bluuebunny/crossref_metadata_2025 huggingface dataset:
Miocene mammal reveals a Mesozoic ghost lineage on insular New Zealand, southwest Pacific
Authors: Worthy Trevor H. , Tennyson Alan J. D. , Archer Michael , Musser Anne M. , Hand Suzanne J. , Jones Craig , Douglas Barry J. , McNamara James A. , Beck Robin M. D.
Published: December 2006

New Zealand (NZ) has long been upheld as the archetypical example of a land where the biota evolved without nonvolant terrestrial mammals. Their absence before human arrival is mysterious, because NZ was still attached to East Antarctica in the Early Cretaceous when a variety of terrestrial mammals occupied the adjacent Australian portion of Gondwana. Here we report discovery of a nonvolant mammal from Miocene (19–16 Ma) sediments of the Manuherikia Group near St Bathans (SB) in Central Otago, South Island, NZ. A partial relatively plesiomorphic femur and two autapomorphically specialized partial mandibles represent at least one mouse-sized mammal of unknown relationships. The material implies the existence of one or more ghost lineages, at least one of which (based on the relatively plesiomorphic partial femur) spanned the Middle Miocene to at least the Early Cretaceous, probably before the time of divergence of marsupials and placentals >125 Ma. Its presence in NZ in the Middle Miocene and apparent absence from Australia and other adjacent landmasses at this time appear to reflect a Gondwanan vicariant event and imply persistence of emergent land during the Oligocene marine transgression of NZ. Nonvolant terrestrial mammals disappeared from NZ some time since the Middle Miocene, possibly because of late Neogene climatic cooling.
Adaptive reconfiguration of fractal small-world human brain functional networks
Authors: Bassett Danielle S. , Meyer-Lindenberg Andreas , Achard Sophie , Duke Thomas , Bullmore Edward
Published: December 2006

Brain function depends on adaptive self-organization of large-scale neural assemblies, but little is known about quantitative network parameters governing these processes in humans. Here, we describe the topology and synchronizability of frequency-specific brain functional networks using wavelet decomposition of magnetoencephalographic time series, followed by construction and analysis of undirected graphs. Magnetoencephalographic data were acquired from 22 subjects, half of whom performed a finger-tapping task, whereas the other half were studied at rest. We found that brain functional networks were characterized by small-world properties at all six wavelet scales considered, corresponding approximately to classical δ (low and high), θ, α, β, and γ frequency bands. Global topological parameters (path length, clustering) were conserved across scales, most consistently in the frequency range 2–37 Hz, implying a scale-invariant or fractal small-world organization. Dynamical analysis showed that networks were located close to the threshold of order/disorder transition in all frequency bands. The highest-frequency γ network had greater synchronizability, greater clustering of connections, and shorter path length than networks in the scaling regime of (lower) frequencies. Behavioral state did not strongly influence global topology or synchronizability; however, motor task performance was associated with emergence of long-range connections in both β and γ networks. Long-range connectivity, e.g., between frontal and parietal cortex, at high frequencies during a motor task may facilitate sensorimotor binding. Human brain functional networks demonstrate a fractal small-world architecture that supports critical dynamics and task-related spatial reconfiguration while preserving global topological parameters.
Delay of T cell senescence by caloric restriction in aged long-lived nonhuman primates
Authors: Messaoudi Ilhem , Warner Jessica , Fischer Miranda , Park Buyng , Hill Brenna , Mattison Julie , Lane Mark A. , Roth George S. , Ingram Donald K. , Picker Louis J. , Douek Daniel C. , Mori Motomi , Nikolich-Žugich Janko
Published: December 2006

Caloric restriction (CR) has long been known to increase median and maximal lifespans and to decreases mortality and morbidity in short-lived animal models, likely by altering fundamental biological processes that regulate aging and longevity. In rodents, CR was reported to delay the aging of the immune system (immune senescence), which is believed to be largely responsible for a dramatic increase in age-related susceptibility to infectious diseases. However, it is unclear whether CR can exert similar effects in long-lived organisms. Previous studies involving 2- to 4-year CR treatment of long-lived primates failed to find a CR effect or reported effects on the immune system opposite to those seen in CR-treated rodents. Here we show that long-term CR delays the adverse effects of aging on nonhuman primate T cells. CR effected a marked improvement in the maintenance and/or production of naïve T cells and the consequent preservation of T cell receptor repertoire diversity. Furthermore, CR also improved T cell function and reduced production of inflammatory cytokines by memory T cells. Our results provide evidence that CR can delay immune senescence in nonhuman primates, potentially contributing to an extended lifespan by reducing susceptibility to infectious disease.
Opposite patterns of hemisphere dominance for early auditory processing of lexical tones and consonants
Authors: Luo Hao , Ni Jing-Tian , Li Zhi-Hao , Li Xiao-Ou , Zhang Da-Ren , Zeng Fan-Gang , Chen Lin
Published: December 2006

In tonal languages such as Mandarin Chinese, a lexical tone carries semantic information and is preferentially processed in the left brain hemisphere of native speakers as revealed by the functional MRI or positron emission tomography studies, which likely measure the temporally aggregated neural events including those at an attentive stage of auditory processing. Here, we demonstrate that early auditory processing of a lexical tone at a preattentive stage is actually lateralized to the right hemisphere. We frequently presented to native Mandarin Chinese speakers a meaningful auditory word with a consonant-vowel structure and infrequently varied either its lexical tone or initial consonant using an odd-ball paradigm to create a contrast resulting in a change in word meaning. The lexical tone contrast evoked a stronger preattentive response, as revealed by whole-head electric recordings of the mismatch negativity, in the right hemisphere than in the left hemisphere, whereas the consonant contrast produced an opposite pattern. Given the distinct acoustic features between a lexical tone and a consonant, this opposite lateralization pattern suggests the dependence of hemisphere dominance mainly on acoustic cues before speech input is mapped into a semantic representation in the processing stream.
Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins
Authors: Li Liang , Mustafi Debarshi , Fu Qiang , Tereshko Valentina , Chen Delai L. , Tice Joshua D. , Ismagilov Rustem F.
Published: December 2006

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a “hybrid” droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as ≈140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in ≈10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, ≈1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.
A route for fructose utilization by
Escherichia coli
involving the fucose regulon
Authors: Kornberg Hans , Lourenco Christopher
Published: December 2006

Fructose can be taken up by
Escherichia coli
via a variety of membrane-spanning proteins that recognize sugars with the 3,4,5-
d
-
arabino
-hexose configuration. Here, we describe a mutant that is devoid of those proteins but takes up fructose via the FucP carrier normally used for the transport of α-
l
-fucose; this implies that the fructose is taken up in the α- or β-fructopyranose form. For growth, the assimilated fructose is sequentially phosphorylated by ATP and (
i
) manno(fructo)kinase, to form fructose 6-phosphate, and (
ii
) phosphofructokinase, to form fructose 1,6-bisphosphate, which is a member of central routes of glycolysis and gluconeogenesis. The mutation that confers on the organism the ability to take up fructose via the fucose regulon was located as a deletion of the
fucA
gene with consequent induction of the proton-linked fucose transporter, FucP.
Canopy leaf area constrains [CO
2
]-induced enhancement of productivity and partitioning among aboveground carbon pools
Authors: McCarthy Heather R. , Oren Ram , Finzi Adrien C. , Johnsen Kurt H.
Published: December 2006

Net primary productivity (NPP) is enhanced under future atmospheric [CO
2
] in temperate forests representing a broad range of productivity. Yet questions remain in regard to how elevated [CO
2
]-induced NPP enhancement may be affected by climatic variations and limiting nutrient resources, as well as how this additional production is distributed among carbon (C) pools of different longevities. Using 10 years of data from the Duke free-air CO
2
enrichment (Duke FACE) site, we show that spatially, the major control of NPP was nitrogen (N) availability, through its control on canopy leaf area index (
L
). Elevated CO
2
levels resulted in greater
L
, and thus greater NPP. After canopy closure had occurred, elevated [CO
2
] did not enhance NPP at a given
L
, regardless of soil water availability. Additionally, using published data from three other forest FACE sites and replacing
L
with leaf area duration (
L
D
) to account for differences in growing season length, we show that aboveground NPP responded to [CO
2
] only through the enhancement of
L
D
. For broadleaf forests, the fraction of aboveground NPP partitioned to wood biomass saturated with increasing
L
D
and was not enhanced by [CO
2
], whereas it linearly decreased for the conifer forest but was enhanced by [CO
2
]. These results underscore the importance of resolving [CO
2
] effects on
L
to assess the response of NPP and C allocation. Further study is necessary to elucidate the mechanisms that control the differential allocation of C among aboveground pools in different forest types.
Sometimes area counts more than number
Authors: Hurewitz Felicia , Gelman Rochel , Schnitzer Brian
Published: December 2006

Using an interference paradigm, we show across three experiments that adults' order judgments of numbers, sizes, or combined area of dots in pairs of arrays occur spontaneously and automatically, but at different speeds and levels of accuracy. Experiment 1 used circles whose sizes varied between but not within arrays. Variation in circle size interfered with judgments of which array had more circles. Experiment 2 used displays in which circle size varied within and between arrays. Between-array differences in the amount of “circle stuff” (area occupied by circles) interfered with judgments of number. Experiment 3 examined whether variation in number also interferes with judgments of area. Interference between discrete and continuous stimulus dimensions occurred in both directions, although it was stronger from the continuous to the discrete than vice versa. These results bear on interpretations of studies with infants and preschoolers wherein subjects respond on the basis of continuous quantity rather than discrete quantity. In light of our results with adults, these findings do not license the conclusion that young children cannot represent discrete quantity. Absent data on attentional hierarchies and speed of processing, it is premature to conclude that infant and child quantity processes are fundamentally different from that of adults.
Three-dimensional equilibria in axially symmetric tokamaks
Authors: Garabedian Paul R.
Published: December 2006

The NSTAB and TRAN computer codes have been developed to study equilibrium, stability, and transport in fusion plasmas with three-dimensional (3D) geometry. The numerical method that is applied calculates islands in tokamaks like the Doublet III-D at General Atomic and the International Thermonuclear Experimental Reactor. When bifurcated 3D solutions are used in Monte Carlo computations of the energy confinement time, a realistic simulation of transport is obtained. The significance of finding many 3D magnetohydrodynamic equilibria in axially symmetric tokamaks needs attention because their cumulative effect may contribute to the prompt loss of α particles or to crashes and disruptions that are observed. The 3D theory predicts good performance for stellarators.
CD27 mediates interleukin-2-independent clonal expansion of the CD8+T cell without effector differentiation
Authors: Carr James M. , Carrasco Marlene J. , Thaventhiran James E. D. , Bambrough Paul J. , Kraman Matthew , Edwards Alexander D. , Al-Shamkhani Aymen , Fearon Douglas T.
Published: December 2006

The clonal expansion of antigen-specific CD8+T cells in response to microbial infections is essential for adaptive immunity. Although IL-2 has been considered to be primarily responsible for this process, quantitatively normal expansion occurs in the absence of IL-2 receptor signaling. Here, we show that ligating CD27 on CD8+T cells that have been stimulated through the T cell receptor causes their expansion in the absence of IL-2 by mediating two distinct cellular processes: enhancing cell cycling and promoting cell survival by maintaining the expression of IL-7 receptor α. This pathway for clonal expansion of the CD8+T cell is not associated with the development of a capacity either for production of IFN-γ or for cytotoxic T lymphocyte function and, therefore, is uncoupled from differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-γ-producing capability by the CD8+T cell, suggesting that CD27 signaling may suppress effector differentiation. Finally, CD8+T cells that have been stimulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to a viral challengein vivo. Thus, the TCR/CD27 pathway enables the CD8+T cell to replicate by a process of self-renewal, which may contribute to the continuous generation of new effector CD8+T cells in persistent viral infections.
Loss of linker histone H1 in cellular senescence
Authors: Funayama Ryo , Saito Motoki , Tanobe Hiroko , Ishikawa Fuyuki
Published: December 2006

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)–tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA–binding proteins.
JunB is required for endothelial cell morphogenesis by regulating core-binding factor β
Authors: Licht Alexander H. , Pein Oliver T. , Florin Lore , Hartenstein Bettina , Reuter Hendrik , Arnold Bernd , Lichter Peter , Angel Peter , Schorpp-Kistner Marina
Published: December 2006

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis.
Neural crest–derived cells with stem cell features can be traced back to multiple lineages in the adult skin
Authors: Wong Christine E. , Paratore Christian , Dours-Zimmermann María T. , Rochat Ariane , Pietri Thomas , Suter Ueli , Zimmermann Dieter R. , Dufour Sylvie , Thiery Jean Paul , Meijer Dies , Beermann Friedrich , Barrandon Yann , Sommer Lukas
Published: December 2006

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell–like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest–derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.
UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle
Authors: Miller Rachel K. , Qadota Hiroshi , Landsverk Megan L. , Mercer Kristina B. , Epstein Henry F. , Benian Guy M.
Published: December 2006

Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion–like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of β-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.
Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts
Authors: Chou Ming-Lun , Chu Chiung-Chih , Chen Lih-Jen , Akita Mitsuru , Li Hsou-min
Published: December 2006

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation.
Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation
Authors: Casazza Joseph P. , Betts Michael R. , Price David A. , Precopio Melissa L. , Ruff Laura E. , Brenchley Jason M. , Hill Brenna J. , Roederer Mario , Douek Daniel C. , Koup Richard A.
Published: December 2006

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.
Functional Characterization and Molecular Cloning of the K+-dependent Na+/Ca2+ Exchanger in Intact Retinal Cone Photoreceptors
Authors: Paillart Christophe , Winkfein Robert J. , Schnetkamp Paul P.M. , Korenbrot Juan I.
Published: December 2006

Light-dependent changes in cytoplasmic free Ca2+ are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na+/Ca2+ exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca2+ measured with optical probes. The exchanger in cones is K+ dependent and operates both in forward and reverse modes. In the reverse mode, the K+ dependence of the exchanger is described by binding to a single site with K1/2 about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods.
Thermal Care of Functional Dyspepsia Based on Bicarbonate‐Sulphate‐Calcium Water: A Sequential Clinical Trial
Authors: Rocca Giuseppe , Dioni Federico , Rocca Nadia , Oliveri Filippo , Brunetto Maurizia R. , Bonino Ferruccio
Published: December 2006

Drug treatment of functional dyspepsia is often unsatisfactory. We assessed the efficacy of a bicarbonate‐sulphate‐calcium thermal water cycle of 12 days, in patients with functional dyspepsia. Patients with functional dyspepsia were sent by their general practitioners to 12 days of treatment with thermal water, 200–400 ml in the morning, at temperature of 33°C (91.4 F) and were evaluated on a strict intention to treat basis. Four efficacy endpoints were analyzed as follows: (i) reduction of the global symptoms score, (ii) reduction of intensity to a level not interfering with everyday activities, (iii) specific efficacy on ulcer‐like or dysmotility‐like dyspepsia and (iv) esophageal or abdominal‐associated symptoms. Statistical significance was reached for all three primary outcomes after the first 29 consecutive patients. Thermal water reduced the global symptom score, reduced intensity of symptoms to a level not interfering with everyday activity, but was unable to completely suppress all symptoms. A parallel effect emerged for ulcer‐like and dyspepsia‐like subgroups. The effect on heartburn and abdominal symptoms was not significant, suggesting a specific effect of the water on the gastric and duodenal wall. The Roma II criteria identify a natural kind of dyspepsia that improves with thermal water. Ulcer‐like and dysmotility‐like are not therapeutically distinguishable subgroups. Patients with dominant esophageal or abdominal symptoms should receive a different therapy. Sequential methods are very effective for the evaluation of traditional care practices and should be considered preliminary and integrative to randomized controlled trials in this context.
Identification of a novel gene, URE2, that functionally complements a urease-negative clinical strain of Cryptococcus neoformans
Authors: Varma Ashok , Wu Shaoxi , Guo Ningru , Liao Wanqing , Lu Guxia , Li Anshen , Hu Yonglin , Bulmer Glenn , Kwon-Chung Kyung J.
Published: December 2006

A urease-negative serotype A strain ofCryptococcus neoformans(B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. TheURE1gene encoding urease failed to complement the mutant phenotype. Urease-positive clones of B-4587 obtained by complementing with a genomic library of strain H99 harboured an episomal plasmid containing DNA inserts with homology to thesudAgene ofAspergillus nidulans. The gene harboured by these plasmids was namedURE2since it enabled the transformants to grow on media containing urea as the sole nitrogen source while the transformants with an empty vector failed to grow. Transformation of strain B-4587 with a plasmid construct containing a truncated version of theURE2gene failed to complement the urease-negative phenotype. Disruption of the nativeURE2gene in a wild-type serotype A strain H99 and a serotype D strain LP1 ofC. neoformansresulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that theURE2gene product is involved in the utilization of urea by the organism. Virulence in mice of the urease-negative isolate B-4587, the urease-positive transformants containing the wild-type copy of theURE2gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain ofC. neoformansregardless of the infection route. Virulence of theURE2disruption stain of H99 was slightly reduced compared to the wild-type strain in the intravenous model but was significantly attenuated in the inhalation model. These results indicate that the importance of urease activity in pathogenicity varies depending on the strains ofC. neoformansused and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such asURE2that have otherwise been annotated as hypothetical proteins in genomic databases.
Topology of the outer-membrane secretin PilQ from Neisseria meningitidis
Authors: Frye Stephan A. , Assalkhou Reza , Collins Richard F. , Ford Robert C. , Petersson Christoffer , Derrick Jeremy P. , Tønjum Tone
Published: December 2006

Neisseria meningitidisis the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure–function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N- and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretinin vivo.
Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention
Authors: Lucas Claressa E. , Brown Ellen , Fields Barry S.
Published: December 2006

Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, which displays reduced intracellular replication, was able to establish itself and persist in the presence of amoebae. Thus, type IV pili and the pre-pilin peptidase facilitate L. pneumophila colonization of biofilms but are not required in the presence of a host for intracellular replication.
MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis
Authors: Casaz Paul , Garrity-Ryan Lynne K. , McKenney David , Jackson Caroline , Levy Stuart B. , Tanaka S. Ken , Alekshun Michael N.
Published: December 2006

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo.
Bacterial adhesion to and viability on positively charged polymer surfaces
Authors: Terada Akihiko , Yuasa Atsushi , Kushimoto Takashi , Tsuneda Satoshi , Katakai Akio , Tamada Masao
Published: December 2006

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than −7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than −8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80 % of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60 % of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than −8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.
The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae
Authors: Marques Marta , Mojzita Dominik , Amorim Maria A. , Almeida Teresa , Hohmann Stefan , Moradas-Ferreira Pedro , Costa Vítor
Published: December 2006

Turnover of damaged molecules is considered to play a key role in housekeeping of cells exposed to oxidative stress, and during the progress of ageing. In this work, global changes in the transcriptome were analysed during recovery of yeast cells after H2O2stress. Regarding induced genes, those associated with protein fate were the most significantly over-represented. In addition to genes encoding subunits of the 20S proteasome, genes related to vacuolar proteolysis (PEP4andLAP4), protein sorting into the vacuole, and vacuolar fusion were found to be induced. The upregulation ofPEP4gene expression was associated with an increase in Pep4p activity. The induction of genes related to proteolysis was correlated with an increased protein turnover after H2O2-induced oxidation. Furthermore, protein degradation and the removal of oxidized proteins decreased in Pep4p-deficient cells. Pep4p activity also increased during chronological ageing, and cells lacking Pep4p displayed a shortened lifespan associated with higher levels of carbonylated proteins.PEP4overexpression prevented the accumulation of oxidized proteins, but did not increase lifespan. These results indicate that Pep4p is important for protein turnover after oxidative damage; however, increased removal of oxidized proteins is not sufficient to enhance lifespan.
Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases
Authors: Valášková Vendula , Baldrian Petr
Published: December 2006

Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65 % loss of dry mass within 98 days, and it produced endo-1,4-β-glucanase (EG), endo-1,4-β-xylanase, endo-1,4-β-mannanase, 1,4-β-glucosidase (BG), 1,4-β-xylosidase, 1,4-β-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and β-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6–2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed β-xylosidase and endoxylanase activities. The K
m for carboxymethylcellulose was 3.5 g l−1, with the highest activity at pH 3.5 and 70 °C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K
m for p-nitrophenyl-1,4-β-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 °C, and the enzyme was competitively inhibited by glucose (K
i=5.8 mM). The fungus produced mainly β-glucosidase and β-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and β-xylosidase were found in fungus-colonized wood.
LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host
Authors: Matsunaga James , Werneid Kristian , Zuerner Richard L. , Frank Ami , Haake David A.
Published: December 2006

Leptospirosis is a widespread zoonosis caused by invasive spirochaetes belonging to the genus Leptospira. Pathogenic leptospires disseminate via the bloodstream to colonize the renal tubules of reservoir hosts. Little is known about leptospiral outer-membrane proteins expressed during the dissemination stage of infection. In this study, a novel surface-exposed lipoprotein is described; it has been designated LipL46 to distinguish it from a previously described 31 kDa peripheral membrane protein, P31LipL45, which is exported as a 45 kDa probable lipoprotein. The lipL46 gene encodes a 412 aa polypeptide with a 21 aa signal peptide. Lipid modification of cysteine at the lipoprotein signal peptidase cleavage site FSISC is supported by the finding that Leptospira interrogans intrinsically labels LipL46 during incubation in medium containing [14C]palmitate. LipL46 appears to be exported to the leptospiral outer membrane as a 46 kDa lipoprotein, based on Triton X-114 solubilization and phase partitioning studies, which included the outer and inner membrane controls LipL32 and LipL31, respectively. Surface immunoprecipitation and whole-cell ELISA experiments indicate that LipL46 is exposed on the leptospiral surface. Immunohistochemistry studies demonstrated expression of LipL46 by leptospires found in the bloodstream of acutely infected hamsters. Leptospires expressing LipL46 were also found in the intercellular spaces of the liver, within splenic phagocytes, and invading the glomerular hilum of the kidney. Infection-associated expression is supported by the finding that LipL46 is a major antigen recognized by sera from infected hamsters. These findings indicate that LipL46 may be important in leptospiral dissemination, and that it may serve as a useful serodiagnostic antigen.
Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501
Authors: Xie Zhihong , Dou Yuetang , Ping Shuzheng , Chen Ming , Wang Guoying , Elmerich Claudine , Lin Min
Published: December 2006

Pseudomonas stutzeristrain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties ofnifLandnifAmutants as well as the physical interaction between NifL and NifA proteins. AnifLmutant strain that carried a mutation non-polar onnifAexpression retained nitrogenase activity. Complementation with a plasmid containing onlynifLled to a decrease in nitrogenase activity in both the wild-type and thenifLmutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.
Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin
Authors: Sun Yuhui , Hong Hui , Samborskyy Markiyan , Mironenko Tatiana , Leadlay Peter F. , Haydock Stephen F.
Published: December 2006

Meridamycin is a non-immunosuppressant, FKBP-binding macrocyclic polyketide, which has major potential as a neuroprotectant in a range of neurodegenerative disorders including dementia, Parkinson's disease and ischaemic stroke. A biosynthetic cluster predicted to encode biosynthesis of meridamycin was cloned from the prolific secondary-metabolite-producing strainStreptomycessp. DSM 4137, not previously known to produce this compound, and specific gene deletion was used to confirm the role of this cluster in the biosynthesis of meridamycin. The meridamycin modular polyketide synthase consists of 14 extension modules distributed between three giant multienzyme proteins. The terminal module is flanked by a highly unusual cytochrome P450-like domain. The characterization of the meridamycin biosynthetic locus in this readily manipulated streptomycete species opens the way to the engineering of new, altered meridamycins of potential therapeutic importance.
The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation
Authors: Nakasugi Kenlee , Svenson Charles J. , Neilan Brett A.
Published: December 2006

The gene slr0388 was previously annotated to encode a hypothetical protein in Synechocystis sp. strain PCC 6803. When a positively phototactic strain of this cyanobacterium was insertionally inactivated at slr0388, the mutants were not transformable, and appeared to aggregate as a result of increased bundling of type IV pili. Also, these mutants were rendered non-phototactic compared to the wild-type. Quantitative real-time PCR revealed a 3.5-fold increase in pilA1 transcript levels in the mutant over wild-type cells, while there were no changes in the level of pilT1 and comA transcripts. Supernatant from mutant liquid culture contained more PilA1 protein, confirmed by mass spectrometric analysis, compared to the wild-type cells, which corresponded to the increase in pilA1 transcripts. The increase in PilA1 subunits may contribute to the bundling morphology of pili that was observed, which in turn may act to retard DNA uptake by hindering the retraction of pili. This gene is therefore proposed to be designated comF, as it possesses a phosphoribosyltransferase domain, a distinguishing feature of other ComF proteins of naturally transformable heterotrophic bacteria. This report is the second of a competence-related gene from Synechocystis sp. strain PCC 6803, the product of which does not show homology to other well-studied type IV pili proteins.
Variable expression of immunoreactive surface proteins of Propionibacterium acnes
Authors: Lodes Michael J. , Secrist Heather , Benson Darin R. , Jen Shyian , Shanebeck Kurt D. , Guderian Jeffrey , Maisonneuve Jean-François , Bhatia Ajay , Persing David , Patrick Sheila , Skeiky Yasir A. W.
Published: December 2006

Despite accumulating data implicatingPropionibacterium acnesin a variety of diseases, its precise role in infection remains to be determined.P. acnesantigen-specific CD4+T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens fromP. acnesexpression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein ofStreptococcus equiand have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of theCorynebacterium diphtheriae htaAgene from thehmuABC transport locus, although only one of these (PA-21693) is encoded within anhmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4+T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role ofP. acnesin inflammatory acne and other diseases.