tetrapods.
Rapid Thinning of Parts of the Southern Greenland Ice Sheet
Authors: Krabill W. , Frederick E. , Manizade S. , Martin C. , Sonntag J. , Swift R. , Thomas R. , Wright W. , Yungel J.
Published: July 2002

Aircraft laser-altimeter surveys over southern Greenland in 1993 and 1998 show three areas of thickening by more than 10 centimeters per year in the southern part of the region and large areas of thinning, particularly in the east. Above 2000 meters elevation the ice sheet is in balance but thinning predominates at lower elevations, with rates exceeding 1 meter per year on east coast outlet glaciers. These high thinning rates occur at different latitudes and at elevations up to 1500 meters, which suggests that they are caused by increased rates of creep thinning rather than by excessive melting. Taken as a whole, the surveyed region is in negative balance.
A Functional Model for O-O Bond Formation by the O
2
-Evolving Complex in Photosystem II
Authors: Limburg Julian , Vrettos John S. , Liable-Sands Louise M. , Rheingold Arnold L. , Crabtree Robert H. , Brudvig Gary W.
Published: July 2002

The formation of molecular oxygen from water in photosynthesis is catalyzed by photosystem II at an active site containing four manganese ions that are arranged in di-μ-oxo dimanganese units (where μ is a bridging mode). The complex [H
2
O(terpy)Mn(O)
2
Mn(terpy)OH
2
](NO
3
)
3
(terpy is 2,2′:6′,2"-terpyridine), which was synthesized and structurally characterized, contains a di-μ-oxo manganese dimer and catalyzes the conversion of sodium hypochlorite to molecular oxygen. Oxygen-18 isotope labeling showed that water is the source of the oxygen atoms in the molecular oxygen evolved, and so this system is a functional model for photosynthetic water oxidation.
Exon Shuffling by L1 Retrotransposition
Authors: Moran John V. , DeBerardinis Ralph J. , Kazazian Haig H.
Published: July 2002

Long interspersed nuclear elements (LINE-1s or L1s) are the most abundant retrotransposons in the human genome, and they serve as major sources of reverse transcriptase activity. Engineered L1s retrotranspose at high frequency in cultured human cells. Here it is shown that L1s insert into transcribed genes and retrotranspose sequences derived from their 3′ flanks to new genomic locations. Thus, retrotransposition-competent L1s provide a vehicle to mobilize non-L1 sequences, such as exons or promoters, into existing genes and may represent a general mechanism for the evolution of new genes.
Chemical Transformations in Individual Ultrasmall Biomimetic Containers
Authors: Chiu Daniel T. , Wilson Clyde F. , Ryttsén Frida , Strömberg Anette , Farre Cecilia , Karlsson Anders , Nordholm Sture , Gaggar Anuj , Modi Biren P. , Moscho Alexander , Garza-López Roberto A. , Orwar Owe , Zare Richard N.
Published: July 2002

Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.
CH
5
+
: The Infrared Spectrum Observed
Authors: White Edmund T. , Tang Jian , Oka Takeshi
Published: July 2002

Protonated methane, CH
5
+
, has unusual vibrational and rotational behavior because its three nonequivalent equilibrium structures have nearly identical energies and its five protons scramble freely. Although many theoretical papers have been published on the quantum mechanics of the system, a better understanding requires spectral data. A complex, high-resolution infrared spectrum of CH
5
+
corresponding to the C–H stretching band in the 3.4-micrometer region is reported. Although no detailed assignment of the individual lines was made, comparison with other carbocation spectra strongly suggests that the transitions are due to CH
5
+
.
The Role of Area 17 in Visual Imagery: Convergent Evidence from PET and rTMS
Authors: Kosslyn S. M. , Pascual-Leone A. , Felician O. , Camposano S. , Keenan J. P. , L. W. , Thompson  , Ganis G. , Sukel K. E. , Alpert N. M.
Published: July 2002

Visual imagery is used in a wide range of mental activities, ranging from memory to reasoning, and also plays a role in perception proper. The contribution of early visual cortex, specifically Area 17, to visual mental imagery was examined by the use of two convergent techniques. In one, subjects closed their eyes during positron emission tomography (PET) while they visualized and compared properties (for example, relative length) of sets of stripes. The results showed that when people perform this task, Area 17 is activated. In the other, repetitive transcranial magnetic stimulation (rTMS) was applied to medial occipital cortex before presentation of the same task. Performance was impaired after rTMS compared with a sham control condition; similar results were obtained when the subjects performed the task by actually looking at the stimuli. In sum, the PET results showed that when patterns of stripes are visualized, Area 17 is activated, and the rTMS results showed that such activation underlies information processing.
Electrodeposited Ceramic Single Crystals
Authors: Switzer Jay A. , Shumsky Mark G. , Bohannan Eric W.
Published: July 2002

Single-crystal films are essential for devices because the intrinsic properties of the material, rather than its grain boundaries, can be exploited. Cubic bismuth oxide has the highest known oxide ion mobility, which makes it useful for fuel cells and sensors, but it is normally only stable from 729° to 825°C. The material has not been previously observed at room temperature. Single-crystal films of the high-temperature cubic polymorph of bismuth oxide were epitaxially electrodeposited from an aqueous solution onto single-crystal gold substrates. The 35.4 percent lattice mismatch was accommodated by forming coincidence lattices in which the bismuth oxide film was rotated in relation to the gold substrate. These results provide a method for producing other nonequilibrium phases that cannot be accessed by traditional thermal processing.
Evolution of a Protein Fold in Vitro
Authors: Cordes Matthew H. J. , Walsh Nathan P. , McKnight C. James , Sauer Robert T.
Published: July 2002

A “switch” mutant of the Arc repressor homodimer was constructed by interchanging the sequence positions of a hydrophobic core residue, leucine 12, and an adjacent surface polar residue, asparagine 11, in each strand of an intersubunit β sheet. The mutant protein adopts a fold in which each β strand is replaced by a right-handed helix and side chains in this region undergo significant repacking. The observed structural changes allow the protein to maintain solvent exposure of polar side chains and optimal burial of hydrophobic side chains. These results suggest that new protein folds can evolve from existing folds without drastic or large-scale mutagenesis.
A Steric Mechanism for Inhibition of CO Binding to Heme Proteins
Authors: Kachalova Galina S. , Popov Alexander N. , Bartunik Hans D.
Published: July 2002

The crystal structures of myoglobin in the deoxy- and carbon monoxide–ligated states at a resolution of 1.15 angstroms show that carbon monoxide binding at ambient temperatures requires concerted motions of the heme, the iron, and helices E and F for relief of steric inhibition. These steps constitute the main mechanism by which heme proteins lower the affinity of the heme group for the toxic ligand carbon monoxide.
Chemokine Up-Regulation and Activated T Cell Attraction by Maturing Dendritic Cells
Authors: Tang H. Lucy , Cyster Jason G.
Published: July 2002

Langerhans' cells migrating from contact-sensitized skin were found to up-regulate expression of macrophage-derived chemokine (MDC) during maturation into lymph node dendritic cells (DCs). Naı̈ve T cells did not migrate toward MDC, but antigen-specific T cells rapidly acquired MDC responsiveness in vivo after a subcutaneous injection of antigen. In chemotaxis assays, maturing DCs attracted activated T cells more strongly than naı̈ve T cells. These studies identified chemokine up-regulation as part of the Langerhans' cell maturation program to immunogenic T cell–zone DC. Preferential recruitment of activated T cells may be a mechanism used by maturing DCs to promote encounters with antigen-specific T cells.
Nutritional Genomics: Manipulating Plant Micronutrients to Improve Human Health
Authors: DellaPenna Dean
Published: July 2002

The nutritional health and well-being of humans are entirely dependent on plant foods either directly or indirectly when plants are consumed by animals. Plant foods provide almost all essential vitamins and minerals and a number of other health-promoting phytochemicals. Because micronutrient concentrations are often low in staple crops, research is under way to understand and manipulate synthesis of micronutrients in order to improve crop nutritional quality. Genome sequencing projects are providing novel approaches for identifying plant biosynthetic genes of nutritional importance. The term “nutritional genomics” is used to describe work at the interface of plant biochemistry, genomics, and human nutrition.
Association of BRCA1 with the hRad50-hMre11-p95 Complex and the DNA Damage Response
Authors: Zhong Qing , Chen Chi-Fen , Li Shang , Chen Yumay , Wang Chuan-Cheng , Xiao Jun , Chen Phang-Lang , Sharp Z. Dave , Lee Wen-Hwa
Published: July 2002

BRCA1
encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in
BRCA1
but was restored by transfection of wild-type
BRCA1
. Ectopic expression of wild-type, but not mutated,
BRCA1
in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.
Insights into Editing from an Ile-tRNA Synthetase Structure with tRNA
Ile
and Mupirocin
Authors: Silvian Laura F. , Wang Jimin , Steitz Thomas A.
Published: July 2002

Isoleucyl–transfer RNA (tRNA) synthetase (IleRS) joins Ile to tRNA
Ile
at its synthetic active site and hydrolyzes incorrectly acylated amino acids at its editing active site. The 2.2 angstrom resolution crystal structure of
Staphylococcus aureus
IleRS complexed with tRNA
Ile
and Mupirocin shows the acceptor strand of the tRNA
Ile
in the continuously stacked, A-form conformation with the 3′ terminal nucleotide in the editing active site. To position the 3′ terminus in the synthetic active site, the acceptor strand must adopt the hairpinned conformation seen in tRNA
Gln
complexed with its synthetase. The amino acid editing activity of the IleRS may result from the incorrect products shuttling between the synthetic and editing active sites, which is reminiscent of the editing mechanism of DNA polymerases.
Switchable Tackiness and Wettability of a Liquid Crystalline Polymer
Authors: Crevoisier Guillaume de , Fabre Pascale , Corpart Jean-Marc , Leibler Ludwik
Published: July 2002

The spreading velocity of liquids on the surface of a liquid crystalline polymer can be tremendously affected by a slight temperature change. Indeed, a bulk transition between a highly ordered smectic and an isotropic phase induces a sharp change from a rigid to a soft behavior, with consequent effects on the tack properties of the liquid crystalline polymer and on the dewetting dynamics of a liquid on its surface.
All-Optical Magnetic Resonance in Semiconductors
Authors: Kikkawa J. M. , Awschalom D. D.
Published: July 2002

A scheme is proposed wherein nuclear magnetic resonance (NMR) can be induced and monitored using only optical fields. In analogy to radio-frequency fields used in traditional NMR, circularly polarized light creates electron spins in semiconductors whose hyperfine coupling could tip nuclear moments. Time-resolved Faraday rotation experiments were performed in which the frequency of electron Larmor precession was used as a magnetometer of local magnetic fields experienced by electrons in n-type gallium arsenide. Electron spin excitation by a periodic optical pulse train appears not only to prepare a hyperpolarized nuclear moment but also to destroy it resonantly at magnetic fields proportional to the pulse frequency. This resonant behavior is in many ways supportive of a simple model of optically induced NMR, but a curious discrepancy between one of the observed frequencies and classic NMR values suggests that this phenomenon is more complex.
Global Carbon Sinks and Their Variability Inferred from Atmospheric O
2
and δ
13
C
Authors: Battle M. , Bender M. L. , Tans P. P. , White J. W. C. , Ellis J. T. , Conway T. , Francey R. J.
Published: July 2002

Recent time-series measurements of atmospheric O
2
show that the land biosphere and world oceans annually sequestered 1.4 ± 0.8 and 2.0 ± 0.6 gigatons of carbon, respectively, between mid-1991 and mid-1997. The rapid storage of carbon by the land biosphere from 1991 to 1997 contrasts with the 1980s, when the land biosphere was approximately neutral. Comparison with measurements of δ
13
CO
2
implies an isotopic flux of 89 ± 21 gigatons of carbon per mil per year, in agreement with model- and inventory-based estimates of this flux. Both the δ
13
C and the O
2
data show significant interannual variability in carbon storage over the period of record. The general agreement of the independent estimates from O
2
and δ
13
C is a robust signal of variable carbon uptake by both the land biosphere and the oceans.
Similar Requirements of a Plant Symbiont and a Mammalian Pathogen for Prolonged Intracellular Survival
Authors: LeVier K. , Phillips R. W. , Grippe V. K. , Roop R. M. , II  , Walker G. C.
Published: July 2002

Brucella abortus
, a mammalian pathogen, and
Rhizobium meliloti
, a phylogenetically related plant symbiont, establish chronic infections in their respective hosts. Here a highly conserved
B. abortus
homolog of the
R. meliloti bacA
gene, which encodes a putative cytoplasmic membrane transport protein required for symbiosis, was identified. An isogenic
B. abortus bacA
mutant exhibited decreased survival in macrophages and greatly accelerated clearance from experimentally infected mice compared to the virulent parental strain. Thus, the
bacA
gene product is critical for the maintenance of two very diverse host-bacterial relationships.
Convergence of the Secretory Pathways for Cholera Toxin and the Filamentous Phage, CTXϕ
Authors: Davis Brigid M. , Lawson Elise H. , Sandkvist Maria , Ali Afsar , Sozhamannan Shanmuga , Waldor Matthew K.
Published: July 2002

Virulence of
Vibrio cholerae
depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXφ. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.
N-Cadherin, a Cell Adhesion Molecule Involved in Establishment of Embryonic Left-Right Asymmetry
Authors: Garcı́a-Castro Martı́n I. , Vielmetter Eva , Bronner-Fraser Marianne
Published: July 2002

Within the bilaterally symmetric vertebrate body plan, many organs develop asymmetrically. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is one of the earliest proteins to be asymmetrically expressed in the chicken embryo and that its activity is required during gastrulation for proper establishment of the left-right axis. Blocking N-cadherin function randomizes heart looping and alters the expression of Snail and Pitx2, later components of the molecular cascade that regulate left-right asymmetry. However, the expression of other components of this cascade (Nodal and Lefty) was unchanged after blocking N-cadherin function, suggesting the existence of parallel pathways in the establishment of left-right morphogenesis. Here, the results suggest that N-cadherin-mediated cell adhesion events are required for establishment of left-right asymmetry.
Like Attracts Like: Getting RNA Processing Together in the Nucleus
Authors: Lewis Joe D. , Tollervey David
Published: July 2002

Structures visible within the eukaryotic nucleus have fascinated generations of biologists. Recent data show that these structures form in response to gene expression and are highly dynamic in living cells. RNA processing and assembly require many factors but the nucleus apparently lacks any active transport system to deliver these to the RNAs. Instead, processing factors move by diffusion but are concentrated by transient association with functionally related components. At sites of high activity this gives rise to visible structures, with components in dynamic equilibrium with the surrounding nucleoplasm. Processing factors are recruited from this pool by cooperative binding to RNA substrates.
Carbon Nanotube-Based Nonvolatile Random Access Memory for Molecular Computing
Authors: Rueckes Thomas , Kim Kyoungha , Joselevich Ernesto , Tseng Greg Y. , Cheung Chin-Li , Lieber Charles M.
Published: July 2002

A concept for molecular electronics exploiting carbon nanotubes as both molecular device elements and molecular wires for reading and writing information was developed. Each device element is based on a suspended, crossed nanotube geometry that leads to bistable, electrostatically switchable ON/OFF states. The device elements are naturally addressable in large arrays by the carbon nanotube molecular wires making up the devices. These reversible, bistable device elements could be used to construct nonvolatile random access memory and logic function tables at an integration level approaching 10
12
elements per square centimeter and an element operation frequency in excess of 100 gigahertz. The viability of this concept is demonstrated by detailed calculations and by the experimental realization of a reversible, bistable nanotube-based bit.
Forming Electrical Networks in Three Dimensions by Self-Assembly
Authors: Gracias David H. , Tien Joe , Breen Tricia L. , Hsu Carey , Whitesides George M.
Published: July 2002

Self-assembly of millimeter-scale polyhedra, with surfaces patterned with solder dots, wires, and light-emitting diodes, generated electrically functional, three-dimensional networks. The patterns of dots and wires controlled the structure of the networks formed; both parallel and serial connections were generated.
Mate Selection and the Evolution of Highly Polymorphic Self/Nonself Recognition Genes
Authors: Grosberg Richard K. , Hart Michael W.
Published: July 2002

Multicellular organisms use the products of highly polymorphic genes to distinguish self from conspecific nonself cells or tissues. These allorecognition polymorphisms may regulate somatic interactions between hosts and pathogens or between competitors (to avoid various forms of parasitism), as well as reproductive interactions between mates or between gametes (to avoid inbreeding). In both cases, rare alleles may be advantageous, but it remains unclear which mechanism maintains the genetic polymorphism for specificity in self/nonself recognition. Contrary to earlier reports, we show that mate selection cannot be a strong force maintaining allorecognition polymorphism in two colonial marine invertebrates. Instead, the regulation of intraspecific competitive interactions appears to promote the evolution of polymorphisms in these species.
Promiscuity and the Primate Immune System
Authors: Nunn Charles L. , Gittleman John L. , Antonovics Janis
Published: July 2002

The behavioral and ecological factors involved in immune system evolution remain poorly explored. We present a phylogenetic analysis of white blood cell counts in primates to test three hypotheses related to disease risk: increases in risk are expected with group size or population density, exposure to soil-borne pathogens, and mating promiscuity. White blood cell counts were significantly greater in species where females have more mating partners, indicating that the risk of sexually transmitted disease is likely to be a major factor leading to systematic differences in the primate immune system.
Mechanisms of Ordering in Striped Patterns
Authors: Harrison Christopher , Adamson Douglas H. , Cheng Zhengdong , Sebastian John M. , Sethuraman Srinivasan , Huse David A. , Register Richard A. , Chaikin P. M.
Published: July 2002

We have studied the ordering dynamics of the striped patterns of a single layer of cylindrical block copolymer microdomains in a thin film. By tracking disclinations during annealing with time-lapse atomic force microscopy, we observe a dominant mechanism of disclination annihilation involving three or four disclinations (quadrupoles). Pairwise disclination annihilation events are suppressed as a result of the topological constraints in this system. The kinetic scaling laws with exponents observed here are consistent with topologically allowed annihilation events involving multiple disclinations. The results provide insight into two-dimensional pattern formation and may lead to the successful application of block copolymer lithography.
Molybdenum Nanowires by Electrodeposition
Authors: Zach Michael P. , Ng Kwok H. , Penner Reginald M.
Published: July 2002

Metallic molybdenum (Mo
o
) wires with diameters ranging from 15 nanometers to 1.0 micrometers and lengths of up to 500 micrometers (0.5 millimeters) were prepared in a two-step procedure. Molybdenum oxide wires were electrodeposited selectively at step edges and then reduced in hydrogen gas at 500°C to yield Mo
o
. The hemicylindrical wires prepared by this technique were self-uniform, and the wires prepared in a particular electrodeposition (in batches of 10
5
to 10
7
) were narrowly distributed in diameter. Wires were obtained size selectively because the mean wire diameter was directly proportional to the square root of the electrolysis time. The metal nanowires could be embedded in a polystyrene film and lifted off the graphite electrode surface. The conductivity and mechanical resiliency of individual embedded wires were similar to those of bulk molybdenum.
Natal Homing in a Marine Fish Metapopulation
Authors: Thorrold Simon R. , Latkoczy Christopher , Swart Peter K. , Jones Cynthia M.
Published: July 2002

Identifying natal origins of marine fishes is challenging because of difficulties in conducting mark-recapture studies in marine systems. We used natural geochemical signatures in otoliths (ear bones) to determine natal sources in weakfish (
Cynoscion regalis
), an estuarine-spawning marine fish, in eastern North America. Spawning site fidelity ranged from 60 to 81%, comparable to estimates of natal homing in birds and anadromous fishes. These data were in contrast to genetic analyses of population structure in weakfish. Our findings highlight the need for consideration of spatial processes in fisheries models and have implications for the design of marine reserves in coastal regions.
Two-State Allosteric Behavior in a Single-Domain Signaling Protein
Authors: Volkman Brian F. , Lipson Doron , Wemmer David E. , Kern Dorothee
Published: July 2002

Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.
Upregulation of
ERG
Genes in
Candida
Species by Azoles and Other Sterol Biosynthesis Inhibitors
Authors: Henry Karl W. , Nickels Joseph T. , Edlind Thomas D.
Published: July 2002

Infections due to
Candida albicans
are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation of
ERG11
, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in
Candida
species. RNA analysis revealed that
ERG11
expression in
C. albicans
is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two- to fivefold increase in
ERG11
RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth.
ERG11
upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal
ERG11
upregulation was reversed. Azole-dependent upregulation was not limited to
ERG11
: five of five
ERG
genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly,
ERG11
upregulation occurred following treatment of
C. albicans
cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for global
ERG
upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent
ERG11
upregulation was demonstrated in three additional
Candida
species (
C. tropicalis
,
C. glabrata
, and
C. krusei
), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.
The EmrR Protein Represses the
Escherichia coli emrRAB
Multidrug Resistance Operon by Directly Binding to Its Promoter Region
Authors: Xiong A. , Gottman A. , Park C. , Baetens M. , Pandza S. , Matin A.
Published: July 2002

EmrR negatively regulates the transcription of the multidrug resistance pump-encoding operon,
emrRAB
, by binding to its regulatory region. The binding site spans the promoter and the downstream sequence up to the transcriptional start site of the operon. Structurally unrelated drugs that induce the pump interfere with this binding.
Expression and Characterization of Recombinant Human-Derived
Pneumocystis carinii
Dihydrofolate Reductase
Authors: Ma Liang , Kovacs Joseph A.
Published: July 2002

Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of
Pneumocystis carinii
pneumonia. While the rat-derived
P. carinii
DHFR has been well characterized, kinetic studies of human-derived
P. carinii
DHFR, which differs from rat-derived
P. carinii
DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived
P. carinii
DHFR. The 618-bp coding sequence of the human-derived
P. carinii
DHFR gene was expressed in
Escherichia coli
. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the

K
m

values for dihydrofolate and NADPH were 2.7 ± 0.3 and 14.0 ± 4.3 μM, respectively, which are similar to those reported for rat-derived
P. carinii
DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived
P. carinii
DHFR, with

K
i

values of 0.28 ± 0.08 and 0.065 ± 0.005 μM, respectively, while trimetrexate and methotrexate were potent inhibitors, with

K
i

values of 0.23 ± 0.03 and 0.016 ± 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived
P. carinii
DHFR.
Mefloquine Pharmacokinetic-Pharmacodynamic Models: Implications for Dosing and Resistance
Authors: Simpson Julie A. , Watkins Emmeline R. , Price Ric N. , Aarons Leon , Kyle Dennis E. , White Nicholas J.
Published: July 2002

Antimalarial resistance develops and spreads when spontaneously occurring mutant malaria parasites are selected by concentrations of antimalarial drug which are sufficient to eradicate the more sensitive parasites but not those with the resistance mutation(s). Mefloquine, a slowly eliminated quinoline-methanol compound, is the most widely used drug for the treatment of multidrug-resistant falciparum malaria. It has been used at doses ranging between 15 and 25 mg of base/kg of body weight. Resistance to mefloquine has developed rapidly on the borders of Thailand, where the drug has been deployed since 1984. Mathematical modeling with population pharmacokinetic and in vivo and in vitro pharmacodynamic data from this region confirms that, early in the evolution of resistance, conventional assessments of the therapeutic response ≤28 days after treatment underestimate considerably the level of resistance. Longer follow-up is required. The model indicates that initial deployment of a lower (15-mg/kg) dose of mefloquine provides a greater opportunity for the selection of resistant mutants and would be expected to lead more rapidly to resistance than de novo use of the higher (25-mg/kg) dose.
Bactericidal Activities of Antibiotics against Intracellular
Francisella tularensis
Authors: Maurin M. , Mersali N. F. , Raoult D.
Published: July 2002

MICs of many antibiotics for
Francisella tularensis
are low in axenic medium, whereas only aminoglycosides, tetracyclines, and fluoroquinolones are useful in treating tularemic patients. In an in vitro cell system, only these antibiotics, rifampin, and telithromycin were bactericidal against intracellular
F. tularensis
. These results correlate better with clinical data than MIC data do.
Substitutions in the Eyelet Region Disrupt Cefepime Diffusion through the
Escherichia coli
OmpF Channel
Authors: Simonet Valérie , Malléa Monique , Pagès Jean-Marie
Published: July 2002

The
Escherichia coli
OmpF porin is a nonspecific channel involved in the membrane translocation of small hydrophilic molecules and especially in the passage of β-lactam antibiotics. In order to understand the dynamic of charged-compound uptake through bacterial porins, specific charges located in the
E. coli
OmpF channel were mutated. Substitutions G119D and G119E, inserting a protruding acidic side chain into the pore, decreased cephalosporin and colicin susceptibilities. Cefepime diffusion was drastically altered by these mutations. Conversely, substitutions R132A and R132D, changing a residue located in the positively charged cluster, increased the rate of cephalosporin uptake without modifying colicin sensitivity. Modelling approaches suggest that G119E generates a transverse hydrogen bond dividing the pore, while the two R132 substitutions stretch the channel size. These charge alterations located in the constriction area have differential effects on cephalosporin diffusion and substantially modify the profile of antibiotic susceptibility.
Delavirdine Susceptibilities and Associated Reverse Transcriptase Mutations in Human Immunodeficiency Virus Type 1 Isolates from Patients in a Phase I/II Trial of Delavirdine Monotherapy (ACTG 260)
Authors: Demeter L. M. , Shafer R. W. , Meehan P. M. , Holden-Wiltse J. , Fischl M. A. , Freimuth W. W. , Para M. F. , Reichman R. C.
Published: July 2002

The development of human immunodeficiency virus type 1 resistance to delavirdine (DLV) was studied in subjects receiving DLV monotherapy. Phenotypic resistance developed in 28 of 30 subjects within 8 weeks. K103N and Y181C, which confer nonnucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance, were the predominant reverse transcriptase mutations. P236L, which confers DLV resistance but hypersensitivity to other NNRTIs, developed in <10% of isolates.
In Vitro Activities of Daptomycin, Vancomycin, Linezolid, and Quinupristin-Dalfopristin against Staphylococci and Enterococci, Including Vancomycin- Intermediate and -Resistant Strains
Authors: Rybak Michael J. , Hershberger Ellie , Moldovan Tabitha , Grucz Richard G.
Published: July 2002

The in vitro activity of daptomycin was compared with those of vancomycin, linezolid, and quinupristin-dalfopristin against a variety (
n
= 203) of gram-positive bacteria, including methicillin-resistant
Staphylococcus aureus
and
S. epidermidis
(MRSA and MRSE, respectively), vancomycin-resistant enterococci (VRE), and vancomycin-intermediate
S. aureus
(VISA). Overall, daptomycin was more active against all organisms tested, except
Enterococcus faecium
and VISA, against which its activity was similar to that of quinupristin-dalfopristin. In time-kill studies with MRSA, MRSE, VRE, and VISA, daptomycin demonstrated greater bactericidal activity than all other drugs tested, killing ≥3 log CFU/ml by 8 h. Daptomycin may be a potential alternative drug therapy for multidrug-resistant gram-positive organisms and warrants further investigation.
Vancomycin and Oxacillin Synergy for Methicillin-Resistant Staphylococci
Authors: Domaracki Beth E. , Evans Ann M. , Venezia Richard A.
Published: July 2002

An increase in oxacillin activity was observed against methicillin-resistant coagulase-negative staphylococci (MRCNS) and methicillin-resistant
Staphylococcus aureus
(MRSA) in the presence of a sub-MIC of vancomycin. Vancomycin and oxacillin were synergistic against 14 of 21 strains of MRCNS and MRSA. A pattern of enhanced killing was also supported by time-kill studies. These results suggest that combinations of sub-MICs of vancomycin and oxacillin may have therapeutic benefits against methicillin-resistant staphylococci.
Activity of the Novel Immunomodulatory Compound Tucaresol against Experimental Visceral Leishmaniasis
Authors: Smith Aden C. , Yardley Vanessa , Rhodes John , Croft Simon L.
Published: July 2002

Tucaresol, a novel immunomodulator, was inactive againstLeishmania donovaniamastigotes in both peritoneal and bone marrow macrophages in vitro at concentrations between 100 and 1 μM, with toxicity to macrophages and parasites at 300 μM. However, againstL. donovaniin BALB/c mice at doses between 80 and 1.25 mg/kg of body weight administered once daily by the oral route during days 7 to 11 of infection, an optimal dose of 5 mg/kg produced a 43.8 to 62.4% suppression of liver amastigotes, with significantly reduced activity at the extremes of the dose range. This response was not related to levels of infection. No interaction with the standard pentavalent antimonial sodium stibogluconate (Pentostam) was observed during this period of infection. The optimum dose of 5 mg/kg was ineffective when administered during the first week of infection and was most effective against the liver infection when administered during weeks 2 to 3 of infection (42.3 to 46.8% inhibition) and against the splenic infection when administered during week 6 of infection (59.5% inhibition). The optimum dose of tucaresol againstL. donovaniin C57BL/6 mice was 5 mg/kg, which produced a 40.8 to 48.7% suppression of liver amastigotes when administered in a range of 80 to 1.25 mg/kg during days 7 to 11 of infection. The drug had no activity againstL. donovaniinfections in C.B-17scidmice when the same regimen was used.
In Vitro Activities of Membrane-Active Peptides Alone and in Combination with Clinically Used Antimicrobial Agents against
Stenotrophomonas maltophilia
Authors: Giacometti A. , Cirioni O. , Del Prete M. S. , Barchiesi F. , Fortuna M. , Drenaggi D. , Scalise G.
Published: July 2002

The in vitro activities of buforin II, cecropin P1, and magainin II, alone and in combination with six clinically used antimicrobial agents, against 12 clinical isolates of
Stenotrophomonas maltophilia
were investigated. Antimicrobial activities were measured by MIC and time-kill studies. The isolates were susceptible to the peptides at concentrations in the range of 0.50 to 16 μg/ml. Synergy was observed when the peptides were combined with polymyxin E, meropenem, ceftazidime, piperacillin, and clarithromycin.
Molecular Basis of High-Level Ciprofloxacin Resistance in
Neisseria gonorrhoeae
Strains Isolated in Denmark from 1995 to 1998
Authors: Su Xiaohong , Lind Inga
Published: July 2002

In Denmark surveillance of the in vitro susceptibility to ciprofloxacin of
Neisseria gonorrhoeae
was established in 1990. The proportion of
N. gonorrhoeae
strains with decreased susceptibility or resistance to ciprofloxacin (MIC ≥ 0.06 μg/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains rose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC ≥ 1 μg/ml), 81% were highly resistant (MIC ≥ 4 μg/ml). Thirty-five
N. gonorrhoeae
strains (40 isolates) for which ciprofloxacin MICs were 4 to 32 μg/ml were investigated for the frequency and patterns of mutations within the
gyrA
and
parC
genes. The quinolone resistance-determining regions of the
gyrA
and
parC
genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to Asn, was found in 19 strains (54%). The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the
gyrA
and
parC
genes, originating from different geographical areas over 3 years, had the same PFGE patterns after
Spe
I as well as
Nhe
I digestion (only one strain with one band difference in the
Nhe
I pattern), suggesting that a resistant clone had spread worldwide. The results from this study strongly suggest that double
gyrA
mutations plus a
parC
mutation(s) play an important role in the development of high-level fluoroquinolone resistance in
N. gonorrhoeae
.
SHV-1 β-Lactamase Is Mainly a Chromosomally Encoded Species-Specific Enzyme in
Klebsiella pneumoniae
Authors: Chaves José , Ladona Margarita G. , Segura Concepción , Coira Amparo , Reig Roser , Ampurdanés Coral
Published: July 2002

The nature of the SHV-1 β-lactamase gene was analyzed in 97 epidemiologically unrelated
Klebsiella pneumoniae
strains isolated from clinical samples. β-Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of β-lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples with
K. pneumoniae
TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with
Eco
RI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 β-lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 β-lactamase originated from the
K. pneumoniae
chromosome.
Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein
Authors: Rudgers Gary W. , Huang Wanzhi , Palzkill Timothy
Published: July 2002

To overcome the antibiotic resistance mechanism mediated by β-lactamases, small-molecule β-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in β-lactamases no longer sensitive to β-lactamase inhibitors. On the basis of the structure of β-lactamase inhibitor protein (BLIP), novel peptide inhibitors of β-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 β-lactamase (
K

i

= 0.3 nM). The cocrystal structure of TEM-1 β-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 β-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 β-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 β-lactamase, the peptide also inhibits a class A β-lactamase and a class C β-lactamase that are not inhibited by BLIP. The crystal structures of class A and C β-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of β-lactamases and PBPs.
Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of
Cryptosporidium parvum
Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations
Authors: Nelson Richard G. , Rosowsky Andre
Published: July 2002

A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of
Cryptosporidium parvum
(J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153–165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from
Escherichia coli
. Fifty percent inhibitory concentrations (IC
50
s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC
50
s in the 1 to 10 μM range against one or both
C. parvum
enzymes and thus were not substantially different from trimethoprim (IC
50
s, ca. 4 μM). Another 25 compounds had IC
50
s of <1.0 μM, and 9 of these had IC
50
s of <0.1 μM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC
50
s, >10 μM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019–1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187–195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for
C. parvum
versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular
C. parvum
oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 μM) and hypoxanthine (100 μM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC
50
s of <3 μM, and five of these had IC
50
s of <0.3 μM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of
C. parvum
in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable
C. parvum
infections.