bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in spider assemblages along grassland and savanna grazing gradients in northern Australia
Authors: Churchill T. B. , Ludwig J. A.
Published: March 2005

Spiders are beneficial predators that respond to those land uses that modify their habitat. Cattle grazing is an extensive land use across northern Australian rangelands, yet the impact of grazing on spider habitat is poorly understood. In this study, we evaluated the way in which spider assemblages varied between grassland and savanna habitats in the Victoria River District of the Northern Territory, and also between dry and wet seasons in the savanna habitat. We also investigated changes in spider assemblages, and some common taxa, with distance from cattle watering points, which was used as a surrogate for a gradient in grazing intensity. Spiders were sampled using pitfall traps and sweep nets along two distance from water-grazing gradients. The first gradient was in savanna on calcareous red loam soils at Kidman Springs, and the second was in grassland on cracking black clays at Mount Sanford. Ordinations revealed that spider assemblages differed between grassland and savanna, and between the late-wet and late-dry seasons in savanna. Spider assemblages also markedly changed along the two distance-grazing gradients in response to habitat changes. For example, orb weavers in the family Araneidae increased in abundance as the cover of perennial grasses and litter increased with distance from water; this response was consistent for both grassland and savanna gradients. Patch type was also important to the distribution of spiders. For example, more individuals of Habronestes sp. were caught when the local habitat was patches of perennial grasses rather than patches of annual grasses and litter, bare ground, or tree-shrub canopy. These results suggest that spiders are good indicators of the habitat changes that may occur with grazing in the rangelands of Australia.
Regional variation of the archaeology in western New South Wales
Authors: Witter D. C.
Published: March 2005

There is considerable variation in the Aboriginal archaeology of western New South Wales. This is demonstrated by differences in the stone artefacts found on the open campsites that are common and distributed over all landscapes throughout western NSW. Other site types may occur in particular regions and show differences from one region to another. Eight archaeological regions are proposed. These are at the same scale as the Interim Biogeographic Regionalisation for Australia, and many of the boundaries are the same. The archaeological regions represent the accumulation of archaeological materials on the landscape and Aboriginal knowledge about how to cope in various situations. There is little relationship between the archaeological regional boundaries and the published tribal boundaries. The condition and preservation of the archaeology is closely related to the nature of landscape change since European arrival.
Potential economic benefits to the Australian wool industry from improving pastures in the hill country of eastern New South Wales
Authors: Vere D. T. , Campbell M. H.
Published: March 2005

Large areas of undeveloped pastures in non-arable hill country in eastern New South Wales could be improved by aerial methods. The potential economic benefits of pasture improving a portion of the hill country area were evaluated in terms of the economic welfare changes in the Australian wool industry. Hill country wool producers and international wool consumers would derive nearly all of the benefits from this process. Other Australian and international wool producers would lose economic welfare because of the reduced price from increased regional wool production. For conservative anticipated levels of pasture improvement adoption, the overall benefits to the Australian wool industry had 15-year mean present values of between $30.2 million and $122.1 million, depending on whether the introduced pasture was predominantly perennial grass or legume based. The value of this analysis is that it provides improved economic information for planning investment in new pasture technologies in the Australian livestock industries.
Optimum time for sampling floristic diversity in tropical eucalypt woodlands of northern Queensland
Authors: Neldner V. J. , Kirkwood A. B. , Collyer B. S.
Published: March 2005

The timing of vegetation sampling in highly seasonal environments is one of the critical factors in determining the proportion of the flora captured in a single sampling. Four sites were located within a 20 km radius of Mareeba, north Queensland and sampled every three months for three years. The sites were located in a variety of eucalypt communities and across an altitudinal range from 380 to 840 m above sea level. In these eucalypt communities experiencing highly seasonal rainfall typical of the tropical savannas, vegetation sampling in the early dry season (May) maximises the diversity of flora recorded. The ANOVA analysis showed a significant effect of month of sampling for the number of ground taxa recorded (P < 0.005). There was significant variation (P < 0.005) in species diversity between the sites but in all four study sites the May sampling recorded greater than 84% of the total recorded flora, whereas the November samplings accounted for between 21% and 56% of the flora. This supports the experience of other researchers that a May sampling is near optimum for sampling the ground layer floristic diversity in tropical eucalypt woodlands.
Most vegetation survey and mapping data are of necessity collected at less than ideal times of the year due to access and resource issues. Care must be exercised in using data collected in the dry season, as only a limited proportion of the total ground flora is likely to be recorded. Studies designed to capture the full florisitic inventory of species present in these highly seasonal environments need to budget resources and plan to access these environments in the late wet season.
Mapping the interaction surface of a membrane protein: Unveiling the conformational switch of phospholamban in calcium pump regulation
Authors: Zamoon J. , Nitu F. , Karim C. , Thomas D. D. , Veglia G.
Published: March 2005

We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN/SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (μs-to-ms) equilibrium between two conformational states in the cytoplasmic domain of PLN. These results support the hypothesis that SERCA attracts the cytoplasmic domain of PLN away from the lipid surface, shifting the preexisting equilibrium of PLN conformers toward a structure that is poised to interact with the regulatory target. EPR shows that this conformational switch behaves similarly in micelles and lipid membranes. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.
Identification of the mismatch repair genes
PMS2
and
MLH1
as p53 target genes by using serial analysis of binding elements
Authors: Chen Jiguo , Sadowski Ivan
Published: March 2005

The ability to determine the global location of transcription factor binding sites
in vivo
is important for a comprehensive understanding of gene regulation in human cells. We have developed a technology, called serial analysis of binding elements (SABE), involving subtractive hybridization of chromatin immunoprecipitation-enriched DNA fragments followed by the generation and analysis of concatamerized sequence tags. We applied the SABE technology to search for p53 target genes in the human genome, and have identified several previously described p53 targets in addition to numerous potentially novel targets, including the DNA mismatch repair genes
MLH1
and
PMS2
. Both of these genes were determined to be responsive to DNA damage and p53 activation in normal human fibroblasts, and have p53-response elements within their first intron. These two genes may serve as a sensor in DNA repair mechanisms and a critical determinant for the decision between cell-cycle arrest and apoptosis. These results also demonstrate the potential for use of SABE as a broadly applicable means to globally identify regulatory elements for human transcription factors
in vivo
.
A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells
Authors: Winter-Vann Ann M. , Baron Rudi A. , Wong Waihay , dela Cruz June , York John D. , Gooden David M. , Bergo Martin O. , Young Stephen G. , Toone Eric J. , Casey Patrick J.
Published: March 2005

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1
H
-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.
Target-directed proteolysis at the ribosome
Authors: Henrichs Tanja , Mikhaleva Natasha , Conz Charlotte , Deuerling Elke , Boyd Dana , Zelazny Adrian , Bibi Eitan , Ban Nenad , Ehrmann Michael
Published: March 2005

Target directed proteolysis allows specific processing of proteins
in vivo
. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed.
An
Arabidopsis
transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants
Authors: Iwata Yuji , Koizumi Nozomu
Published: March 2005

Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the
Arabidopsis
genome identified
AtbZIP60
, which was induced by tunicamycin.
AtbZIP60
encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60ΔC) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane.
AtbZIP60
was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60ΔC clearly activated any of three
BiP
and two
calnexin
promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60ΔC also appeared to induce the expression of
AtbZIP60
through an ER stress-response element-like sequence in the promoter of
AtbZIP60
. These characteristics of AtbZIP60 imply a signal transduction pathway of the ER stress response unique to plants.
A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells
Authors: Song Hui , Wang Renxiao , Wang Shaomeng , Lin Jiayuh
Published: March 2005

This study focused on the screening of small-molecule inhibitors that target signal transducers and activators of transcription 3 (Stat3) in human breast carcinoma. The constitutive activation of Stat3 is frequently detected in human breast cancer cell lines as well as clinical breast cancer specimens and may play an important role in the oncogenesis of breast carcinoma. Activated Stat3 may participate in oncogenesis by stimulating cell proliferation, promoting tumor angiogenesis, and resisting apoptosis. Because a variety of human cancers are associated with constitutively active Stat3, Stat3 represents an attractive target for cancer therapy. In this study, of the nearly 429,000 compounds screened by virtual database screening, chemical samples of top 100 compounds identified as candidate small-molecule inhibitors of Stat3 were evaluated by using Stat3-dependent luciferase reporter as well as other cell-based assays. Through serial functional evaluation based on our established cell-based assays, one compound, termed STA-21, was identified as the best match for our selection criteria. Further investigation demonstrated that STA-21 inhibits Stat3 DNA binding activity, Stat3 dimerization, and Stat3-dependent luciferase activity. Moreover, STA-21 reduces the survival of breast carcinoma cells with constitutive Stat3 signaling but has minimal effect on the cells in which constitutive Stat3 signaling is absent. Together, these results demonstrate that STA-21 inhibits breast cancer cells that express constitutively active Stat3.
Bmpr1a
and
Bmpr1b
have overlapping functions and are essential for chondrogenesis
in vivo
Authors: Yoon Byeong S. , Ovchinnikov Dmitry A. , Yoshii Isaac , Mishina Yuji , Behringer Richard R. , Lyons Karen M.
Published: March 2005

Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation
in vitro
. However, the
in vivo
role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors
Bmpr1a
or
Bmpr1b
in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of
Sox9
,
L-Sox5
, and
Sox6
expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation
in vivo
.
Regulation of inducible nitric oxide synthase by aggresome formation
Authors: Kolodziejska Katarzyna E. , Burns Alan R. , Moore Robert H. , Stenoien David L. , Eissa N. Tony
Published: March 2005

Misfolding and aggregation of proteins play an important part in the pathogenesis of several genetic and degenerative diseases. Recent evidence suggests that cells have evolved a pathway that involves sequestration of aggregated proteins into specialized “holding stations” called aggresomes. Here we show that cells regulate inducible NO synthase (iNOS), an important host defense protein, through aggresome formation. iNOS aggresome formation depends on a functional dynein motor and the integrity of the microtubules. The iNOS aggresome represents a “physiologic aggresome” and thus defines a new paradigm for cellular regulation of protein processing. This study indicates that aggresome formation in response to misfolded proteins may merely represent an acceleration of an established physiologic regulatory process for specific proteins whose regulation by aggresome formation is deemed necessary by the cell.
Functional complementation of
Trypanosoma brucei
RNA
in vitro
editing with recombinant RNA ligase
Authors: Gao Guanghan , Simpson Agda M. , Kang Xuedong , Rogers Kestrel , Nebohacova Martina , Li Feng , Simpson Larry
Published: March 2005

The ≈20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains ≈16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2.
Leishmania tarentolae
REL1 and REL2 and
Trypanosoma brucei
REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a
Baculovirus
system. When these proteins were added to mitochondrial lysates from
T. brucei
procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of
in vitro
-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the
T. brucei
recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation
in vitro
provides a powerful tool for molecular dissection of the editing reaction.
Characterizing wildfire regimes in the United States
Authors: Malamud Bruce D. , Millington James D. A. , Perry George L. W.
Published: March 2005

Wildfires statistics for the conterminous United States (U.S.) are examined in a spatially and temporally explicit manner. We use a high-resolution data set consisting of 88,916 U.S. Department of Agriculture Forest Service wildfires over the time period 1970-2000 and consider wildfire occurrence as a function of ecoregion (land units classified by climate, vegetation, and topography), ignition source (anthropogenic vs. lightning), and decade. For the conterminous U.S., we (
i
) find that wildfires exhibit robust frequency-area power-law behavior in 18 different ecoregions; (
ii
) use normalized power-law exponents to compare the scaling of wildfire-burned areas between ecoregions, finding a systematic change from east to west; (
iii
) find that wildfires in the eastern third of the U.S. have higher power-law exponents for anthropogenic vs. lightning ignition sources; and (
iv
) calculate recurrence intervals for wildfires of a given burned area or larger for each ecoregion, allowing for the classification of wildfire regimes for probabilistic hazard estimation in the same vein as is now used for earthquakes.
Beneficial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress
Authors: de Nigris Filomena , Williams-Ignarro Sharon , Lerman Lilach O. , Crimi Ettore , Botti Chiara , Mansueto Gelsomina , D'Armiento Francesco P. , De Rosa Gaetano , Sica Vincenzo , Ignarro Louis J. , Napoli Claudio
Published: March 2005

Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases the expression of oxidation-sensitive responsive genes (such as ELK-1 and
p
-JUN) in the endothelium. Evidence suggests that polyphenolic antioxidants contained in the juice derived from the pomegranate can contribute to the reduction of oxidative stress and atherogenesis. The aim of the present study was to evaluate the effects of intervention with pomegranate juice (PJ) on oxidation-sensitive genes and endothelial NO synthase (eNOS) expression induced by high shear stress
in vitro
and
in vivo
. Cultured human coronary artery endothelial cells (EC) exposed to high shear stress
in vitro
and hypercholesterolemic mice were used in this study. PJ concentrate reduced the activation of redox-sensitive genes (ELK-1 and
p
-JUN) and increased eNOS expression (which was decreased by perturbed shear stress) in cultured EC and in atherosclerosis-prone areas of hypercholesterolemic mice. Moreover, oral administration of PJ to hypercholesterolemic mice at various stages of disease reduced significantly the progression of atherosclerosis. This experimental study indicates that the proatherogenic effects induced by perturbed shear stress can be reversed by chronic administration of PJ. This approach may have implications for the prevention or treatment of atherosclerosis and its clinical manifestations.
σ-π molecular dielectric multilayers for low-voltage organic thin-film transistors
Authors: Yoon Myung-Han , Facchetti Antonio , Marks Tobin J.
Published: March 2005

Very thin (2.3-5.5 nm) self-assembled organic dielectric multilayers have been integrated into organic thin-film transistor structures to achieve sub-1-V operating characteristics. These new dielectrics are fabricated by means of layer-by-layer solution phase deposition of molecular silicon precursors, resulting in smooth, nanostructurally well defined, strongly adherent, thermally stable, virtually pinhole-free, organosiloxane thin films having exceptionally large electrical capacitances (up to ≈2,500 nF·cm
-2
), excellent insulating properties (leakage current densities as low as 10
-9
A·cm
-2
), and single-layer dielectric constant (
k
)of ≈16. These 3D self-assembled multilayers enable organic thin-film transistor function at very low source-drain, gate, and threshold voltages (<1 V) and are compatible with a broad variety of vapor- or solution-deposited p- and n-channel organic semiconductors.
Methods to improve the detection of mild cognitive impairment
Authors: Shankle William R. , Romney A. Kimball , Hara Junko , Fortier Dennis , Dick Malcolm B. , Chen James M. , Chan Timothy , Sun Xijiang
Published: March 2005

We examined whether the performance of the National Institute of Aging's Consortium to Establish a Registry for Alzheimer's Disease's 10-word list (CWL), part of the consortium's neuropsychological battery, can be improved for detecting Alzheimer's disease and related disorders early. We focused on mild cognitive impairment (MCI) and mild dementia because these stages often go undetected, and their detection is important for treatment. Using standardized diagnostic criteria combined with history, physical examination, and cognitive, laboratory, and neuroimaging studies, we staged 471 community-dwelling subjects for dementia severity by using the Clinical Dementia Rating Scale. We then used correspondence analysis (CA) to derive a weighted score for each subject from their item responses over the three immediate- and one delayed-recall trials of the CWL. These CA-weighted scores were used with logistic regression to predict each subject's probability of impairment, and receiver operating characteristic analysis was used to measure accuracy. For MCI vs. normal, accuracy was 97% [confidence interval (C.I.) 97-98%], sensitivity was 94% (C.I. 93-95%), and specificity was 89% (C.I. 88-91%). For MCI/mild dementia vs. normal, accuracy was 98% (C.I. 98-99%), sensitivity was 96% (C.I. 95-97%), and specificity was 91% (C.I. 89-93%). MCI sensitivity was 12% higher (without lowering specificity) than that obtained with the delayed-recall total score (the standard method for CWL interpretation). Optimal positive and negative predictive values were 100% and at least 96.6%. These results show that CA-weighted scores can significantly improve early detection of Alzheimer's disease and related disorders.
A central role of
Arabidopsis thaliana
ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins
Authors: Hackbusch Jana , Richter Klaus , Müller Judith , Salamini Francesco , Uhrig Joachim F.
Published: March 2005

The organization of living cells is based on networks of interacting molecules. Systematic analysis of protein interactions of 3-aa loop extension (TALE) homeodomain proteins, fundamental regulators of plant meristem function and leaf development, revealed a highly connected, complex network. The network includes nine members of
Arabidopsis thaliana
ovate family proteins (AtOFPs), a plant-specific protein family, indicating a close functional connection to TALE homeodomain proteins. Evidence is provided that AtOFP1 is an essential pleiotropic developmental regulator. AtOFP1 and AtOFP5 are shown to associate with the cytoskeleton and to regulate subcellular localization of TALE homeodomain proteins, suggesting a previously unrecognized control mechanism in plant development.
Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the
GRM6
gene encoding mGluR6
Authors: Dryja Thaddeus P. , McGee Terri L. , Berson Eliot L. , Fishman Gerald A. , Sandberg Michael A. , Alexander Kenneth R. , Derlacki Deborah J. , Rajagopalan Aruna S.
Published: March 2005

We report three unrelated patients with mutations in the
GRM6
gene that normally encodes the glutamate receptor mGluR6. This neurotransmitter receptor has been shown previously to be present only in the synapses of the ON bipolar cell dendrites, and it mediates synaptic transmission from rod and cone photoreceptors to this type of second-order neuron. Despite the synaptic defect, best visual acuities were normal or only moderately reduced (20/15 to 20/40). The patients were night blind from an early age, and when maximally dark-adapted, they could perceive lights only with an intensity equal to or slightly dimmer than that normally detected by the cone system (i.e., 2-3 log units above normal). Electroretinograms (ERGs) in response to single brief flashes of light had clearly detectable a-waves, which are derived from photoreceptors, and greatly reduced b-waves, which are derived from the second-order inner retinal neurons. ERGs in response to sawtooth flickering light indicated a markedly reduced ON response and a nearly normal OFF response. There was no subjective delay in the perception of suddenly appearing white vs. black objects on a gray background. These patients exemplify a previously unrecognized, autosomal recessive form of congenital night blindness associated with a negative ERG waveform.
CD4+T cells mediate antibody-independent acquired immunity to pneumococcal colonization
Authors: Malley Richard , Trzcinski Krzysztof , Srivastava Amit , Thompson Claudette M. , Anderson Porter W. , Lipsitch Marc
Published: March 2005

Acquired immunity toStreptococcus pneumoniae(pneumococcus) has long been assumed to depend on the presence of anticapsular antibodies. We found, however, that colonization with live pneumococci of serotypes 6B, 7F, or 14 protected mice against recolonization by any of the serotypes and that protection from acquisition of a heterologous or homologous strain did not depend on anticapsular antibody. Further, intranasal immunization by live pneumococcal colonization or by a killed, nonencapsulated whole-cell vaccine protected antibody-deficient mice against colonization, suggesting independence of antibodies to any pneumococcal antigens. Protection by intranasal immunization with whole-cell vaccine was completely abrogated in T cell-deficient mice, and in mice that were congenitally deficient in CD4+T cells or depleted of these cells at the time of challenge. In contrast, mice congenitally deficient in, or depleted of, CD8+T cells were fully protected. Protection in this model was observed beyond 2 months after immunization, arguing against innate or nonspecific immune mechanisms. Thus, we find that immunity to pneumococcal colonization can be induced in the absence of antibody, independent of the capsular type, and this protection requires the presence of CD4+T cells at the time of challenge.
Quantification of cyclic and linear flows in plants
Authors: Joliot Pierre , Joliot Anne
Published: March 2005

A method was developed to quantify the fraction of photosystem I (PSI) centers that operate according to the cyclic or linear mode, respectively. P
700
and plastocyanin oxidation were analyzed under a weak far-red excitation (approximately eight photons per s
-1
per PSI) that induces P
700
oxidation in ≈20 s and ≈3 s in dark-adapted and preilluminated leaves, respectively. This finding implies that, in dark-adapted leaves, most of the electrons formed on the stromal side of PSI are transferred back to PSI through an efficient cyclic chain, whereas in preilluminated leaves, electrons are transferred to NADP and then to the Benson-Calvin cycle. Preillumination thus induces a transition from the cyclic to the linear mode. A reverse transition occurs in the dark in a time that increases with the duration and intensity of preillumination. After a ≈10-min illumination under strong light that activates the Benson-Calvin cycle, the transition from the linear to the cyclic mode is completed in >1 h (
t
1/2
≈ 30 min). The fraction of PSI involved in the cyclic process in dark-adapted leaves can be close to 100%. An apparent equilibrium constant of ≈4 between P
700
and plastocyanin was measured during the course of the far-red illumination. This value is much lower than that computed from the midpoint redox potential of the two carriers (≈30). These results are interpreted assuming that chloroplasts include isolated compartments defined on the basis of the structural organization of the photosynthetic chain proposed by Albertsson [Albertsson, P. A. (2001)
Trends Plant Sci.
6, 349-354].
Temperature-sensitive protein–DNA dimerizers
Authors: Hauschild Karl E. , Metzler Renee E. , Arndt Hans-Dieter , Moretti Rocco , Raffaelle Marni , Dervan Peter B. , Ansari Aseem Z.
Published: March 2005

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide–YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein–DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a “switch”-like property on the protein–DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.
Pathogen effector protein screening in yeast identifies
Legionella
factors that interfere with membrane trafficking
Authors: Shohdy Nadim , Efe Jem A. , Emr Scott D. , Shuman Howard A.
Published: March 2005

Legionella pneumophila
invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how
L. pneumophila
modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying
L. pneumophila
genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four
L. pneumophila
DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from
Legionella
into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies
L. pneumophila
effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.
Mammalian motor neurons corelease glutamate and acetylcholine at central synapses
Authors: Nishimaru Hiroshi , Restrepo Carlos Ernesto , Ryge Jesper , Yanagawa Yuchio , Kiehn Ole
Published: March 2005

Motor neurons (MNs) are the principal neurons in the mammalian spinal cord whose activities cause muscles to contract. In addition to their peripheral axons, MNs have central collaterals that contact inhibitory Renshaw cells and other MNs. Since its original discovery >60 years ago, it has been a general notion that acetylcholine is the only transmitter released from MN synapses both peripherally and centrally. Here, we show, using a multidisciplinary approach, that mammalian spinal MNs, in addition to acetylcholine, corelease glutamate to excite Renshaw cells and other MNs but not to excite muscles. Our study demonstrates that glutamate can be released as a functional neurotransmitter from mammalian MNs.
Sheep gut passage and survival of Mediterranean shrub seeds
Authors: Manzano Pablo , Malo Juan E. , Peco Begoña
Published: March 2005

Although viable seeds of Mediterranean dry-fruited shrubs are found in herbivore dung, the ecological importance of this observation is still not well understood. We analysed seed retrieval percentages, defecation time and germinability after sheep gut passage for the five most common shrub species of an area in central Spain (Retama sphaerocarpa, Cytisus scoparius, Halimium umbellatum subsp. viscosum, Cistus ladanifer and Lavandula stoechas subsp. pedunculata). Five ewes were fed seeds, and their dung was collected regularly during the following week. Seeds were hand-sorted from dung subsamples and tested for germinability. The defecated seeds were clustered in time, with a majority retrieved in the 24–40 h period, although over 1% of the seeds were retained in the gut for more than 72 h. Data suggested a possible link between seed size and retrieval, with medium-sized seeds less damaged (16–23%) than larger and smaller seeds (10–12%), although only a small number of species were studied. Germination results showed an increased percentage of germination after gut passage for H. umbellatum (x2 test, P<0.05) and a marginally significant difference for C. scoparius (P<0.1). Soft-seeded L. stoechas did not germinate after gut passage. The results indicate a potential role of herbivore endozoochory for the long-distance dispersal of dry-fruited shrubs and their potential colonization of distant sites.
Longevity of seeds stored in a genebank: species characteristics
Authors: Walters Christina , Wheeler Lana M. , Grotenhuis Judith M.
Published: March 2005

Seeds of different species are believed to have characteristic shelf lives, although data confirming this are scarce, and a mechanistic understanding of why this should be remains elusive. We have quantified storage performance of c. 42,000 seed accessions, representing 276 species, within the USDA National Plant Germplasm System (NPGS) collection, as well as a smaller experiment of 207 cultivars from 42 species. Accessions from the NPGS collection were harvested between 1934 and 1975, and had relatively high initial germination percentages that decreased at a variable rate during storage at both 5 and –18°C. Germination time courses, which represent the average performance of the species, were fitted to Avrami kinetics, to calculate the time at which germination characteristically declined to 50% (P50). These P50 values correlated with other longevity surveys reported in the literature for seeds stored under controlled conditions, but there was no correlation among these studies and seed persistence observed in the classic buried seed experiment by Duvel. Some plant families had characteristically short-lived (e.g. Apiaceae and Brassicaceae) or long-lived (e.g. Malvaceae and Chenopodiaceae) seeds. Also, seeds from species that originated from particular localities had characteristically short (e.g. Europe) or long (e.g. South Asia and Australia) shelf lives. However, there appeared to be no correlation between longevity and dry matter reserves, soluble carbohydrates and parameters relating to soil persistence or resource allocation. Although data from this survey support the hypothesis that some species tend to survive longer than others in a genebank environment, there is little information on the attributes of the seed that affect its storage performance.
Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE
Authors: Honda Akira , Al-Awar Omayma S. , Hay Jesse C. , Donaldson Julie G.
Published: March 2005

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16–amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.
Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation
Authors: Mansukhani Alka , Ambrosetti Davide , Holmes Greg , Cornivelli Lizbeth , Basilico Claudio
Published: March 2005

Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with β-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt–β-catenin pathway.
Golgi positioning
Authors: Barr Francis A. , Egerer Johannes
Published: March 2005

One of the characteristics of the mammalian Golgi is its position adjacent to the nucleus. This characteristic is maintained through the action of the microtubule (MT) minus end–directed motor dynein and MT-associated proteins (MAPs). Recent findings suggest that GMAP-210, a member of the golgin family of proteins, may help to link Golgi membranes and vesicles with the MT cytoskeleton. However, there are good grounds to doubt that either GMAP-210 or its yeast homologue Rud3p is a MAP. Instead, they appear to function in vesicle trafficking events at the Golgi together with the GTPase ARF1 and a small membrane protein, Erv14. As such, the interesting question of how the Golgi interacts with MTs may well remain open to further investigation.
Genotype-level variation in lifetime breeding success, litter size and survival of sheep in scrapie-affected flocks
Authors: Chase-Topping Margo E. , Kruuk Loeske E. B. , Lajous Daniel , Touzeau Suzanne , Matthews Louise , Simm Geoff , Foster James D. , Rupp Rachel , Eychenne Francis , Hunter Nora , Elsen Jean-Michel , Woolhouse Mark E. J.
Published: March 2005

Five different sheep flocks with natural outbreaks of scrapie were examined to determine associations between individual performance (lifetime breeding success, litter size and survival) and scrapie infection or PrP genotype. Despite different breed composition and forces of infection, consistent patterns were found among the flocks. Regardless of the flock, scrapie-infected sheep produced on average 34 % fewer offspring than non-scrapie-infected sheep. The effect of scrapie on lifetime breeding success appears to be a function of lifespan as opposed to fecundity. Analysis of litter size revealed no overall or genotype differences among the five sheep flocks. Survival, however, depends on the individual's scrapie status (infected or not) and its PrP genotype. Susceptible genotypes appear to perform less well in lifetime breeding success and life expectancy even if they are never affected with clinical scrapie. One possible explanation for these results is the effect of pre-clinical scrapie. Additional evidence supporting this hypothesis is discussed.
Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins
Authors: Canto Tomas , Palukaitis Peter
Published: March 2005

The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubles.
Gene organization and sequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome
Authors: Lauzon Hilary A. M. , Jamieson Peter B. , Krell Peter J. , Arif Basil M.
Published: March 2005

Two distinct nucleopolyhedrovirus species of the eastern spruce budworm, Choristoneura fumiferana, exist in a symbiont-like relationship. C. fumiferana defective nucleopolyhedrovirus (CfDEFNPV) only infects C. fumiferana larvae per os in the presence of C. fumiferana nucleopolyhedrovirus Ireland strain (CfMNPV), but is infective when injected into the haemolymph. CfDEFNPV synergizes CfMNPV in per os infections and CfMNPV is always the predominant progeny. This study was undertaken to report the genomic makeup and organization of CfDEFNPV in an attempt to identify its defect and understand its synergistic role. The genome was mapped, sequenced, characterized and compared to other baculoviruses. The CfDEFNPV genome was 131 160 nt long with 149 putative open reading frames (ORFs) and a G+C content of 45·8 mol%. Homologues of all 62 conserved lepidopteran baculovirus genes were found including those implicated in per os infectivity, p74, per os infectivity factor (pif) and pif-2. Although no obvious deletions were observed to explain the defect, two ORFs, Cfdef79 and Cfdef99 (inhibitor of apoptosis-4), contained potential deletions. Cfdef50 (late expression factor-10)/Cfdef51 (vp1054) and Cfdef76/Cfdef77 (telokin-like protein) had large overlaps and a potential homologue to ac105/he65 was split. Four baculovirus repeat ORFs were present, as were two unique genes, but no enhancins were identified. CfDEFNPV contained 13 homologous regions, each with one to five palindromes. Comparison with fully sequenced baculovirus genomes identified CfDEFNPV as a group I NPV with the closest average amino acid identity to Epiphyas postvittana NPV, followed by Orgyia pseudotsugata MNPV and CfMNPV, with its closest matches being to individual Anticarsia gemmatalis MNPV gene sequences.
Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein σNS
Authors: Tourís-Otero Fernando , Martínez-Costas José , Vakharia Vikram N. , Benavente Javier
Published: March 2005

The avian reovirus non-structural proteinσNS has previously been shown to bind single-stranded (ss) RNAin vitroin a sequence-independent manner. The results of the present study further reveal thatσNS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting thatσNS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show thatσNS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAsin vivo. Removal of RNA from the complexes generates aσNS protein form that sediments between 4·5 and 7 S, suggesting that RNA-freeσNS associates into small oligomers. Expression and purification of recombinantσNS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-freeσNS exists as homodimers and homotrimers; (ii) the minimum RNA size forσNS binding is between 10 and 20 nt; (iii)σNS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletionσNS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entireσNS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus proteinσNS are compared with those of its mammalian reovirus counterpart.